UNIVERSIDADE ESTADUAL PAULISTA – UNESP 

CAMPUS DE JABOTICABAL 

 

 

 

 

 

XAC4296: A multidomain and exclusive 

Xanthomonadaceae protein related to chromosome 

segregation, cell division, and bacterial fitness  

 

 

 

Amanda Carolina Paulino de Oliveira 

Bióloga 

 

 

 

 

 

 

 

 

 

 

2021  



UNIVERSIDADE ESTADUAL PAULISTA – UNESP 

CAMPUS DE JABOTICABAL 

 

 

 

 

 

 

 

XAC4296: A multidomain and exclusive 

Xanthomonadaceae protein related to chromosome 

segregation, cell division, and bacterial fitness  

 

 

 

Discente: Amanda Carolina Paulino de Oliveira 

Orientador: Dr. Alessandro de Mello Varani 

Coorientador: Prof. Dr. Henrique Ferreira 

Coorientador: Dr. Rafael Marini Ferreira 

 

 

Tese apresentada à Faculdade de Ciências 
Agrárias e Veterinárias – Unesp, Câmpus de 
Jaboticabal, como parte das exigências para a 
obtenção do título de Doutor em Microbiologia 
Agropecuária 

 

 

 

 

 

2021  



Ficha catalográfica 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  



 



CURRICULAR DATA OF THE AUTHOR 

 

AMANDA CAROLINA PAULINO DE OLIVEIRA - born on February 18, 1993 

in Santa Adelia, São Paulo, Brazil. Bachelor's degree in Biology at the Faculty of 

Agricultural and Veterinary Sciences - Unesp/FCAV, Jaboticabal Campus (2012-

2015). Participated in the "Intralab" extension project related to chemical waste 

management (2012). Developed the scientific research at the Laboratory of 

Biochemistry and Molecular Biology (LBM): "PATHOGENICITY AND VIRULENCE 

EVALUATION FOR THE MUTANT mlt and mltB OF Xanthomonas citri subsp. citri" 

under the supervision of Prof. Dr. Jesus Aparecido Ferro (2013-2015). In 2016, joined 

the Agricultural and Livestock Microbiology Graduation Program, Faculty of 

Agricultural and Veterinary Sciences - Unesp/FCAV at the Technology Department, 

Laboratory of Biochemistry and Molecular Biology (LBM) under the supervision of Prof. 

Dr. Alessandro de Mello Varani and Dr. Rafael Marini Ferreira. The dissertation project 

was developed as a fellow at the Coordination for the Improvement of Higher Education 

Personnel (CAPES). Participated as a collaborator of the "BIGA PROJECT" 

(Bioinformatics, Genomic and Associated - CAPES). In the same period, joined a 

science degree in Biology from the Faculty of Agricultural and Veterinary Sciences - 

Unesp/FCAV, FCAV (2016-2018). In August 2018, Joined the doctorate in the 

Agricultural and Livestock Microbiology Graduation Program, Faculty of Agricultural 

and Veterinary Sciences - Unesp/FCAV at the Department of Agricultural and 

Environmental Biotechnology, Laboratory of Biochemistry and Molecular Biology 

(LBM) under the supervision of Prof. Dr. Alessandro de Mello Varani, Prof. Dr. 

Henrique Ferreira and Dr. Rafael Marini Ferreira. Participated as a collaborator in 

research projects at the Laboratory of Bacterial Genetics (LGB), Biochemistry and 

Microbiology Department, Instituto de Biociências, Universidade Estadual Paulista, 

Rio Claro, Brazil. The thesis project was developed as a fellow at the Coordination for 

the Improvement of Higher Education Personnel (CAPES). 

  



Acknowledgments 

 

To God for the beautiful life, for the possibilities in my life, and for guiding me in 

all choices. 

To my wonderful family for the love and support in my choices. Thanks for never 

measuring efforts to conclude my studies. For belief in my dreams and my potential. 

 To my fiancé Túlio and his family for the patience and incentive for this work. 

 To my advisors, Dr. Alessandro de Mello Varani, Rafael Marini Ferreira, and Dr. 

Henrique Ferreira, for the opportunity, patience, and support for this work. Thank you 

for teaching me and for providing my personal and professional development. 

 To the professors Dr. Jesus Aparecido Ferro and Dra. Maria Inês Tiraboschi 

Ferro for the access to the Laboratory of Biochemistry and Molecular Biology and 

contribution for this work. 

 To all Unesp/FCAV employees to execute my project. The excellent graduation 

program "Agricultural and Livestock Microbiology Graduation Program" for accepting 

me as a course council member. 

 To my friends of the Laboratory of Biochemistry and Molecular Biology and 

Technology Department, for the coffees, brunch, company, making my day. 

 To Dr. Henrique Ferreira for the opportunity and for teaching me. Thank you for 

being disponible to the laboratory dependences and contributing to this work. 

 To the Centro de Recursos Biológicos (CREBIO) to contribute to my experience, 

personal and professional, and help conclude this work. 

 To all who contributed directly or indirectly to the execution of this work. Those 

long years would not be easy without you. My most sincere thank you. 

This study was financed in part by the Coordenação de Aperfeiçoamento de 

Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. 

 

 

 

 

 

 

 



Sumário 

Summary ..................................................................................................................... 8 

RESUMO................................................................................................................... 11 

ABSTRACT ............................................................................................................... 13 

LIST OF ABBREVIATIONS ....................................................................................... 15 

LIST OF TABLES ..................................................................................................... xvii 

LIST OF FIGURES .................................................................................................. xviii 

APPENDIX ............................................................................................................. xxvii 

1. Introduction ......................................................................................................... 28 

2. LITERATURE REVIEW ...................................................................................... 31 

2.1. Xanthomonas genus ................................................................................. 31 

2.2. Xanthomonas citri and Citrus canker ........................................................ 32 

2.3. The main Xanthomonas Virulence factors ................................................ 35 

2.4. The possible role of XAC4296 in the Xanthomonas carbohydrate 

metabolism ............................................................................................................ 37 

2.5. Epimerases enzymes and their connection to XAC4296 ....................... 39 

2.6. Epimerases as multifunctional enzymes of the bacterial metabolism .... 43 

2.7. Metabolism, cell shape, and chromosome segregation ............................ 46 

2.8. The role of Lytic transglycosylases in cell shape by interacting with 

Peptidoglycan Metabolism .................................................................................... 50 

2.9. Multidomain and multifunctional enzymes (MFEs) .................................... 59 

2.10. Gene fusion and new multidomain proteins .............................................. 62 

3. MAIN AND SPECIFIC OBJECTIVES.................................................................. 64 

4. MATERIALS AND METHODS ............................................................................ 65 

4.1. In silico analysis ........................................................................................ 65 

4.2. Strains and growth conditions ................................................................... 66 

4.3. RNA extraction and cDNA synthesis from XAC4296 ................................ 67 



4.4. Mutant construction ................................................................................... 67 

4.5. Cloning XAC4296 at pMAJIIc .................................................................... 68 

4.6. Pathogenicity assay .................................................................................. 68 

4.7. Ex vivo growth curves ............................................................................... 69 

4.8. In planta growth curves ............................................................................. 69 

4.9. Microscopy ................................................................................................ 70 

4.10. Fluorescence microscopy ......................................................................... 70 

4.11. Statistics .................................................................................................... 71 

5. RESULTS ........................................................................................................... 72 

5.1. In silico analyzes of XAC4296 ................................................................... 72 

5.2. X. citri expresses XAC4296 gene in Citrus sinensis L. Osbeck ................ 81 

5.3. Pathogenicity assay supports the relation between XAC4296 and citrus 

canker progression................................................................................................. 82 

5.4. Ex planta bacterial growth indicates XAC4296 is not an essential gene ... 85 

5.5. Δ4296 cells displayed abnormal nucleoid distribution, chains, and short 

filaments ................................................................................................................. 87 

5.6. XAC4296 is required for proper cell wall synthesis in Xanthomonas ........ 90 

5.7. Δ4296 phenotype can be suppressed with nutrient supplementation ....... 93 

5.7.1. Sucrose ..................................................................................................... 94 

5.7.2. Glutamic acid ............................................................................................ 97 

5.8. XAC4296 is a cytoplasmic protein .......................................................... 100 

6. DISCUSSION ................................................................................................... 102 

7. CONCLUSIONS ............................................................................................... 109 

9. APPENDIX ........................................................................................................ 132 

 



10 

 

Certificado da Comissão Interna de Biossegurança  

 

A Comissão Técnica Nacional de Biossegurança aprovou o desenvolvimento 

do projeto intitulado “XAC4296: A multidomain and exclusive 

Xanthomonadaceae protein related to chromosome segregation, cell division, 

and bacterial fitness” 

  



11 

 

RESUMO 

Os micro-organismos apresentam um repertório limitado e 

extremamente adaptável de genes. Muitos destes genes codificam para 

proteínas contendo um único domínio ou multidomínios proteicos variáveis, 

sendo que em ambos os casos, esses domínios proteicos podem ser 

combinados de diferentes maneiras, e assim formar o repertório enzimático 

total de um genoma. A bactéria fitopatogênica Xanthomonas citri subsp. citri 

306 (X. citri), agente etiológico do cancro cítrico tipo A (CC) e considerada uma 

das doenças mais devastadoras da citricultura, apresenta um repertório de 

proteínas multidomínios ainda pouco explorado. Neste sentido, estudos 

recentes demonstraram que as proteínas multidomínios pertencentes a 

superfamília das Transglicosilases Líticas (LTs), apresentam um papel 

importante para a biologia de X. citri. As LTs, estão relacionadas com o 

metabolismo do peptidoglicano, portanto, apresentando uma função importante 

relacionada com a síntese, remodelagem e degradação da parede celular 

bacteriana. Em particular, dentre as 14 LTs presentes no genoma de X. citri, 

uma é exclusiva para este gênero, e não caracterizada experimentalmente, 

denominada XAC4296 (XAC_RS21660). Neste trabalho, empregamos 

métodos in-silico, mutação sítio dirigida e caracterização funcional, com a 

finalidade avaliar o papel que XAC4296 pode desempenhar em X. citri. Nossos 

resultados indicam que a proteína XAC4296 apresenta uma estrutura 

multidomíno, formada por dois módulos estruturalmente distintos: o primeiro 

módulo transglicosilase (PG_binding1, SLT_2), e o segundo módulo epimerase 

(aldose-1-epimerase). Análises filogenéticas e de contexto genômico indicam 

que XAC4296 provavelmente foi originada por um evento de fusão gênica a 

partir do ancestral comum para Xanthomonas, Stenotrophomonas e 

Pseudoxanthomonas. Análises funcionais indicam que o gene XAC4296 é 

expresso durante a interação patógeno-hospedeiro, porém, não é essencial 

para X. citri ou desenvolvimento do CC, mas influi no ‘fitness’ bacteriano. Além 

disso, cepas de X. citri sem o gene XAC4296 apresentaram células 

filamentosas e em formato de correntes, com sua massa cromossômica 

dispersa, porém, ainda apresentando a formação de constrição de septo, desta 

forma, sugerindo, primeiro o erro de segregação cromossômica e, 

consequentemente, divisão celular, indicando que a função LT presente neste 



12 

 

gene possa estar relacionada com este fenótipo observado. Em contrapartida, 

este fenótipo mutante foi completamente restaurado utilizando sacarose como 

fonte de carbono, e ácido glutâmico, como fonte de nitrogênio, sugerindo que o 

módulo epimerase da proteína XAC4296 possa contribuir para o metabolismo 

basal, impactando paralelamente, na divisão celular. Neste sentido foi possível 

elaborar a hipótese que, na ausência do gene XAC4296 e de precursores das 

vias metabólicas basais, a produção de piruvato é alterada, levando ao colapso 

dos mecanismos de segregação cromossômica e divisão celular. Além disso, o 

fenótipo mutante foi intensificado na presença do antibiótico ampicilina, 

atingindo 100% das células, sugerindo que como função adicional, a proteína 

XAC4296 contribui para a resistência a antibióticos β-lactâmicos em X. citri. A 

expressão da fusão mCherry-XAC4296 mostrou a proteína em ambiente 

citoplasmático, porém a presença de peptídeo sinal sugere uma localização 

secundária no periplasma, corroborando a hipótese de multifuncionalidade. 

Esses dados ressaltam o papel das proteínas multidomínio como mecanismo 

de geração de diversidade enzimática, aumentando sua funcionalidade sem 

ocasionar expansão genômica, trazendo novos conhecimentos sobre o papel 

que enzimas multidomínios podem desempenhar em X. citri. 

 

 

Palavras chave: Cancro cítrico, proteínas multidomínios, transglicosilases, 

epimerase, mCherry.  



13 

 

ABSTRACT 

Microorganisms have a limited and highly adaptable repertoire of genes capable 

of encoding proteins containing a single or variable multi-domains that can be 

combined in different ways to form the enzymatic repertoire. The 

phytopathogenic bacteria Xanthomonas citri subsp. citri (X. citri) 

(Xanthomonadaceae family), the etiological agent of Citrus Canker (CC), 

presents a collection of multi-domain and multi-functional enzymes (MFEs) that 

remains to be explored. For instance, recent studies have shown that multi-

domain proteins belonging to the superfamily of Lytic Transglycosylases (LTs) 

play an essential role in X. citri biology. The LT are enzymes that act on the 

metabolism of the peptidoglycan and bacterial cell wall. In particular, among the 

14 LTs present in the genome of X. citri, one is exclusive to this genus and has 

not yet been experimentally characterized, called XAC4296 (XAC_RS21660), 

and apart from the Transglycosylase SLT_2 and Peptidoglycan binding-like 

domains, contains an unexpected epimerase domain linked to the central 

metabolism; therefore, resembling a canonical MFE. In this work, we 

experimentally characterized XAC4296 revealing its role as an MFE in X. citri, 

demonstrating their probable gene fusion origin before the closely-related 

Xanthomonadaceae members, Xanthomonas, Xyella, Stenotrophomonas, and 

Pseudoxanthomonas genera differentiation. Interestingly, it is likely that due to 

the extensive genome reduction, the Xyella genus have lost its XAC4296 

homolog. In X. citri, the XAC4296 is expressed during plant-pathogen 

interaction, and the Δ4296 shows an impact on CC progression. Moreover, the 

Δ4296 exhibited chromosome segregation and cell division errors, and 

sensitivity to ampicillin, suggesting not only the LT activity but also that 

XAC4296 may also contribute to resistance to β-lactams. However, both Δ4296 

phenotypes are partially or wholly restored when the mutant is supplemented 

with sucrose or glutamic acid as a carbon and nitrogen source, respectively, 

supporting the epimerase domain's functional relationship with the central 

carbon and cell wall metabolism.  In this sense, it was possible to hypothesize 

that, when there is a “lack” of the XAC4296 gene and precursors of the basal 

metabolic pathways, the production of pyruvate is altered, leading to the 

collapse of the mechanisms of chromosomal segregation and cell division. 

Furthermore, The expression of the mCherry-XAC4296 fusion showed the 



14 

 

protein in a cytoplasmic environment, but the presence of a signal peptide 

suggests a secondary location in the periplasm, corroborating the hypothesis of 

multifunctionality. Taken together, these results elucidate the role of XAC4296 

as an MFE in X. citri, also bringing new insights into the evolution of multi-

domain proteins and antimicrobial resistance in the Xanthomonadaceae family. 

 

Keywords: Citrus canker, multidomain protein, transglycosylase, epimerase, 

mCherry.  



15 

 

LIST OF ABBREVIATIONS 

 

ampC     adenosine monophosphate 

Bp      Base pairs 

CC      Citrus Canker 

DPI      Days post inoculation  

Embden-Meyerhof-Parnas  EM 

Entner-Doudoroff    ED 

Fructose 6-phosphate   F6P 

ftsZ      Filamenting temperature-sensitive  

galE      UDPgalactose-4-epimerase  

gdhZ      Glutamate dehydrogenase 

galK      Galactokinase  

galM      Aldose-1-epimerase  

galT      Galactose-1-phosphateuridylyl transferase  

Glucose-6-phosphate   G6P 

GMP      5'-guanylic acid 

GTFs      Peptidoglycan glycosyltransferases  

kidO      NADH-binding oxidoreductase  

LB      Luria-Bertani 

Lipid II  [GlcNAc-(1,4)-MurNAc-(pentapeptide)-

pyrophosphoril-undecaprenol] 

LTs      LyticTransglycosylases 

manA      mannose-6-phosphate isomerase  

MFEs      Multifunctional enzymes 

NA      Nutrient agar 

NAG      N-acetylmuramic acid 

NAM      N-acetylglucosamine 

O.D.     Optical density 

opgH      Glucans biosynthesis glucosyltransferase H, 

PBPs      Penicillin-Binding Proteins 



16 

 

PEP      Phosphoenolpyruvate  

PG      Peptidoglycan 

pgcA      Phosphoglucomutase,  

pgm      Phosphoglycerate mutase,  

pykA      Pyruvate kinase 

PG_Binding1    Peptidoglycan-binding 

SLT_2     Transglycosylase SLT domain 

Sma      Stenotrophomonas maltophilia 

T3SS      Type 3 secretion system 

T4SS      Type 4 secretion system 

T6SS      Type 6 secretion system 

UDP-GalNAc    uridine-diphosphate-N-acetylgalactosamine 

ugtP      Diacylglycerol glucosyltransferase,  

X. citri     Xanthomonas citri subsp. citri 306 

XfaB  Xanthomonas fuscans subsp. aurantifolii 

strain B 

XfaC  Xanthomonas fuscans subsp. aurantifolii 

strain C  

XMP      5'-xanthylic acid  

3-phosphoglycerate   3PG 

2-phosphoglycerate   2PG 

 

 



xvii 

 

LIST OF TABLES 

 

TABLE 1. LIST OF DOMAINS FOUND IN LYTIC TRANSGLYCOSYLASE (LTS) (2018). DOMAIN 

INFORMATION WAS OBTAINED ON INTERPRO DATABASES. SOURCE: DIK ET AL., 2017; 

OLIVEIRA ET AL., 2018. .......................................................................................... 53 

TABLE 2. LYTIC  TRANSGLYCOSYLASE (LTS) AND BIOSYNTHETIC PEPTIDOGLYCAN 

TRANSGLYCOSYLASE ARE FOUND IN XANTHOMONAS CITRI (X. CITRI). THE SEARCH FOR LT 

GENES WAS PERFORMED ON THE GENBANK DATABASE AT NCBI USING THE BLAST TOOL 

(ALTSCHUL ET AL., 1997), AND CONSENSUS ANALYSIS WAS PERFORMED USING CLUSTALX 

(LARKIN ET AL., 2007) AND INTERPROSCAN (FINN ET AL., 2017) TOOLS USING THE 

CLASSIFICATION PROPOSED BY DIK ET AL. (2017). LT XAC4296 (XAC_RS21660) IS AN 

EXCLUSIVE PROTEIN IN XANTHOMONADACEAE, IT SHOWS THE DOMAINS SLT_2 AND 

PG_BINDING1 FROM THE 3B LT FAMILY AND AN ADDITIONAL EPIMERASE DOMAIN. 

PROTEINS VIRB1 (XAC_RS13315 AND XAC_RS22400) AND HPA2 (XAC_RS02185) 

WERE FOUND AFTER A NEW BLAST SEARCH. ADAPTED FROM OLIVEIRA ET AL., 2018. 56 

TABLE 3. LIST OF STRAINS AND PLASMIDS USED IN THIS STUDY. ....................................... 66 

TABLE 4. CITRUS CANKER QUANTIFICATION OF “PERA RIO” ORANGE LEAVES (CITRUS SINENSIS 

L. OSBECK) BY SPRAY METHOD. STRAINS WERE INOCULATED IN A TOTAL OF 65 LEAVES. 

THE NUMBER OF LESIONS WAS QUANTIFIED IN THE UNDERSIDE OF THE LEAVES. .......... 83 

TABLE 5. QUANTITATIVE MORPHOLOGICAL INVESTIGATION FOR XANTHOMONAS CITRI (X. CITRI) 

CONSTRUCTIONS ON RICH MEDIUM (NB). ................................................................. 88 

TABLE 6. QUANTITATIVE MORPHOLOGICAL INVESTIGATION FOR XANTHOMONAS CITRI (X. CITRI) 

CONSTRUCTIONS ON RICH MEDIUM (NB) WITH AMPICILLIN 20µG/ML. .......................... 92 

TABLE 7. LIVE-DEAD QUANTIFICATION FOR XANTHOMONAS CITRI (X. CITRI) CONSTRUCTIONS 

ON RICH MEDIUM WITH AMPICILLIN 20µG/ML. ............................................................ 93 

TABLE 8. QUANTITATIVE MORPHOLOGICAL INVESTIGATION FOR XANTHOMONAS CITRI (X. CITRI) 

CONSTRUCTIONS ON RICH MEDIUM (NB) WITH SUCROSE AND AMPICILLIN 20µG/ML. ..... 97 

TABLE 9. QUANTITATIVE MORPHOLOGICAL INVESTIGATION FOR XANTHOMONAS CITRI (X. CITRI 

) CONSTRUCTIONS ON RICH MEDIUM (NB) WITH GLUTAMIC ACID OR AMPICILLIN 20µG/ML.

 .......................................................................................................................... 100 

 

  



xviii 

 

LIST OF FIGURES 

 

FIGURE 1. DOMAIN ARCHITECTURE OF XAC4296 (WP_011052877), SHOWING THE LTS 

DOMAIN ASSOCIATED WITH 3B FAMILY: MLTB2 (PFAM13406) AND PEPTIDOGLYCAN 

BINDING (PFAM01471) DOMAINS, AND THE ALDOSE 1-EPIMERASE (PFAM01263) DOMAIN. 

THE LIPOPROTEIN SIGNAL PEPTIDE (SEC/SPII) IS SHOWN AS A RED DIAMOND. THE 

PHOSPHATE-BINDING RESIDUES AND CATALYTIC RESIDUES ARE SHOWN AS GREEN AND 

ORANGE CIRCLES ON THE EPIMERASE DOMAIN. THIS FIGURE WAS GENERATED USING THE 

PROSITE MYDOMAINS TOOL (HTTPS://PROSITE.EXPASY.ORG/CGI-

BIN/PROSITE/MYDOMAINS/). .................................................................................... 30 

FIGURE 2. COLONIES OF XANTHOMONAS CITRI SPP. CULTIVATED IN THE LABORATORY. 

COLONIES ARE USUALLY YELLOW DUE TO ‘XANTHOMONADIN’ PIGMENT PRODUCTION. 

SOURCE: 

HTTPS://WWW.APSNET.ORG/EDCENTER/DISANDPATH/PROKARYOTE/PDLESSONS/PAGES/

CITRUSCANKER.ASPX ............................................................................................ 31 

FIGURE 3. CITRUS GENOTYPES RESISTANCE AND SUSCEPTIBILITY SCALE TO CITRUS CANKER 

A. ADAPTED FROM FERRASA ET AL., 2020. .............................................................. 33 

FIGURE 4. CITRUS CANKER DISEASE INFECTION AND SYMPTOMS. A: SCANNING ELECTRON 

MICROSCOPY OF INFECTIONS BY XANTHOMONAS CITRI (X. CITRI) ON THE ABAXIAL LEAF 

SURFACE OF GRAPEFRUIT (CITRUS PARADISI). BACTERIAL EGRESS FROM A STOMATAL 

OPENING. YELLOW ARROWS INDICATE X. CITRI; RED ARROWS INDICATE STOMATAL 

OPENING. B: CITRUS CANKER LESIONS ARE RAISED WITH A CORK-LIKE APPEARANCE, 

SURROUNDED BY A YELLOW HALO. C: CITRUS CANKER SYMPTOMS IN LEAVES (C), 

BRANCHES (D), AND FRUITS (E) OF CITRIC PLANTS. SOURCE: GRAHAM ET AL., 2003 AND 

FUNDECITRUS (HTTPS://WWW.FUNDECITRUS.COM.BR/DOENCAS/CANCRO). ................. 34 

FIGURE 5. GLYCOLYTIC PATHWAYS FROM BACTERIA: EMBDEN-MEYERHOF-PARNAS (EM OR 

GLYCOLYSIS) AND ENTNER-DOUDOROFF. THE RED ARROW SHOWS THE LINK BETWEEN 

THE EM AND ED PATHWAYS. .................................................................................. 38 

FIGURE 6. EPIMERIZATION REACTION CATALYZES THE INTERCONVERSION OF ALPHA (Α) AND 

BETA (Β)-ANOMERS OF SUGARS: THE INVERSION OF THE CONFIGURATION OF AN 

ASYMMETRICAL SUBSTITUTION ON CARBON IN SUGARS. X CORRESPONDS TO GROUPS -

OH OR NH. SOURCE: ALLARD, 2001. ..................................................................... 39 

FIGURE 7. UDP-GALACTOSE 4-EPIMERASE (GALE GENE) EPIMERIZATION BY A TRANSIENT 

KETO INTERMEDIATE.1. AT THE FIRST STEP OCCURS THE ABCTRACTION OF THE 4-



xix 

 

HYDROXYL PROTON BY AN ENZYMATIC BASE AND AN ABSTRACTION OF A HYDRIDE FROM 

THE C4 POSITION OF THE SUGAR TO THE C4 POSITION ON NAD+. 2. NADH IS FORMED. 

3. A PROTON SHUTTLE MECHANISM IS CREATED, FROM SERINE (SER124) TO TYR149 AND 

A TRANSIENT KETO SUGAR IS FORMED BUT IT HAS NO CHIRALITY AT THE C4 POSITION. 4. 

THE KETO SUGAR IS NOT RELEASED BY THE ENZYME AND REMAINS BOUND, THE NADH 

TRANSFERS THE HYDRIDE BACK TO THE C4 OF THE SUGAR, BUT THIS TIME TO THE 

OPPOSITE FACE, WITH INVERSION OF CONFIGURATION AT C4 OF THE SUGAR. THE PROTON 

EXTRACTED BY TYR149 (OR SER124) IS TRANSFERRED BACK TO THE SUGAR. BLUE 

ARROWS INDICATE NAD+ REACTIONS. SOURCE: ALLARD ET AL. (2001). .................... 40 

FIGURE 8. D-RIBULOSE-5-PHOSPHATE 3-EPIMERASE (RPEASE) EPIMERIZATION BY 

ABSTRACTION/PROTONS ADDITION. THE EPIMERIZED STEREOCENTER IN A POSITION OF A 

KETO GROUP HAS LED TO THE CATALYTIC MECHANISM IN WHICH DEPROTONATION (1) AND 

REPROTONATION (3) TAKE PLACE VIA AN ENE-DIOLATE INTERMEDIATE (2). THE KETO 

GROUP IS SITUATED ON THE C2 CARBON, AND THE STEREOCENTRE AT C3 IS INVERTED. 

SOURCE: ALLARD ET AL. (2001). ............................................................................ 41 

FIGURE 9. UDP-N-ACETYLGLUCOSAMINE 2-EPIMERASE: EPIMERIZATION BY NUCLEOTIDE 

ELIMINATION AND RE-ADDITION. 1. THE FIRST STEP ENTERS WITH THE INITIAL ELIMINATION 

OF UDP FROM UDP-GLCNAC GENERATES THE INTERMEDIATE 2-ACETAMIDOGLUCAL. 2. 

THE ELIMINATION OF UDP WAS TRIGGERED BY A CATIONIC ELIMINATION. 3. READDITION 

OF UDP WITH PROTONATION OF THE C2 ATOM AT THE OPPOSITE FACE. 4. THE SYN 

ADDITION OF UDP GIVES THE PRODUCT UDP-MANNAC. SOURCE: ALLARD ET AL. (2001).

 ............................................................................................................................ 42 

FIGURE 10. EPIMERIZATION BY CARBON-CARBON BOND CLEAVAGE AS CARRIED OUT BY L-

RIBULOSE-5-PHOSPHATE 4-EPIMERASE. B REPRESENTS THE BASE, AND HB THE 

PROTONATED BASE. 1. THE FIRST STEP STARTS WITH AN ABSTRACTION OF THE PROTON 

FROM THE C4 HYDROXYL GROUP, FOLLOWED BY THE C3–C4 BOND CLEAVAGE. 2. 

DIHYDROXYACETONE ENDIOLATE IS GENERATED 3. GLYCOLALDEHYDE IS GENERATED. 4. 

THE C–C GLYCOLALDEHYDE BOND MUST BE ROTATED BY 180° TO ALLOW THE INVERSION 

OF STEREOCHEMISTRY AT C4 AFTER THE REGENERATION OF THE C3–C4 BOND. SOURCE: 

ALLARD ET AL. (2001). .......................................................................................... 42 

FIGURE 11. GALACTOSE MUTAROTASE EPIMERIZATION BY MUTAROTATION. THE GALACTOSE 

MUTAROTASE LINEAR FORM (A) HAS A KETO GROUP AT C1, WHICH SUGGESTS THE 

INTERCONVERSION BETWEEN THE Α (B) AND Β (C) FORMS IS QUITE RAPID. SOURCE: 

ALLARD ET AL. (2001). .......................................................................................... 43 



xx 

 

FIGURE 12. THE LELOIR PATHWAY OF D-GALACTOSE METABOLISM IN STREPTOCOCCUS 

THERMOPHILUS. STARTING FROM THE UPPER SIDE OF THE PANEL, THE LACTOSE IS 

CONVERTED IN Β-D-GALACTOSE BY THE Β-GALACTOSIDASE; Β-D-GALACTOSE CAN 

MUTAROTATE SPONTANEOUSLY TO THE Α-ANOMER (Α-D-GALACTOSE) AT A SLOW RATE, 

THE ENZYME ALDOSE-1-EPIMERASE (GALM) CONVERTS THE Β-D-GALACTOSE INTO Α-D-

GALACTOSE BEFORE PHOSPHORYLATION (5); THE GALACTOKINASE (GALK) CONVERTS 

THE Α-D-GALACTOSE IN GALACTOSE-1-PHOSPHATE; THE GALACTOSE-1-

PHOSPHATEURIDYLYL TRANSFERASE (GALT) CONVERTS GALACTOSE-1-PHOSPHATE IN 

UDP GALACTOSE; THE UDPGALACTOSE-4-EPIMERASE (GALE) CONVERTS THE UDP 

GALACTOSE IN UDP GLUCOSE. ALDOSE-1-EPIMERASE LINKS THE ENZYMES OF LACTOSE 

AND GALACTOSE METABOLISM INTO A COMMON PATHWAY. SOURCE: ADAPTED FROM 

SØRENSEN ET AL., 2016. ....................................................................................... 45 

FIGURE 13. MODEL LINKING CELL WALL INTEGRITY AND CHROMOSOME MORPHOLOGY IN WILD-

TYPE CELLS OF BACILLUS SUBTILIS AND ΔMANA. CELL WALL (GREEN) AND CHROMOSOME 

(RED) IN WILD TYPE AND ΔMANA CELLS. IN WILD-TYPE CELLS, THE NUCLEOID 

ORGANIZATION AND SEGREGATION ARE COORDINATED WITH CELL WALL SYNTHESIS AND 

ELONGATION. IN THE ABSENCE OF MANA, THE NORMAL EXTENSION OF THE CELL WALL IS 

BLOCKED, AS INDICATED BY THE DISAPPEARANCE OF HELICAL SIDEWALL STAINING. THE 

NUCLEOID IS DETACHED FROM CELL WALL COMPONENTS AND THE SYNCHRONIZATION IS 

LOST BETWEEN CELL GROWTH AND DNA REPLICATION AND SEGREGATION, RESULTING IN 

THE FORMATION OF POLYPLOID CELLS. SOURCE: ELBAZ; BEN-YEHUDA, 2010 (2010). 47 

FIGURE 14. THE MAINTENANCE AND SYNTHESIS OF PEPTIDOGLYCAN ARE PERFORMED IN 

THREE STEPS. A: THE FIRST STEP INCLUDES UDP-MURNAC PENTAPEPTIDE CONVERSION 

FROM UDP-GLCNAC IN THE CYTOPLASM, FOLLOWED BY LIPID I AND LIPID II PRODUCTION. 

IN THE CYTOPLASMIC MEMBRANE'S EXTRACELLULAR LEAFLET, LIPID II IS POLYMERIZED, 

CROSS-LINKED, AND PROCESSED BY TRANSGLYCOSYLASE (B), TRANSPEPTIDASE (C), 

AND CARBOXYPEPTIDASE ACTIVITIES BY PENICILLIN-BINDING PROTEINS (PBPS). THE 

PBPS ACTS BY REDUCING THE ENDS OF THE N-ACETYLMURAMIC ACID (M) OF THE 

NASCENT LIPID-LINKED PEPTIDOGLYCAN STRAND, WHICH IS LIKELY TRANSFERRED ONTO 

THE C-4 CARBON OF THE N-ACETYLGLUCOSAMINE. THE OUTER MEMBRANE IS NOT 

SHOWN. ABBREVIATIONS: GLCNAC, N-ACETYLGLUCOSAMINE; MURNAC, N-

ACETYLMURAMIC ACID. ADAPTED FROM SCHEFFERS AND PINHO (2005). ................... 51 

FIGURE 15. LYTIC TRANSGLYCOSYLASE (LTS) DOMAIN ARCHITECTURE AND FAMILY 

ORGANIZATION. MODEL LTS CLASSIFIED IN ESCHERICHIA COLI K12 (ECK4384, 



xxi 

 

ECK2958, ECK1181, ECK0211, ECK2556, ECK2809, ECK2696, ECK1083, 

ECK0626), PSEUDOMONAS AERUGINOSA PAO1 (PA3020, PA1812, PA 3764, 

PA2865, PA 1222, PA4444, PA4001,1171, PA3992, PA2963, PA4000), 

STENOTROPHOMONAS MALTOPHILIA KJ (SMLT4007, SMLT0994, SMLT3434, SMLT0155, 

SMLT4052, SMLT4650, SMLT1034, SMLT4051) AND NEISSERIA GONORRHOEAE 

FA1090 (NGO2135, NGO1033, NGO0608, NGO5004, NGO2048, NGO0626, 

NGO2038, NGO1728) ACCORDING TO THE PRESENCE OF DOMAINS AND PUTATIVE 

FUNCTION. DOMAINS WERE ASSIGNED BASED ON INTERPRO DATABASE: SLT 

(IPR008258), LYSM (IPR018392), DUF3393 (IPR023664), SBP_BAC_3 

(IPR001638), PG_BINDING_1 (IPR002477), MLTA (IPR034654), 3D (IPR034654), 

SLT_2 (IPR031304), PHAGE_LYSOZYME (IPR023347), YCEG (IPR003770), DPBB_1 

(IPR007112), SPOR (IPR007730). SOURCE: DIK ET AL. (2017). ........................... 52 

FIGURE 16. CHEMICAL REACTION PERFORMED BY LYTIC  TRANSGLYCOSYLASE (LTS). THIS 

REACTION IS DEFINED AS THE BREAKING DOWN OF POLYSACCHARIDES IN THE GLYCOSIDIC 

LIGATION BETWEEN ACID RESIDUES NAG-NAM, WHICH GOES THROUGH AN 

OXOCARBENIUM, WHICH INTERCEPTS GLUCOSAMINE 6-HYDROXYL GROUP, RESULTING IN 

THE FORMATION OF MUROPEPTIDES CONTAINING A 1,6 ANHYDROUS LIGATION IN THE 

MURAMIC ACID RESIDUE. SOURCE: DIK ET AL., 2017................................................. 54 

FIGURE 17. XAC4296 (XAC_RS21660) DOMAIN ARCHITECTURE IN XANTHOMONADACEAE 

AND NON-XANTHOMONADACEAE. A. XAC4296 PRESENTS THE DOMAIN SIGNATURE OF 

THE 3B TRANSGLYCOSYLASE FAMILY (SLT_2 AND PB_BINDING_1) AND AN ADDITIONAL 

ALDOSE-1-EPIMERASE (EP - IPR011013) DOMAIN RELATED TO CARBOHYDRATE 

METABOLISM. B. SEARCH FOR XAC4296 IN XANTHOMONADACEAE SHOWS THE 

EXCLUSIVE XAC4296 DOMAIN ARCHITECTURE TO THE GENUS, AND IN NON-

XANTHOMONADACEAE, THE TRANSGLYCOSYLASE AND EP DOMAINS APPEAR AS TWO 

SEPARATED GENES. SOURCE: OLIVEIRA ET AL., 2018. .............................................. 58 

FIGURE 18. SCHEMATIC REPRESENTATION OF PROTEINS IN BACTERIA. A. SINGLE DOMAIN 

PROTEINS (BLUE, ORANGE, AND YELLOW) PERFORM A SINGLE FUNCTION, EACH DOMAIN 

CATALYZES ONE REACTION. B. MULTIDOMAIN PROTEIN (BLUE AND ORANGE), EACH DOMAIN 

CATALYZES ONE REACTION, AND THE SINGLE-DOMAIN PROTEIN (YELLOW) PERFORMS A 

SINGLE FUNCTION. C. MULTIDOMAIN PROTEIN (BLUE AND ORANGE) PERFORMS MULTIPLE 

FUNCTIONS, NOW, THE DOMAINS MIGHT BE CATALYZING TWO STEPS, SINGLE-DOMAIN 

PROTEIN (YELLOW) PERFORMS A SINGLE FUNCTION. ADAPT FROM VOGEL ET AL., 2004.

 ............................................................................................................................ 60 



xxii 

 

FIGURE 19. SCHEMATIC REPRESENTATION OF GENE FUSION MECHANISM. A) GENE 

INSERTION/DELETION (YELLOW AND ORANGE BLOCKS) IN A GENOME STRETCH. B) 

MAINTENANCE OF NEW GENE INSERTION IN THE GENOME STRETCH (ORANGE BLOCK) AND 

GENE FISSION BETWEEN THE TWO NEXT GENES IN THE GENOME STRETCH (BLUE BLOCKS). 

C) EXCHANGE OF DOMAIN IS THE SUBSTITUTION OF ONE DOMAIN FOR ANOTHER. ADAPT 

FROM PARSEK ET AL., 2006. .................................................................................. 63 

FIGURE 20. GENOME CONTEXT, PROTEIN DOMAIN, AND STRUCTURE OF XAC4296. A: 

GENOME CONTEXT OF XAC4296 FROM XANTHOMONAS CITRI (X. CITRI) GENOME. B: 

PROTEIN DOMAIN AND STRUCTURE OF XAC4296. XAC4296 HAS 720 AA WITH THE LT 

DOMAIN ASSOCIATED WITH 3B FAMILY: TRANSGLYCOSYLASE SLT DOMAIN (SLT_2) 

(PF01464.) AND PEPTIDOGLYCAN BINDING (PG_BINDING_1) (PF01471) DOMAINS, AND 

THE ALDOSE 1-EPIMERASE (PF01263) DOMAIN, RELATED TO CARBOHYDRATE 

METABOLISM. C: MOLECULAR MODELING CARTOON REPRESENTATION OF XAC4296 

DOMAINS. ............................................................................................................. 73 

FIGURE 21. ALIGNMENT BETWEEN THE FIRST XAC4296 MODULE FORMED BY SLT_2 AND 

PG_BINDING1. ALIGNMENT WAS PERFORMED USING THE 3D MODELS AT PROTEIN DATA 

BANK (PDB). 5AO8 AND 5ANZ: SLTB3 OF PSEUDOMONAS AERUGINOSA; 1LTM: THE 

SOLUBLE LYTIC TRANSGLYCOSYLASE SLT35 FROM ESCHERICHIA COLI; 1D0K: THE 

SLT35, 4ANR AND 5O8X: THE SLTB1 FROM PSEUDOMONAS AERUGINOSA. .............. 74 

FIGURE 22. ALIGNMENT BETWEEN THE SECOND XAC4296 MODULE FORMED BY ALDOSE 1-

EPIMERASE DOMAIN. 2HTA: THE MUTAROTASE YEAD FROM SALMONELLA TYPHIMURIUM; 

1JOV: THE HI1317 FROM HAEMOPHILUS INFLUENZAE; 2CIQ AND 2CIR: THE YMR099C 

FROM SACCHAROMYCES CEREVISIAE S288C; 3K25: THE SLR1438 FROM 

SYNECHOCYSTIS SP. PCC 6803. ........................................................................... 75 

FIGURE 23. 3D STRUCTURE OF THE FIRST MODULE OF XAC4296 FROM XANTHOMONAS CITRI 

(X. CITRI) (CONTAINING THE LT DOMAINS PG_BINDING1 AND SLT_2), WAS PERFORMED 

USING SLTB3 FROM PSEUDOMONAS AERUGINOSA, IN COMPLEX WITH NAG-NAM-

PENTAPEPTIDE (5AO8) AS A MODEL (FIGURE 23A). LIGAND NAG-NAM IS DEPICTED AS 

CAPPED STICKS AND COLORED IN LIGHT BLUE FOR C ATOMS, RED FOR O ATOMS, AND 

WHITE FOR N ATOMS. THE BLUE ARROW INDICATES Α-HELIX IN THE SECONDARY 

STRUCTURE OF THE PROTEIN. A RED AMINO ACID IN THE STRUCTURE INDICATES THE 

CATALYTIC SITE ACID GLU-139. THE MODEL WAS CREATED USING THE CHIMERA 

SOFTWARE VERSION 1.15 (PETTERSEN ET A., 2004). ............................................... 76 



xxiii 

 

FIGURE 24. 3D STRUCTURE OF A PUTATIVE MUTAROTASE (XAC4296 SECOND MODULE) FROM 

XANTHOMONAS CITRI (X. CITRI), WITH YEAD FROM SALMONELLA TYPHIMURIUM AS A 

MODEL. CATALYTIC AND SUBSTRATE-POSITIONING RESIDUES: ASP-204 AND GLU-264 

(GREEN AND RED AMINO ACID, RESPECTIVELY). THE BLACK ARROW INDICATES THE Β-

SHEET AMONG THE PROTEIN. THE MODEL WAS CREATED USING SOFTWARE CHIMERA 

VERSION 1.15 (PETTERSEN ET A., 2004). ................................................................ 77 

FIGURE 25. MAXIMUM-LIKELIHOOD PHYLOGENETIC TREE OF XAC4296 HOMOLOGS ACROSS 

XANTHOMONADACEAE FAMILY SUPPORTS THE XAC4296 POTENTIAL ORIGIN BEFORE 

XANTHOMONAS, XYLELLA, PSEUDOXANTHOMONAS, AND STENOTROPHOMONAS 

DIFFERENTIATION. THE CLOSELY-RELATED XYLELLA GENUS LOST THE XAC4296 

HOMOLOG.  B.  PHYLOGENETIC CONSTRUCTION OF THE XANTHOMONADACEAE FAMILY 

PHYLOGROUP FORMED BY XANTHOMONAS, XYLELLA, PSEUDOXANTHOMONAS, AND 

STENOTROPHOMONAS (BASED ON (BANSAL ET AL., 2021). THE RED ARROW INDICATES 

THE POTENTIAL GENE FUSION EVENT THAT ORIGINATED THE XAC4296 ANCESTOR. .... 79 

FIGURE 26. GENETIC ORGANIZATION OF XAC4296 ACROSS XANTHOMONAS, 

PSEUDOXANTHOMONAS, AND STENOTROPHOMONAS SPP. THE CENTRAL GRAY 

RECTANGLE REPRESENTS THE XAC4296 POSITION IN EACH XANTHOMONADALES 

GENOME. .............................................................................................................. 80 

FIGURE 27. 1% AGAROSE GEL SHOWING AMPLIFICATION OF XAC4296 GENE IN X. CITRI. (M) 

1KB FERMENTAS DNA LADDER MARKER. (1) PCR PRODUCT OF GENOMIC DNA (~2400 

BP) USED AS A POSITIVE CONTROL (NON-INFECTING CONDITION). (2,3,4) PCR PRODUCT 

OF X. CITRI CDNA OBTAINED FROM TOTAL RNA OF 5 DAYS INFECTED CITRUS SINENSIS L. 

OSBECK WITH X. CITRI EXPRESSING IN VITRO. .......................................................... 81 

FIGURE 28. PATHOGENICITY TEST BY SPRAY METHOD. CITRUS CANKER DISEASE 

PROGRESSION IN "PERA RIO" SWEET ORANGE LEAVES SPRAYED WITH 108 CFU/ML 

BACTERIAL SUSPENSION OF X. CITRI AND MUTANT Δ4296  REPRESENTATIVE LEAVES OF 

EACH TREATMENT WITH THE HIGHEST DISEASE SEVERITY 25 DAYS POST INOCULATION ARE 

PRESENTED. ......................................................................................................... 83 

FIGURE 30. PATHOGENICITY TEST BY INFILTRATION METHOD. CITRUS CANKER DISEASE 

PROGRESSION IN LEAVES OF DIFFERENT CITRUS VARIETIES INFILTRATED WITH 106 

CFU/ML BACTERIAL SUSPENSION OF X. CITRI, Δ4296, Δ4296C AND AUTOCLAVED TAP 

WATER AS NEGATIVE CONTROL AFTER 25 DAYS POST OF INOCULATION. ON EACH LEAF, X. 

CITRI AND Δ4296C WERE INFILTRATED ON THE LEFT-HAND CENTRAL VEIN, WHILE 



xxiv 

 

MUTANTTHE Δ4296 AND NEGATIVE CONTROL WERE INFILTRATED ON THE RIGHT-HAND 

SIDE. .................................................................................................................... 84 

FIGURE 30. COMPARISON OF THE GROWTH CURVES OF EX PLANTA BACTERIAL GROWTH AND 

IN PLANTA BACTERIAL GROWTH. (A) EX PLANTA BACTERIAL GROWTH CURVE PERFORMED 

ON RICH MEDIUM NB FOR 72 H. (B) EX PLANTA BACTERIAL GROWTH WAS ACHIEVED ON 

XVM2 DEFINED MEDIUM FOR 72 H. (C) IN PLANTA BACTERIAL GROWTH CURVE. ERROR 

BARS INDICATE THE STANDARD ERROR OF THREE INDEPENDENT TECHNICAL REPLICATES.

 ............................................................................................................................ 86 

FIGURE 31. PHASE-CONTRAST IMAGES SHOWING THE MORPHOLOGY OF XANTHOMONAS CITRI 

(X. CITRI) (A) AND Δ4296 (B, C) STRAINS GROWN TO EXPONENTIAL PHASE IN NB MEDIUM. 

RED ARROWS INDICATE SEPT CONSTRICTION IN SHORT FILAMENTS (MAGNIFICATION OF 

100X) — SCALE=5 µM. .......................................................................................... 87 

FIGURE 32. MORPHOLOGICAL ANALYSIS AND CHROMOSOMAL SEGREGATION OF 

XANTHOMONAS CITRI (X. CITRI), Δ4296, AND Δ4296C STRAINS. THE FIGURE SHOWS 

MICROSCOPY PHASE CONTRAST, DAPI, AND OVERLAY OF THE TWO FILTERS, 

RESPECTIVELY, FOR A: X. CITRI WT; B: Δ4296 AND C: Δ4296C (MAGNIFICATION OF 

100X). WHITE ARROWS INDICATE NUCLEOID DISTRIBUTION; RED ARROWS INDICATE 

SEPTUM CONSTRICTION (MAGNIFICATION OF 100X), -SCALE=5 µM. ............................ 89 

FIGURE 33. EX PLANTA BACTERIAL GROWTH CURVES PERFORMED ON RICH MEDIUM AND 

AMPICILLIN 20µG/ML FOR XANTHOMONAS CITRI (X. CITRI), Δ4296, AND Δ4296C. Δ4296 

GROWTH IS AFFECTED IN THE PRESENCE OF AMPICILLIN. ERROR BARS INDICATE THE 

STANDARD ERROR OF THREE INDEPENDENT BIOLOGICAL AND TECHNICAL REPLICATES. 91 

FIGURE 34. MORPHOLOGICAL ANALYSIS OF XANTHOMONAS CITRI (X. CITRI) AND Δ4296 

STRAINS ON NB SUPPLEMENTED WITH AMPICILLIN 20µG/ML. THE FIGURE SHOWS THE 

PHASE CONTRAST, DAPI, AND OVERLAY OF THE TWO FILTERS, RESPECTIVELY, FOR A: 

XANTHOMONAS CITRI (X. CITRI); B: Δ4296 (MAGNIFICATION OF 100X). WHITE ARROWS 

INDICATE THE CHROMOSOME DISTRIBUTION, AND RED ARROWS INDICATE SEPTUM 

CONSTRICTION (MAGNIFICATION OF 100X). SCALE=5µM. .......................................... 91 

FIGURE 35. LIVE-DEAD ASSAY FOR XANTHOMONAS CITRI (X. CITRI) AND MUTANT  Δ4296 

STRAINS. THE FIGURE SHOWS THE PHASE CONTRAST, DAPI, PROPIDIUM IODIDE (IP), AND 

OVERLAY OF THE TWO FILTERS, RESPECTIVELY (MAGNIFICATION OF 100X). SCALE=5µM.

 ............................................................................................................................ 93 

FIGURE 36. EX PLANTA GROWTH CURVE FOR XANTHOMONAS CITRI (X. CITRI), Δ4296 MUTANT, 

AND Δ4296C IN DIFFERENT CONDITIONS. A: NB MEDIUM SUPPLEMENTED WITH SUCROSE 



xxv 

 

0.1%; B: NB MEDIUM SUPPLEMENTED WITH SUCROSE 0.1% (W/V) AND AMPICILLIN 

20µG/ML. ERROR BARS INDICATE THE STANDARD ERROR OF THREE INDEPENDENT 

BIOLOGICAL AND TECHNICAL REPLICATES ................................................................. 95 

FIGURE 37. SHORT FILAMENTS AND CHAIN PHENOTYPE SHOW FULL REVERSION IN THE 

PRESENCE OF SUCROSE AS CARBON SOURCE (A) MORPHOLOGICAL ANALYSIS OF X. CITRI, 

Δ4296, AND Δ4296C STRAINS ON NB SUPPLEMENTED WITH SUCROSE 0.1% (W/V). (B) 

NB SUPPLEMENTED WITH SUCROSE 0.1% (W/V) AND AMPICILLIN 20 µG/ML. THE FIGURE 

SHOWS THE PHASE CONTRAST, DAPI, AND OVERLAY OF THE TWO FILTERS, 

RESPECTIVELY, FOR X. CITRI; Δ4296 AND Δ4296C. WHITE ARROWS INDICATE THE 

CHROMOSOME DISTRIBUTION, AND RED ARROWS INDICATE SEPTUM CONSTRICTION 

(MAGNIFICATION OF 100X), SCALE=5 µM. ................................................................ 96 

FIGURE 38. EX PLANTA GROWTH CURVE FOR XANTHOMONAS CITRI (X. CITRI), Δ4296 MUTANT, 

AND Δ4296C IN DIFFERENT CONDITIONS. A: NB MEDIUM SUPPLEMENTED WITH GLUTAMIC 

ACID 2% (W/V); B: NB MEDIUM SUPPLEMENTED WITH GLUTAMIC ACID 0.1% (W/V) AND 

AMPICILLIN 20µG/ML. ERROR BARS INDICATE THE STANDARD ERROR OF THREE 

INDEPENDENT BIOLOGICAL AND TECHNICAL REPLICATES............................................ 98 

FIGURE 39. SHORT FILAMENTS, CHAIN PHENOTYPE, AND NUCLEOID ORGANIZATION SHOW FULL 

REVERSION OF THE WILD-TYPE PHENOTYPE IN THE PRESENCE OF GLUTAMATE AS CARBON 

SOURCE (A) MORPHOLOGICAL ANALYSIS OF X. CITRI, Δ4296, AND Δ4296C STRAINS ON 

NB SUPPLEMENTED WITH GLUTAMIC ACID 0.1% (W/V). (B) NB SUPPLEMENTED WITH 

GLUTAMIC ACID 0.1% (W/V) AND AMPICILLIN 20 µG/ML. THE FIGURE SHOWS THE PHASE 

CONTRAST, DAPI, AND OVERLAY OF THE TWO FILTERS, RESPECTIVELY, FOR X. CITRI, 

Δ4296 AND Δ4296C (MAGNIFICATION OF 100X). SCALE=5µM. ................................. 99 

FIGURE 40. SUBCELLULAR LOCALIZATION OF XAC4296 FUSED WITH MCHERRY IN X. CITRI-P-

4296 STRAIN. X. CITRI-P WAS USED AS A NEGATIVE CONTROL. THE FIGURE SHOWS PHASE 

CONTRAST (A1), TXRED (A2), AND OVERLAY OF THE TWO FILTERS (A3), RESPECTIVELY 

(100X MAGNIFICATION). SCALE=5µM. ................................................................... 101 

FIGURE 41. POSSIBLE ROLE OF THE MULTIDOMAIN XAC4296 PROTEIN. XAC4296 MIGHT ACT 

AS AN EPIMERASE IN THE CYTOPLASM, CONNECTING THE GLYCOLYSIS AND ENTNER–

DOUDOROFF PATHWAY. XAC4296 ALSO MIGHT ACT AS A TRANSGLYCOSYLASE IN THE 

PERIPLASM. IN BOTH CASES, XAC4296 CONTRIBUTES INDIRECTLY TO BASAL 

METABOLISM, AND ITS DELETION INCREASES CELLS DEFECTIVE PHENOTYPES. ........... 106 

FIGURE 42. POSSIBLE EFFECTS OF THE MULTIDOMAIN XAC4296 DELETION IN X. CITRI. IN 

THE ABSENCE OF THE XAC4296, MAY OCCUR AN IMBALANCE OF METABOLIC PRECURSORS 



xxvi 

 

RELATED TO ANABOLISM PATHWAYS, LEADING TO LATE CELL DIVISION AND CONSEQUENTLY 

CHROMOSOME SEGREGATION ERRORS AND AMPICILLIN RESISTANCE…………………106  

FIGURE 43. POSSIBLE EFFECTS OF THE MULTIDOMAIN XAC4296 DEPLETION IN CELLS 

CULTIVATED WITH AMPICILLIN. IN THE ABSENCE OF XAC4296 MAY OCCUR A BACTERIAL 

DECREASING OF THE POOL OF MUROPEPTIDES IN THE PERIPLASM, CONSEQUENTLY IN THE 

CYTOPLASM, LEADING TO PEPTIDOGLYCAN SYNTHESIS IMBALANCE AND INTERRUPTING 

AMPC TRANSCRIPTION, AND CONSEQUENTLY, INCREASING Β-LACTAMS 

SUSCEPTIBILITY………………………………………………………………………...107 

 

  



xxvii 

 

APPENDIX 

 

Table S1. Oligonucleotides utilized in this study……………………………………….123 

Table S2. Xanthomonadaceae used for comparative genomics. In this table, there are 

2 species for the Pseudoxanthomonas genus, 36 species for the Stenotrophomonas 

genus, and 270 species for the Xanthomonas genus.. 

…………………………………………………………………………………………..…..133 

Figure S1. Comparative analyses of XAC4296 gene nucleotide structure and domains 

localization against the Xanthomonas albilineans strain Xa-FJ1 and GPE PC73 

respective locus, showing the presence of the Epimerase and LT 3B as separated but 

overlapping genes…………………………………………………………………..........143 

Figure S2. Predicted signal peptide for XAC4296 from Xanthomonas citri. The analysis 

was performed using the SignalP 5.0 Server (ARMENTEROS et al., 2019)……….144 

 

 

 

 

 

 



28 

 

1. Introduction 

 

The Xanthomonadaceae order contains many gram-negative phytopathogens 

relevant to agriculture (An et al., 2019). A critical species in this group is the 

phytopathogen Xanthomonas citri subsp. citri 306 wild type (X. citri), the causal agent 

of citrus canker (CC) (Rodriguez-r et al., 2012). CC is a severe disease that affects 

citrus crops and decreases fruit production, leading to economic losses (Gottwald et 

al., 2002). Many efforts to understand the CC mechanisms were made since the 

disease was discovered in the early 1900s (Boch and Bonas, 2010). One of the 

hallmarks that led to several new insights into the plant-pathogen mechanisms was 

related to the analyses of the X. citri genome, revealing the genetic basis of bacterial 

pathogenicity (Ryan et al., 2011). Since then, most studies are focused on 

Xanthomonas pathogenicity mechanisms, like the regulation and secretion of virulence 

factors, such as the Type 3 Secretion System (Büttner and Bonas, 2010; Ryan et al., 

2011). Moreover, genetics studies were also conducted to understand chromosome 

segregation and cell division mechanism, aiming to better picture the cellular biology 

of this phytopathogen(Ucci et al., 2014; Lacerda et al., 2017). However, other possible 

genetic mechanisms related to Xanthomonas pathogenicity remain unknown. For 

instance, the multidomain and multifunctional enzymes (MFEs) role towards bacterial 

cellular biology, virulence, and fitness.  

The MFEs are ubiquitous in prokaryotes (Sriram et al., 2005). These proteins 

generally harbor more than one domain, each exhibiting a distinct function (Hult and 

Berglund, 2007). Therefore, the MFEs may perform multiple physiologically 

biochemical or biophysical functions simultaneously in the cell (Moore, 2004; Vogel et 

al., 2004), and these numerous functionalities might provide evolutionary advantages 

for the cell (Aharoni et al., 2005). For instance, combining multiple functions enables 

the enzyme to catalyze different steps of a single metabolic pathway (Jeffery, 2003). 

In addition, the MFEs can be considered a clever strategy for generating complexity 

from existing proteins without expanding the genome (Aharoni et al., 2005).  

One example of MFEs is the cardiolipin phosphatidylethanolamine synthase 

(CL/PEs), which is involved in unusual phospholipid biosynthesis pathways in the 

Xanthomonas campestris (Moser et al., 2014). While the CL/PEs produces cardiolipin 

(CL) using phosphatidylglycerol and cytidine diphosphate diacylglycerol (CDP‐DAG) 

like a typical ‘eukaryotic’ cardiolipin synthases (Cls), this enzyme can also condense 



29 

 

ethanolamine (EA) and CDP‐DAG to phosphatidylethanolamine (PE), representing an 

unusual PE biosynthesis mechanism in bacteria (Moser et al., 2014). Indeed, the MFEs 

emerged during species evolution and how they gained their multiple functions by gene 

fusion or other recombination events is not yet wholly established (Cheng et al., 2012). 

In sharp contrast to the MFEs, one interesting class of enzymes has gained 

attention by their relation to bacterial fitness and virulence. These are the Lytic 

Transglycosylases (LTs), which are related to cell-wall recycling and cell-wall-antibiotic 

detection (Dik et al., 2017). LTs are also ubiquitous in prokaryotes, showing 

involvement with the peptidoglycan biosynthesis and recycling, cell division, septum 

division allowing cell separation, and insertion of protein complexes like secretion 

systems, flagella, and pili (Höltje, 1995; Koraimann, 2003; Scheurwater and Clarke, 

2008; Uehara and Park, 2008; Scheurwater and Burrows, 2011; Alcorlo et al., 2017; 

Dik et al., 2017). Due to these features, LTs may also play a relevant role in the 

pathogenesis and fitness of many bacterial species, such as Neisseria gonorrhoeae 

(Chan et al., 2012), Burkholderia pseudomallei (Jenkins et al., 2019), and 

Xanthomonas citri (X. citri) (Oliveira et al., 2018).  

Recently, we described the LT's arsenal present in the X. citri genome (16 LTs 

from different families) (Oliveira et al., 2018). Among those, we functionally revealed 

that two LTs from the 3B family: MltB2.1 and MltB2.2, are directly implicated in X. citri 

fitness (Oliveira et al., 2018). We also identified another 3B-like LT, named as 

XAC4296 (NCBI locus_tag: XAC_RS21660). Notably, apart from the Transglycosylase 

SLT 2 (IPR031304) and Peptidoglycan binding-like (IPR002477) domains, XAC4296 

contains an additional and unexpected aldose 1-epimerase domain (IPR015443) 

linked to carbohydrate metabolism, and potentially showing involvement with the 

bacterial cell wall metabolism and biosynthesis of a variety of cell surface 

polysaccharides (Sala et al., 1996) (Figure 1). Interestingly, the XAC4296 gene was 

previously identified exclusively in the Xanthomonas genus (Oliveira et al., 2018). 

Moreover, in silico analyses revealed that XAC4296 appears to have been formed by 

a previous gene fusion event, originated by two independent genes (a 3B family LT 

and D-hexose-6-phosphate mutarotase gene), commonly separated in distinct loci in 

other non-Xanthomonas species (Oliveira et al., 2018). Therefore, XAC4296 

resembles a canonical MFEs, showing a multi-domain architecture. 



30 

 

 

 

Figure 1. Domain architecture of XAC4296 (WP_011052877), showing the LTs domain 

associated with 3B family: MltB2 (pfam13406) and Peptidoglycan binding (pfam01471) 

domains, and the Aldose 1-epimerase (pfam01263) domain. The Lipoprotein signal 

peptide (Sec/SPII) is shown as a red diamond. The phosphate-binding residues and 

catalytic residues are shown as green and orange circles on the epimerase domain. 

This figure was generated using the Prosite MyDomains tool 

(https://prosite.expasy.org/cgi-bin/prosite/mydomains/). 

 
 

In this work, we performed an in silico, fluorescence microscopy and 

pathogenicity assays to investigate the evolution and role of XAC4296 as a putative 

MFE. We also evaluated XAC4296 as a potential X. citri virulence and pathogenicity 

factor. Our results indicate that XAC4296 functions resemble a typical LT, mainly 

related to peptidoglycan biosynthesis. We also unveiled an additional role related to 

carbohydrate metabolism, compatible with epimerase domain, and chromosome 

segregation during cell division. Taken together, these results demonstrate that 

XAC4296 behaves like a classic MFE, showing at least two unrelated and 

mechanistically different roles: a primary role related to enzymatic catalysis and a 

secondary role related to cell structural function.  



31 

 

2. LITERATURE REVIEW 

 

2.1. Xanthomonas genus 

 

Xanthomonas genus (from greek, xanthos = yellow; monas = entity) comprises 

more than 200 bacterial species belonging to the Gammaproteobacteria class. 

Xanthomonas are obligate aerobic, rod-shaped bacteria (1.5–2.0 × 0.5–0.75 µm), 

presenting a single polar flagellum and yellow coloration once cultivated due to the 

production of xanthomonadin (He et al., 2020) (Figure 2).  

 

 

Figure 2. Colonies of Xanthomonas citri spp. cultivated in the laboratory. Colonies are 

usually yellow due to ‘xanthomonadin’ pigment production. Source: 

https://www.apsnet.org/edcenter/disandpath/prokaryote/pdlessons/Pages/CitrusCank

er.aspx 

 

Bacteria belonging to this genus infect economically important monocot and 

dicot cultures and presents many hosts, such as Citrus spp. (lemons, sweet oranges, 

sour oranges, grapefruits, among others), Oryza ssp. (rice), crucifers (broccoli, 

cauliflower, lettuce, radish, and Arabidopsis thaliana) and Manihot esculenta (cassava) 

(Ryan et al., 2011). However, they still possess too high host specificity, therefore, 

each member usually infects only a few or even a single host (Jacques et al., 2016). 

Furthermore, Xanthomonas can epiphytically survive in soil, seeds, and harvest 

remains, interacting with insects and hosts or non-host plants (An et al., 2019). 

Therefore, these bacteria can be disseminated during agricultural practices, such as 



32 

 

pruning, nebulization, rainwater, contaminated soil, and insects (e.g., “Citrus leafminer” 

- Phyllocnistis citrella).  

The Xanthomonas possess several invasion strategies to enter into plant 

tissues: initially, bacteria cells adhere to the host surface using its adhesins, gaining 

access through wounds or hydathodes, systematically spreading on the vascular 

system, or penetrating through stomata, colonizing leaf mesophyll (Ference et al., 

2018). 

Many functional and comparative genomics studies have been developed to 

elucidate the mechanisms related to the adaptation and evolution of Xanthomonas, 

given the vast diversity of plant hosts and host tissues (Lam et al., 2009)(An et al., 

2019). Among the species of the Xanthomonas genus, different species and strains 

can infect citrus plants (e.g., Citrus sinensis). The most common is Xanthomonas 

fuscans subsp. aurantifolii strain B (XfaB) and C (XfaC), both the etiologic agents of 

the citrus cancrosis; Xanthomonas alfalfae subsp. citrumelonis (Xacm) causing citrus 

bacterial spot (Vauterin et al., 1995; Schaad et al., 2006), and X. citri, causal agent of 

the citrus canker (CC) disease (Da Silva et al., 2002). 

 

2.2. Xanthomonas citri and Citrus canker  
 

X. citri is the causal agent of citrus canker (CC) type A, a globally significant 

disease that affects all Citrus varieties, compromising the commerce of fruits and sub-

products worldwide (Gottwald et al., 2002). CC severity varies according to the citrus 

species. In general, while the kumquats (Fortunella spp.) are resistant, the sweet 

oranges (Citrus sinensis L. Osbeck) ‘Bahia’, ‘Hamlin’, ‘Valencia’ and ‘Pera’ shows 

intermediate levels of susceptibility, whereas the ‘Mexican’ lime (Citrus aurantifolia 

(Christm.) Swingle) are considered susceptible to CC (Ferrasa et al., 2020) (Figure 3). 

Therefore, millions of dollars are spent annually on prevention, quarantines, and 

eradication programs for disease control (USDA, 2021).  

 



33 

 

 

 

Figure 3. Citrus genotypes resistance and susceptibility scale to citrus canker A. 

Source: Ferrasa et al., 2020. 

 

The CC infection starts when X. citri attaches to the host tissue (Graham, 1992). 

The pathogen enters the host tissue through wounds or stomatal openings (Figure 4A). 

The CC symptoms start as pinpoint spots. After approximately ten days, young leaves 

show pustules. Eventually, the leaves become corky and crateriform with a raised 

margin, surrounded by a yellow halo, a process known as waker-soaking (Figure 4B) 

(Schubert and Sun, 2003). The CC lesions are readily observable as cork-like lesions 

on both leaf faces, in branches, leaves, and fruits (Figure 4 C, D, and E) (Gottwald et 

al., 2002). 

 



34 

 

 

Figure 4. Citrus canker disease infection and symptoms. A: Scanning electron 

microscopy of infections by Xanthomonas citri (X. citri) on the abaxial leaf surface of 

grapefruit (Citrus paradisi). Bacterial egress from a stomatal opening. Yellow arrows 

indicate X. citri; red arrows indicate stomatal opening. B: Citrus canker lesions are 

raised with a cork-like appearance, surrounded by a yellow halo. C: Citrus canker 

symptoms in leaves (C), branches (D), and fruits (E) of citric plants. Source: Graham 

et al., 2003 and Fundecitrus (https://www.fundecitrus.com.br/doencas/cancro). 

 

The spread of CC occurs by wind or raindrops, always respecting short 

distances within neighboring trees or by contaminated agricultural instruments. In 

general, the disease develops more severely on the side of the tree exposed to wind-

driven rain (Gottwald and Timmer, 1995). In addition, leafminer infestations caused by 

Phyllocnistis citrella can also contribute to the X. citri dissemination (Gottwald et al., 

2002). 

Epidemiological, genomic, and evolutionary studies indicate that CC was 

originated in Southeast Asia (Patané et al., 2019). Nowadays, the disease is also 

present in Japan, Central Africa, South America (Brazil and Argentina), and the United 

States of America (Florida) (Schubert and Sun, 2003; Canteros et al., 2017).  

CC notably affects the American continent (Schubert and Sun, 2003). For 

instance, Brazil is responsible for the largest orange production globally, producing at 

least 14% of the global orange fruits (3.6 million metric tons), and has the most 



35 

 

extensive orange juice production, corresponding to 17% of the global orange juice, 

equivalent to 1.8 million tons (USDA, 2021). Indeed, Brazilian citriculture is one of the 

most important agricultural activities for the country, with production concentrated in 

São Paulo and Minas Gerais states. However, CC infection rate was estimated at 

17.26% in Sao Paulo and Minas Gerais orchards (USDA, 2021), making the country 

one of the centers of CC (Behlau, 2021).  

Unfortunately, there is no treatment for CC, and management and sanitation are 

the only ways to control the disease (Ismail and Zhang, 2004). However, it is still not 

easy to manage the disease in the production fields. For example, one alternative to 

CC control relies on copper-based antimicrobial products; however, indiscriminate use 

gives rise to resistant X. citri strains (Voloudakis et al., 2005; Richard et al., 2017). 

Conversely, copper is considered hazardous to human health and the environment 

(Behlau et al., 2013). Other promising alternatives to control CC, such as alkyl gallates 

(Savietto et al., 2018) and even transgenic citrus varieties (Jia and Wang, 2020; 

Martins et al., 2021) are under scrutiny and development. 

 
 

2.3. The main Xanthomonas Virulence factors  

 

Virulence factors are molecules encoded in the pathogen genome which are 

secreted to enable infection or damage in the host tissue (Casadevall and Pirofski, 

2009). In Xanthomonas, secretion systems (1 to 6) are associated with virulence 

factors related to disease development (Büttner and Bonas, 2010; Alvarez-martinez et 

al., 2021). Moreover, other virulence factors, like adhesins, extracellular 

polysaccharides (EPS), lipopolysaccharides (LPS), and degradative enzymes, such as 

proteases, lipases, and cell wall-degrading enzymes, are also part of the Xanthomonas 

arsenal to ensure efficient multiplication and disease progression (Büttner and Bonas, 

2010).  

The Xanthomonas pathogenicity is mainly determined by the Type 3 Secretion 

System (T3SS), also called injectisome (Cornelis, 2006; Saijo and Schulze-lefert, 

2008). The T3SS appears as a needle-like structure capable of penetrating the plant 

cell wall, connecting the bacterial cell to the cytosol of the plant cell (Wagner et al., 

2018). The main role of T3SS is to inject effector proteins directly into the host cell 



36 

 

cytosol to manipulate plant cellular processes such as basal defense for the benefit of 

the pathogen (Büttner and Bonas, 2010).  

Several effector proteins are involved with CC disease development (Ference 

et al., 2018), such as Xop effectors (Xanthomonas outer proteins) (Jalan et al., 2013), 

and the Transcription activation-like effectors (TALEs), also known as AvrBs3/PthA 

(avirulence and pathogenicity proteins). Both effectors are injected by the T3SS 

machinery into the plant cell. Of those, however, TALEs migrates to the cell nucleus 

and binds to a specific DNA sequence (Boch et al., 2009), acting as a classic 

transcription factor, modulating the host gene expression, presumably by direct 

interaction with the host transcription machinery (Boch and Bonas, 2010; Bogdanove 

et al., 2010). Indeed, TALEs genes are essential for full CC induction, activating host 

susceptibility genes (An et al., 2019). 

X. citri also uses other virulence factors for bacterial survival during epiphytic 

and endophytic growth (Rigano et al., 2007). These are related to the biosynthesis of 

lipopolysaccharides (LPS), extracellular polysaccharides (EPS), and biofilm formation 

(Büttner and Bonas, 2010).  

LPS protects the bacteria against antimicrobial compounds and other external 

stresses, being a component of the outer membrane (Kummerfeld and Teichmann, 

2005). In addition, during the plant-pathogen interactions, the LPS may also protect 

the bacterial cell acting as a PAMP (“Pathogen-Associated Molecular Pattern”), 

controlling the plant defense responses such as pathogenesis-related gene 

expression, oxidative burst, and thickening of the plant cell wall (Dow m., 2000). 

The EPS or xanthan gum is a main feature of the Xanthomonas species, leading 

to the bacterial colonies a mucoid appearance (Becker et al., 1998). The gum gene 

cluster directs xanthan gum production (12 genes: gumB to gumM), a polymer of 

repeating pentasaccharide units with cellulose and trisaccharide side chains (Becker 

et al., 1998; Katzen et al., 1998). The role of xanthan gum is different among 

Xanthomonas strains and hosts (Kemp et al., 2004; Dunger et al., 2007; Rigano et al., 

2007). In X. citri, the xanthan gum is necessary for initial states of CC development, 

displaying direct involvement with bacterial adhesion to host cells and protecting 

against environmental stresses (Rigano et al., 2007; Facincani et al., 2014). In 

contrast, for X. campestris, the xanthan gum induces plant susceptibility by 

suppressing callose deposition (Oa et al., 2006). In addition, xanthan gum might 

increase bacterial colonization in the host since it affects the motility by flagellum-



37 

 

independent movement, the so-called “sliding motility”, and is one of the main 

components of biofilm matrix (Dunger et al., 2007; Malamud et al., 2013). 

Biofilms are a dense surface-associated microorganisms community connected 

to the cell through an extracellular polymeric substance matrix composed of LPS, 

proteins, and nucleic acids, important for greater resistance to antibiotics and host 

immune effectors (Costerton, 1995). In Xanthomonas, the biofilm is formed after the 

initial attachment to the host. This primary function is to protect bacteria against abiotic 

stress, acting as a defense mechanism (An et al., 2019). Biofilms structure, assembly, 

and dispersal are mediated by the quorum-sensing signal molecule diffusible signal 

factor (DSF) (Rigano et al., 2007). 

 

 

2.4. The possible role of XAC4296 in the Xanthomonas carbohydrate 

metabolism 

 

Procaryotes have three main metabolic pathways to degrade monosaccharides: 

the Embden-Meyerhof-Parnas (EM or glycolysis), the Entner-Doudoroff (ED), and the 

pentose phosphate pathways (Chubukov et al., 2014) (Figure 5). Glycolysis takes 

place in the cytosol of the cell and occurs in two phases: the investment phase, where 

adenosine triphosphate (ATP) is consumed and glucose is broken down into two three-

carbon compounds, generating pyruvic acid as a product. The yield phase, where the 

free energy produced in this process is used to form pyruvate. In this process, the cell 

produces four high-energy ATP molecules and two nicotinamide adenine dinucleotide 

(NADH). Each reaction in glycolysis is catalyzed by many enzymes, producing 

intermediate metabolites (Bender, 2013). 

Some organisms do not have the glycolysis pathway or show some variances 

to the classical glycolytic pathways, such as the ED pathway. ED was first discovered 

in Pseudomonas saccharophila (1952) (Entner and Douroff, 1952) and later in E. coli 

(1967) (Eisenberg and Dobrogosz, 1967), then, it was discovered in all domains 

(Eukarya, Bacteria, and Archaea), but most notably in Gram-negative bacteria 

(Conway, 1992). Subsequently, studies performed in Xylella fastidiosa 9a5c showed 

that the ED pathway prevails over the glycolysis pathway in carbohydrate metabolism 

(Facincani et al., 2003). In X. campestris pv. campestris, the central metabolism was 

elucidated using 13C-based metabolic flux and NMR-based isotopologue profiling, 



38 

 

showing the prevalent role of ED pathway over glycolysis and pentose phosphate 

pathways (Schatschneider et al., 2014). 

The ED metabolic pathway converts glucose into pyruvate using alternative 

enzymes and, has a net yield of one ATP, one NADH, and one NADPH per glucose 

molecule processed (Figure 6) (Romano and Conway, 1996). In many cases, bacteria 

perform the ED pathway using gluconate as a carbon source (Conway, 1992). 

 

 

Figure 5. Glycolytic pathways from bacteria: Embden-Meyerhof-Parnas (EM or 
glycolysis) and Entner-Doudoroff. The red arrow shows the link between the EM and 
ED pathways. 

 

Many enzymes catalyze specific steps in these metabolic pathways, such as 

the glucose phosphate isomerase (pgi) which converts glucose-6-phosphate (G6P) 

into fructose 6-phosphate (F6P) in the EM pathway, which is related to the 

carbohydrate degradation (Rose, 1975). The phosphoglycerate mutase isomerases 

convert 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG) through a 2,3-

bisphosphoglycerate intermediate, in the glycolysis via ED and EM pathways, 

respectively (Figure 5, enzyme number 8). Curiously, the X. citri XAC4296 protein (the 

main focus of this study) contains an aldose 1-epimerase (pfam01263) domain, 



39 

 

belonging to the “Isomerases family”, which catalyzes the conversion of one isomeric 

form of a chemical compound to another within one molecule (McDonald et al., 2015). 

Therefore, it is tempting to speculate that the X. citri XAC4296 protein may have a role 

as a pgi-like enzyme, and presumably contribute to EM and ED pathways (Figure 5). 

 

 

2.5. Epimerases enzymes and their connection to XAC4296 

 

As stated in the previous topic, the X. citri XAC4296 contains an epimerase 

domain. The Epimerases are an important class of isomerases, enzymes that performs 

the interconversion of an isomeric form of a chemical compound into another 

(McDonald et al., 2015). The isomerases comprises the following families: Isomerases 

cis-trans (EC 5.2); intramolecular oxidoreductases (EC 5.3); intramolecular 

transferases (EC5.4); and intramolecular lyases (EC 5.5) (McDonald et al., 2015). This 

enzyme catalyzes the inversion of the configuration of an asymmetrically substituted 

carbon in linear or cyclic sugars (Figure 6). 

The epimerases are abundant in procaryotes and the microorganisms might 

benefit from using these enzymes to produce complex carbohydrate polymers, using 

them as biosynthetic building blocks in their cell wall (McNeil et al., 1990).  

 

 

Figure 6. Epimerization reaction catalyzes the interconversion of alpha (α) and beta 

(β)-anomers of sugars: the inversion of the configuration of an asymmetrical 

substitution on carbon in sugars. X corresponds to groups -OH or NH. Source: Allard, 

2001. 

 

Epimerases are usually involved in metabolic pathways such as inversion of D-

alanine and D-glutamate for bacterial cell wall metabolism (Sala et al., 1996), 

biosynthesis of a variety of cell surface polysaccharides, biosynthesis of heparin and 

heparin sulfate (Li et al., 2001), biosynthesis of LPS and capsular sugar precursors 



40 

 

(McNeil et al., 1990) and complex biosynthetic pathways, such as Embden-Meyerhof-

Parnas pathway (Glycolysis) (Figure 6), Entner-Doudoroff (Figure 5), Leloir and others 

that present several chemical steps (Nowitzki et al., 1995; Teige et al., 1995). 

Epimerases are also involved in many chemical steps, such as oxidation, 

acetylation, dehydration and carbohydrate reduction (Allard et al., 2001). The 

carbohydrate epimerization occurs in five ways, as described below (reviewed by 

Allard et al., 2001). 

 

a) Epimerization employing a transitory keto intermediate.  

The UDP-galactose 4-epimerase is the most common example of an enzyme 

with this type of epimerization. It catalyzes UDP-glucose and UDP-galactose 

interconversion by inverting the stereochemistry at the C4 position, utilized in de novo 

biosynthesis of sugar (Figure 7) (Carnell, 1999). In addition, this enzyme performs the 

biological interconversion of galactose and glucose in the Leloir Pathway (Frey, 1996). 

 

 

Figure 7. UDP-galactose 4-epimerase (GALE gene) epimerization by a transient keto 

intermediate.1. At the first step occurs the abctraction of the 4-hydroxyl proton by an 

enzymatic base and an abstraction of a hydride from the C4 position of the sugar to 

the C4 position on NAD+. 2. NADH is formed. 3. A proton shuttle mechanism is 

created, from serine (Ser124) to Tyr149 and a transient keto sugar is formed but it has 

no chirality at the C4 position. 4. The keto sugar is not released by the enzyme and 

remains bound, the NADH transfers the hydride back to the C4 of the sugar, but this 

time to the opposite face, with inversion of configuration at C4 of the sugar. The proton 

extracted by Tyr149 (or Ser124) is transferred back to the sugar. Blue arrows indicate 

NAD+ reactions. Source: Allard et al. (2001). 

 

 

b) Epimerization by abstraction/protons addition  

The enzyme D-ribulose-5-phosphate 3-epimerase is found in the oxidative 

pentose phosphate pathway. This enzyme catalyzes the conversion of D-ribulose 5-



41 

 

phosphate to D-xylulose 5-phosphate. In these reactions, the keto group is located on 

the C2 carbon, and the stereocenter in the C3 is inverted. This mechanism involves 

deprotonation and reprotonation and occurs via an enediolate intermediate (Figure 8) 

(Kopp et al., 1999). 

 

 

Figure 8. D-ribulose-5-phosphate 3-epimerase (RPEase) epimerization by 

abstraction/protons addition. The epimerized stereocenter in a position of a keto group 

has led to the catalytic mechanism in which deprotonation (1) and reprotonation (3) 

take place via an ene-diolate intermediate (2). The keto group is situated on the C2 

carbon, and the stereocentre at C3 is inverted. Source: Allard et al. (2001). 

 

 

c) Epimerization by elimination/nucleotides readdiction. 

The enzyme UDP-N-acetylglucosamine 2-epimerase might be necessary for 

bacteria since it catalyzes the reversible interconversion of UDP-N-acetylglucosamine 

(UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). This mechanism 

occurs by the elimination and readdiction of UDP from the molecule (Figure 9) (Sala et 

al., 1996). 

 



42 

 

 

Figure 9. UDP-N-acetylglucosamine 2-epimerase: epimerization by nucleotide 

elimination and re-addition. 1. The first step enters with the initial elimination of UDP 

from UDP-GlcNAc generates the intermediate 2-acetamidoglucal. 2. The elimination 

of UDP was triggered by a cationic elimination. 3. Readdition of UDP with protonation 

of the C2 atom at the opposite face. 4. The syn addition of UDP gives the product UDP-

ManNAc. Source: Allard et al. (2001). 

 

 

 d) Epimerization by carbon-carbon ligation cleavage. 

The enzyme L-ribulose-5-phosphate 4-epimerase interconverts L-ribulose 5-

phosphate and D-xylulose 5-phosphate by inverting the configuration at the C4 

stereocenter and without the incorporation of solvent-derived oxygen and hydrogen 

(Figure 10) (Salo et al., 1972). This enzyme enhances bacteria to use arabinose as 

the energy source, connecting the arabinose pathway to the pentose phosphate 

pathway (Johnson and Tanner, 1998). 

 

 

Figure 10. Epimerization by carbon-carbon bond cleavage as carried out by L-ribulose-

5-phosphate 4-epimerase. B represents the base, and HB the protonated base. 1. The 

first step starts with an abstraction of the proton from the C4 hydroxyl group, followed 

by the C3–C4 bond cleavage. 2. Dihydroxyacetone endiolate is generated 3. 

Glycolaldehyde is generated. 4. The C–C glycolaldehyde bond must be rotated by 180° 

to allow the inversion of stereochemistry at C4 after the regeneration of the C3–C4 

bond. Source: Allard et al. (2001). 

 

 



43 

 

e) Epimerization by ring-opening epimerization or mutarotation.  

The foremost example of this reaction is the galactose mutarotase, a ubiquitous 

enzyme that converts the β anomer to the α anomer (inversion of chirality at C1) of 

galactose (Figure 11) (Hucho and Wallenfels, 1971). Galactose mutarotase enzymes 

have been reported in bacteria (Thoden et al., 2003), plants (Martyn bailey et al., 1966), 

fungi (Martyn bailey et al., 1966), and mammals (Timson and Ã, 2003), including 

humans (hsGalM) (Thoden and Holden, 2005). It has been suggested that galactose 

mutarotase be included in a superclass of enzymes (Thoden et al., 2003). In the first 

step of the Leloir pathway, these enzymes actuate on galactose metabolism, 

converting β-D- galactose to α-D-galactose, playing an auxiliary role on carbohydrate 

metabolism (Holden et al., 2003). Presumably, X. citri XAC4296 protein, which 

contains the aldose 1-epimerase domain, might perform the epimerization by ring-

opening or mutarotation (Thoden et al., 2003). 

 

 

Figure 11. Galactose mutarotase epimerization by mutarotation. The galactose 

mutarotase linear form (A) has a keto group at C1, which suggests the interconversion 

between the α (B) and β (C) forms is quite rapid. Source: Allard et al. (2001). 

 

 

2.6.  Epimerases as multifunctional enzymes of the bacterial metabolism  

 



44 

 

In cellular metabolism, sugar is converted into a few simple carbohydrates 

(monosaccharides): glucose, fructose, and galactose (Thoden et al., 2003). 

Carbohydrates are central to many essential metabolic pathways, which involve: 

Glycolysis, Gluconeogenesis, Glycogenolysis, Glycogenesis, Pentose phosphate 

pathway, and more specifically in the Fructose metabolism and Galactose metabolism. 

In this context, isomerases classified as epimerases play a central role, contributing to 

sugar interconversions (Allard et al., 2001) (Figure 5).  

For instance, the isomerases from the EM pathway, like the phosphoglucose 

isomerase/phosphoglucoisomerase (PGI) is a cytoplasmic protein that interconverts 

aldehyde-D-glucose 6-phosphate into keto-D-fructose 6-phosphate (Figure 5) (Rose, 

1975). Moreover, the triose-phosphate isomerase (TPI) is an enzyme that catalyzes 

the reversible interconversion of dihydroxyacetone phosphate into D-glyceraldehyde 

3-phosphate (Albery and Knowles, 1976). Finally, the ribose-5-phosphate isomerase 

interconverts ribulose-phosphate 3-epimerase into ribulose-5-phosphate in the Calvin 

cycle and Penthouse phosphate pathway (Nowitzki et al., 1995).  

An example of epimerases in the cell metabolism is present in the Leloir 

pathway (LP), which is an exclusive mechanism to convert galactose to glucose 

without external galactose (Figure 12) (Frey, 1996). This pathway uses as an initial 

substrate D-galactose, which is metabolized to glucose-1-phosphate by four enzymes: 

aldose-1-epimerase or galactose mutarotase (galM), galactokinase (galK), galactose-

1-phosphateuridylyl transferase (galT), and UDPgalactose-4-epimerase (galE) 

(Holden et al., 2003). This metabolic pathway is used as an energy and carbon source 

during the anabolic pathway of carbohydrate metabolism. For instance, it is used to 

synthesize cell wall compounds and exopolysaccharides, where the galactosides are 

required as building blocks (Frey, 1996).  

 

https://en.wikipedia.org/wiki/Cytoplasm


45 

 

 

Figure 12. The Leloir pathway of D-galactose metabolism in Streptococcus 

thermophilus. Starting from the upper side of the panel, the lactose is converted in β-

D-galactose by the β-galactosidase; β-D-galactose can mutarotate spontaneously to 

the α-anomer (α-D-galactose) at a slow rate, the enzyme aldose-1-epimerase (galM) 

converts the β-d-galactose into α-d-galactose before phosphorylation (5); the 

galactokinase (galK) converts the α-D-galactose in galactose-1-phosphate; the 

galactose-1-phosphateuridylyl transferase (galT) converts galactose-1-phosphate in 

UDP galactose; the UDPgalactose-4-epimerase (galE) converts the UDP galactose in 

UDP glucose. Aldose-1-epimerase links the enzymes of lactose and galactose 

metabolism into a common pathway. Source: adapted from Sørensen et al., 2016. 

 

Another example of epimerase from the LP is the galM (aldose 1-epimerase) 

(STH8232_RS07000) from Streptococcus thermophilus, which converts D-glucose 

into L-glucose (Poolman et al., 1990). This enzyme is part of the gal operon, and it was 

characterized by several microorganisms, including E. coli (Bouffard et al., 1994). The 

Aldose 1-epimerase is also active on D-glucose, L-arabinose, D-xylose, D-galactose, 

maltose and lactose pathways. In addition, this enzyme is involved in the hexose 

pathway metabolism, which is also part of the carbohydrate metabolism (Poolman et 

al., 1990). Noteworthy that the aldose 1-epimerase domain is also present in the X. 

citri XAC4296 protein. 

In Saccharomyces cerevisiae, the GAL10 (galE, YBR019C) gene contains a 

mutarotase related domain (galactose mutarotase) and an additional UDP-galactose-



46 

 

4-epimerase domain, which confers this protein twice as many amino acids residues 

(699 aa) as the bacterial or the human protein (LGALS10) (Majumdar et al., 2004). The 

UDP-galactose 4-epimerase catalyzes the conversion between UDP-galactose and 

UDP-glucose in the galactose metabolic pathway (for eukaryotes and prokaryotes) 

(Wilson and Hogness, 1969). In addition, enzymatic assays showed that gal10 has an 

additional aldose 1-epimerase activity. Therefore, it is considered a bifunctional 

enzyme (Majumdar et al., 2004; Scott and Timson, 2007). On the other hand, galE is 

also related as a virulence factor in many bacterial pathogens such as Erwinia 

amylovora (Metzger et al., 1994), Pasteurella multocida (Fernández de henestrosa et 

al., 1997), Vibrio cholera (Nesper et al., 2001), Porphyromonas gingivalis (Nakao et 

al., 2006), Aeromonas hydrophila (Agarwal et al., 2007). In X. campestris pv. 

campestris, galE is also necessary for bacterial full virulence (Li et al., 2014), 

suggesting that the epimerase galE might act beyond the glycolytic pathways.  

Interestingly, in addition to the lytic transglycosylase domain, the XAC4296 

protein from X. citri also bears an epimerase domain presumably related to a 

mutarotase activity, supporting its role as a bifunctional enzyme. Recently, several 

bifunctional proteins were characterized as acting in two or more roles in plants, 

animals, yeast, and prokaryotes, the so-called Multidomain and Multifunctional 

Enzymes (MFEs) (Vogel et al., 2004; Cheng et al., 2012). MFEs may coordinate the 

crosstalk of dissimilar biological processes, such as metabolism, regulatory pathways, 

virulence factors, and virulence (Gancedo and Flores, 2008). Indeed, the study of 

MFEs is important for the understanding of living systems (more details of MFEs are 

shown in the next topics). 

 

2.7. Metabolism, cell shape, and chromosome segregation 

 

Bacteria respond swiftly to nutrient availability and changes in their environment, 

adjusting their shape and size (Wang and Levin, 2009). In conditions of unrestricted 

nutrient availability, bacteria capitalize on the available resources by increasing cell 

size and reproducing more often (Wang and Levin, 2009). During restrict nutrient 

conditions, bacteria balance growth, and cell size, showing a remarkable homogeneity 

across a population, suggesting that the cell growth and division cycles are regulated 

and not random (Sperber and Herman, 2017). 



47 

 

Many efforts were made to understand the relationship between bacterial 

metabolism and cell growth. For instance, the relation between metabolism and cellular 

elongation has been shown using ManA (mannose-6-phosphate isomerase) protein as 

a model (Elbaz and Ben-yehuda, 2010). ManA is responsible for phosphoglucose 

isomerase activity catalyzing glucose-6-phosphate to fructose-6-phosphate 

isomerization (Hansen et al., 2004). Therefore, ManA is directly involved in carbon 

metabolism and impacting cell shape (Elbaz and Ben-yehuda, 2010). For instance, in 

Bacillus subtilis, ManA is needed for the cell to keep its rod shape and be viable in the 

Luria-Bertani (LB) medium. However, ManA is not required for bacterial growth in the 

minimal medium containing glucose as a sole carbon source (Elbaz and Ben-yehuda, 

2010). Studies performed on B. subtilis revealed that ΔmanA mutant shows rounded 

cells and does not grow after changing from a minimum medium to an LB medium, 

also showing atypical nucleoid morphologies (Figure 13) (Elbaz and Ben-yehuda, 

2010). Two hypotheses were proposed to explain this observation. The first postulates 

that ΔmanA mutant offers reduced galactose levels and GalNAc (N-

acetylglucosamine, a component derived from teichoic acid), so the ΔmanA phenotype 

may be due to issues in the synthesis of teichoic acid (Yeom et al., 2009). Moreover, 

UDP-GalNAc (uridine-diphosphate-N-acetylgalactosamine) is a substrate for teichoic 

acid synthesis, generated by UDP-GlcNAc epimerization suggesting that ΔmanA 

mutant might also be hindered in its ability to synthesize UDP-GlcNAc (Young and 

Arias, 1967). 

 

 

Figure 13. Model linking cell wall integrity and chromosome morphology in wild-type 

cells of Bacillus subtilis and ΔmanA. Cell wall (green) and chromosome (red) in wild 

type and ΔmanA cells. In wild-type cells, the nucleoid organization and segregation 

are coordinated with cell wall synthesis and elongation. In the absence of ManA, the 

normal extension of the cell wall is blocked, as indicated by the disappearance of 



48 

 

helical sidewall staining. The nucleoid is detached from cell wall components and the 

synchronization is lost between cell growth and DNA replication and segregation, 

resulting in the formation of polyploid cells. Source: Elbaz; Ben-Yehuda, 2010 (2010). 

 

A first explanation for the round cell and altered chromosomal structure 

phenotype obtained from ΔmanA mutant can be made by observing the Escherichia 

coli phenotype when pyrimidine precursors are limited (Zaritsky et al., 2006). Indeed, 

DNA and peptidoglycan (PG) synthesis might share a precursor, UTP (Uridine 

triphosphate), to generate UDP-GlcNAc and the nucleosides dCTP (deoxycytidine 

triphosphate), and dTTP (deoxythymidine triphosphate), suggesting that cellular 

growth is susceptible to UTP disturbances (Zaritsky, 2015). This observation possibly 

explains the correlation observed between cell growth and DNA replication during a 

stationary state (Zaritsky, 2015). A second possibility that might explain the observed 

results is that in glucose deficient LB growth conditions, ManA contributes significantly 

to converting fructose-6-phosphate to glucose-6-phosphate to feed PG teichoic acid 

paths and DNA synthesis (Elbaz and Ben-yehuda, 2010).  

Taken together, these findings indicate that manA can perform more than one 

function simultaneously in the cell. Therefore, ManA probably regulates some aspects 

of cell envelope biogenesis, although its diffuse location does not explain how this 

mechanism would occur (Elbaz and Ben-yehuda, 2010).  

Studies on genetic interactions between carbon/nitrogen metabolism and cell 

division are abundant, and several examples were demonstrated more deeply in recent 

revision papers (Monahan et al., 2014; Monahan and Harry, 2016). It was observed 

that the loss or inactivation of central metabolism-related genes (e.g., pgm - 

Phosphoglycerate mutase, pgcA- Phosphoglucomutase, ugtP - Diacylglycerol 

glucosyltransferase, opgH- Glucans biosynthesis glucosyltransferase H, and pykA- 

Pyruvate kinase) might result in a more active FtsZ protein activity (Filamenting 

temperature-sensitive mutant Z) that is directly related to bacterial cell division, and 

thus, making cells multiply more frequently (Debarbieux et al., 1997; Hill et al., 2013). 

For instance, in B. subtilis, the pykA deletion, which catalyzes PEP 

(Phosphoenolpyruvate) conversion to pyruvate, activates an ftsZ temperature-

sensitive allele, possibly suggesting that heightened levels of PEP (gluconeogenesis) 

are correlated to observed defects in cell division (Monahan et al., 2014). Also, pykA 

deletion alters ftsz normal regulation, resulting in ∼40% of cells with multiple or 



49 

 

subpolar rings (Monahan et al., 2014). Although a direct relation between PykA and 

ftsZ was not demonstrated, the following experiment showed that PdhA (an enzyme 

that feeds pyruvate on tricarboxylic acid cycle – TCA) super expression promotes ftsZ 

additional polar ring formation on mutant ΔpykA (Monahan et al., 2014). However, 

when exogenous pyruvate was added, ΔpykA phenotype was restored, suggesting 

that pdhA relies on pyruvate resulting from pykA indirect action and not from pykA itself 

to assembly ftsZ ring (Monahan et al., 2014).  

Moreover, the loss of gdhZ (glutamate dehydrogenase), kidO (NADH-binding 

oxidoreductase), and pycK genes may inhibit cell division (Beaufay et al., 2015). The 

gdhZ is the enzyme responsible for converting glutamate and NAD+ in α-ketoglutarate, 

ammonia, and NADH in this microorganism. gdhZ was identified in an interaction assay 

with ftsZ, and gdhZ deletion results in abnormal cell division, resulting in a mix of cell 

populations showing short, standard, and filamentous phenotypes (Beaufay et al., 

2015). Indeed, gdhZ physically interacts in vivo with FtsZ, stimulating FtsZ GTPase 

activity in a glutamate-independent manner (Beaufay et al., 2015). Therefore, gdhZ 

enzymatic activity is required for ftsZ in vivo activity since gdhZ mutant will not stimulate 

in vitro GTPase activity, suggesting that gdhZ might be necessary to stimulate GTP 

hydrolysis (Beaufay et al., 2015).  

The bifunctional regulator KidO is another protein that promotes the 

disassembly of ftsZ (Beaufay et al., 2015). kidO is believed to play a role in conjunction 

with gdhZ to regulate ftsZ disassembly during the cell cycle and under nitrogen limiting 

conditions. kidO deletion resulting phenotypes are not so pronounced as those 

associated with gdhZ deletion; however, the proteins suffer a similar cell cycle 

regulation and co-localize with FtsZ rings (Radhakrishnan et al., 2010; Beaufay et al., 

2015). Therefore, kidO inhibits lateral filament grouping of ftsZ in an NADH-dependent 

manner in vitro, while also is proposed to work in cooperation with gdhZ, using NADH 

produced from gdhZ enzyme activity to inhibit this process (Beaufay et al., 2015).  

Taken together, these observations strongly suggest that enzymes related to 

the central metabolism, such as the epimerases that present a role on the EM and/or 

ED pathways, may indirectly impact the bacterial cell shape and chromosome 

segregation. 

 

 



50 

 

2.8. The role of Lytic transglycosylases in cell shape by interacting with 

Peptidoglycan Metabolism 

 

In addition to the epimerase domain, which may be related to the bacterial 

central metabolism, showing indirect impacts on the cell shape and chromosome 

segregation, the XAC4296 also contains a lytic transglycosylase domain. In Gram-

negative and Gram-positive microorganisms, the bacterial cell wall is the major stress-

bearing and shape-maintaining element, and its integrity is of critical importance to cell 

viability (Denome et al., 1999). Furthermore, the bacterial cell wall needs to endure 

expansion, contraction, and remodeling processes to occur proper division and shape 

and size adjustments. The structure of the cell wall consists of the cross-linked polymer 

Peptidoglycan (PG). This polymer consists of long glycan chains with alternating 1,4-

linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) subunits. 

The chemistry of the glycan chains varies only slightly between different bacteria, 

mostly on stem peptides linked to the carboxyl group of MurNAc (Vollmer and 

Bertsche, 2008). 

PG synthesis can be divided into three different stages (Van Heijenoort, 1998), 

the first step starts in the cytoplasm from precursors UDP-N-acetylmuramyl-

pentapeptide (UDP-MurNAc-pentapeptide) and UDP-N-acetylglucosamine (UDP-

GlcNAc). In the second step, UDP-N-acetylmuramyl-pentapeptide is carried to the 

membrane, where lipid intermediates are synthetized: UDP-N-acetylmuramyl-

pentapeptide is transferred to the membrane acceptor, producing Lipid I ([MurNAc-

(pentapeptide)-pyrophosphoryl-undecaprenol]) (Reviewed by van Heijenoort, 1998). 

GlcNAc from UDP-GlcNAc is then added to lipid I, giving rise to Lipid II [GlcNAc-(1,4)-

MurNAc-(pentapeptide)-pyrophosphoril-undecaprenol]. The third and last step is 

performed on the outside of the membrane through PBPs (Penicillin-Binding Proteins) 

action, which catalyzes transglycosylation and transpeptidation reactions responsible 

for PG glycolytic and peptidic ligations (Figure 14). 

 



51 

 

 

Figure 14. The maintenance and synthesis of peptidoglycan are performed in three 

steps. A: The first step includes UDP-MurNAc pentapeptide conversion from UDP-

GlcNAc in the cytoplasm, followed by Lipid I and Lipid II production. In the cytoplasmic 

membrane's extracellular leaflet, Lipid II is polymerized, cross-linked, and processed 

by transglycosylase (B), transpeptidase (C), and carboxypeptidase activities by 

penicillin-binding proteins (PBPs). The PBPs acts by reducing the ends of the N-

acetylmuramic acid (M) of the nascent lipid-linked peptidoglycan strand, which is likely 

transferred onto the C-4 carbon of the N-acetylglucosamine. The outer membrane is 

not shown. Abbreviations: GlcNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic 

acid. Adapted from Scheffers and Pinho (2005). 

 

Many enzymes are involved in peptidoglycan biosynthesis; for example (a) the 

Peptidoglycan glycosyltransferases (GTFs) polymerize the glycan chains; (b) the 

transpeptidases (TPases) form peptide crosslinks; (c) the PBPs, peptidoglycan 

hydrolases, that are present in multiple variants in all bacteria, these proteins are 

responsible for both the elongation of glycan strands (transglycosylation) and the 

formation of cross-links between the peptides (transpeptidation) of PG; (d) N-

acetylmuramidases (lysozymes); (e) N-acetylglucosaminidases; (f) amidases (g) 

endopeptidases; (h) carboxypeptidases, and (i) the Lytic transglycosylases (LTs), that 

act in peptidoglycan recycling and cell-wall-antibiotics detection (Scheffers and Pinho, 

2005). 

LTs comprise a family of proteins showing a key role in peptidoglycan dynamics 

(Hoeltje et al., 1975). The LTs are organized into 6 distinct families (1, 2, 3, 4, 5, and 



52 

 

6) distributed across 14 subfamilies (1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 2A, 3A, 3B, 4A, 

5A, 6A), and grouped according to their domain structure and function (Dik et al., 2017) 

(Figure 15 and Table 1).  

 

Figure 15. Lytic transglycosylase (LTs) domain architecture and family organization. 

Model LTs classified in Escherichia coli K12 (ECK4384, ECK2958, ECK1181, 

ECK0211, ECK2556, ECK2809, ECK2696, ECK1083, ECK0626), Pseudomonas 

aeruginosa PAO1 (PA3020, PA1812, PA 3764, PA2865, PA 1222, PA4444, 



53 

 

PA4001,1171, PA3992, PA2963, PA4000), Stenotrophomonas maltophilia KJ 

(Smlt4007, Smlt0994, Smlt3434, Smlt0155, Smlt4052, Smlt4650, Smlt1034, 

Smlt4051) and Neisseria gonorrhoeae FA1090 (NGO2135, NGO1033, NGO0608, 

NGO5004, NGO2048, NGO0626, NGO2038, NGO1728) according to the presence of 

domains and putative function. Domains were assigned based on InterPro database: 

SLT (IPR008258), LysM (IPR018392), DUF3393 (IPR023664), SBP_bac_3 

(IPR001638), PG_binding_1 (IPR002477), MltA (IPR034654), 3D (IPR034654), 

SLT_2 (IPR031304), Phage_lysozyme (IPR023347), YceG (IPR003770), DPBB_1 

(IPR007112), SPOR (IPR007730). Source: Dik et al. (2017). 

 

Table 1. List of domains found in Lytic Transglycosylase (LTs) (2018). Domain 

information was obtained on InterPro databases. Source: Dik et al., 2017; Oliveira et 

al., 2018. 

Domain Function (InterproScan) (FINN et al., 2017)  
 

SLT (IPR008258) Related to proteins encoded by bacteriophages for 
2, 3, and 4 secretion systems. This domain 
presents cleavage activity of the β-1,4 glycosidic 
ligation between acid residues N-acetylmuramic 
acid and N-acetylglucosamine, and the formation 
of muropeptides containing a 1,6 anhydro ligation 
in the muramic acid residue. 

LysM (IPR018392) Related to peptidoglycan binding and plant-
pathogen interactions. 

DUF3393 (IPR023664) Unknown domain presenting potential function 
related to murein degradation during the recycling 
of muropeptides and cell elongation and/or cell 
division.  

SBP_bac_3 (IPR001638) Involved in the active transport of solutes across 
the cytoplasmic membrane. 

PG_binding_1 (IPR002477) Related to peptidoglycan binding. 

MltA (IPR034654) Helps binding to peptidoglycan. 

3D (IPR034654) Composed of three conserved residues of aspartic 
acid, presumably related to peptidase activity. 

SLT_2 (IPR031304) Related to SLT domain.  

Phage_lysozyme (IPR023347) Related to breaking down the peptidoglycan, 
hydrolyzing the 1,4-beta ligation between N-
acetylglucosamine and N-acetylmuramic acid in 
heteropolymers. 

YceG (IPR003770) Related to the end of peptidoglycan 
polymerization by endolytic breaking of nascent 
chains. 

DPBB_1 (IPR007112) Related to outer membrane proteins and 
specificity for peptidoglycan. 

SPOR (IPR007730) Related to cell division, morphogenesis, and 
sporulation processes. 

 



54 

 

The LTs show catalytic activity related to peptidoglycan polysaccharide 

fragmentation on glycosidic ligation between amino acid residues NAG-NAM, resulting 

in muropeptides formation containing 1,6-anhydrous ligation on muramic acid residue 

(1,6-anhydromuramic) (Höltje, 1998). These muropeptides are transported to the 

cytoplasm through AmpG transmembrane protein, degraded, and their subproducts 

are used on the Lipids Biosynthesis II pathway or induce the lactamase production 

(Jacobs et al., 1994; Heidrich et al., 2002). Products from the Lipids Biosynthesis II 

pathway are transported from cytoplasm to periplasm, where they will be 

reincorporated on PG metabolism (Barreteau et al., 2008; Bouhss et al., 2008; 

Scheffers and Tol, 2015; Leclercq et al., 2017) (Figure 16). 

 

 

Figure 16. Chemical reaction performed by Lytic transglycosylase (LTs). This reaction 

is defined as the breaking down of polysaccharides in the glycosidic ligation between 

acid residues NAG-NAM, which goes through an oxocarbenium, which intercepts 

glucosamine 6-hydroxyl group, resulting in the formation of muropeptides containing a 

1,6 anhydrous ligation in the muramic acid residue. Source: Dik et al., 2017. 

 

LTs can make space on the PG sacculus, allowing several metabolic processes 

to occur in that space (Scheurwater et al., 2008). For instance, the LT may expand the 

sacculus and, consequently, cell growth by creating PG precursors sites (Höltje, 1998; 

Scheurwater et al., 2008). Together with amidases, they split the septum allowing 

separation during cell division (Heidrich et al., 2002). LTs are also involved in 

endospore germination in Gram-positive cells, facilitating the insertion of macro 

complexes that extend through the PG sacculus, such as T3SS, T4SS, flagella, and 

pili on Gram-negative cells (reviewed in Koraimann, 2003).  

These proteins are autolytic due to their ability to cause complete cellular lysis 

if their activity proceeds uncontrolled (Scheurwater and Clarke, 2008). In general, LTs 

are ubiquitous on PG-producing microorganisms (Blackburn and Clarke, 2001). Still, 

each species shows a different repertoire, with some species showing duplicated 

content, suggesting that LTs might have functional redundancy (Scheurwater and 



55 

 

Clarke, 2008; Dik et al., 2017). It was demonstrated in E. coli that individual deletion in 

each one of the six LTs present on this bacterium genome, and even in all of them at 

the same time, compromised septum cleavage during cell division, resulting in cell 

chains and an increase in permeability of the outer membrane (Heidrich et al., 2002). 

However, no lethal effect was observed with individual LTs in E. coli, adding to the 

hypothesis of functional redundancy among LTs and to other proteins (Heidrich et al., 

2002). Additionally, it has been suggested that some LT functions are essential for the 

bacterium when facing different physiological conditions, indicating that it is not 

possible to knock out all LTs (Scheurwater and Clarke, 2008). 

LTs are also related to pathogenicity and virulence processes. For example, the 

LT EtgA (1G Family) from enteropathogenic Escherichia coli is required for efficient 

T3SS function (García-gómez et al., 2011). The Pseudomonas syringae, LTs HrpH 

(1D Family), HopP1 (4A Family), and HopAJ1 (3B Family) can facilitate the 

translocation of effector proteins by the T3SS (Oh et al., 2007). In X. axonopodis pv. 

glycines, HpaH (1G Family) contributes for virulence and Hyper sensibility Response 

(HR) (Noël et al., 2002). In X. oryzae pv. oryzicola, Hpa2 (1G Family) is related to 

virulence and responsible for translocating effectors by the T3SS, interacting with the 

hrpF translocon (Li et al., 2011). In X. campestris pv. vesicatoria, HpaH (1G Family ) 

can promote T3SS effector protein transport and interacts with T3SS structural 

proteins HrpB1 and HrpB2 and the pilus protein HrpE (Hausner et al., 2017). In X. 

campestris pv. campestris, the membrane-bound lytic transglycosylase (XC_0706) (3A 

Family) is important for cell division, and the Hpa2 (1G Family) affects T3SS effector 

translocation (Wang et al., 2019). 

The X. citri LT content and diversity were already unraveled (Oliveira et al., 

2018). X. citri encodes a total of 17 LTs: 12 belonging to families 1A, 1B, 1C, 1D (two 

copies), 1F (three copies), 1G (2 copies), 3A, 3B (two copies), 5A, 6A, and one which 

is non-categorized and not showing a common LT domain (Table 2). It was also 

demonstrated that the mltB2.1 and mltB2.2 (3B Family) LTs were laterally acquired, 

and using site-directed deletion mutagenesis, they were functionally characterized 

(Oliveira et al., 2018). These mltB genes (mltB2.1 and mltB2.2) are directly related to 

X. citri virulence and affect CC progression, indicating that the acquisition by lateral 

transfer leads to evolutive advantages for the bacterium (Oliveira et al., 2018).



56 

 

Table 2. Lytic Transglycosylase (LTs) and biosynthetic peptidoglycan transglycosylase are found in Xanthomonas citri (X. citri). 

The search for LT genes was performed on the GenBank database at NCBI using the BLAST tool (Altschul et al., 1997), and 

consensus analysis was performed using ClustalX (Larkin et al., 2007) and InterProScan (Finn et al., 2017) tools using the 

classification proposed by Dik et al. (2017). LT XAC4296 (XAC_RS21660) is an exclusive protein in Xanthomonadaceae, it shows 

the domains SLT_2 and