Discovering the Molecular Determinants of Phaeobacter inhibens Susceptibility to Phaeobacter Phage MD18

ABSTRACT Bacteriophages have immense potential as antibiotic therapies and in genetic engineering. Understanding the mechanisms that bacteriophages implement to infect their hosts will allow researchers to manipulate these systems and adapt them to specific bacterial targets. In this study, we isolated a bacteriophage capable of infecting the marine alphaproteobacterium Phaeobacter inhibens and determined its mechanism of infection. Phaeobacter virus MD18, a novel species of bacteriophage isolated in Woods Hole, MA, exhibits potent lytic ability against P. inhibens and appears to be of the Siphoviridae morphotype. The genomic sequence of MD18 displayed significant similarity to another siphophage, the recently discovered Roseobacter phage DSS3P8, but genomic and phylogenetic analyses, assessing host range and a search of available metagenomes are all consistent with the conclusion that Phaeobacter phage MD18 is a novel lytic phage. We incubated MD18 with a library of barcoded P. inhibens transposon insertion mutants and identified 22 genes that appear to be required for phage predation of this host. Network analysis of these genes using genomic position, Gene Ontology (GO) term enrichment, and protein associations revealed that these genes are enriched for roles in assembly of a type IV pilus (T4P) and regulators of cellular morphology. Our results suggest that T4P serve as receptors for a novel marine virus that targets P. inhibens. IMPORTANCE Bacteriophages are useful nonantibiotic therapeutics for bacterial infections as well as threats to industries utilizing bacterial agents. This study identified Phaeobacter virus MD18, a phage antagonist of Phaeobacter inhibens, a bacterium with promising use as a probiotic for aquatic farming industries. Genomic analysis suggested that Phaeobacter phage MD18 has evolved to enhance its replication in P. inhibens by adopting favorable tRNA genes as well as through genomic sequence adaptation to resemble host codon usage. Lastly, a high-throughput analysis of P. inhibens transposon insertion mutants identified genes that modulate host susceptibility to phage MD18 and implicated the type IV pilus as the likely receptor recognized for adsorption. This study marks the first characterization of the relationship between P. inhibens and an environmentally sampled phage, which informs our understanding of natural threats to the bacterium and may promote the development of novel phage technologies for genetic manipulation of this host.

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Discovering the Molecular Determinants of Phaeobacter inhibens susceptibility to Phaeobacter phage MD18

10.1101/2020.04.13.038638 ◽

2020 ◽

Cited By ~ 1

Author(s):

Guillaume Urtecho ◽

Danielle E. Campbell ◽

David M. Hershey ◽

Rachel J. Whitaker ◽

George A. O’Toole

Keyword(s):

Bacterial Infections ◽

Genomic Sequence ◽

Genetic Manipulation ◽

Host Susceptibility ◽

Trna Genes ◽

Type Iv Pilus ◽

Type Iv ◽

Transposon Insertion ◽

Phaeobacter Inhibens ◽

Insertion Mutants

AbstractBacteriophage technologies have immense potential as antibiotic therapies and in genetic engineering. Understanding the mechanisms that bacteriophages implement to infect their hosts will allow researchers to manipulate these systems and adapt them to specific bacterial targets. Here, we isolated a bacteriophage capable of infecting the marine alphaproteobacterium Phaeobacter inhibens and dissected its mechanism of infection. Phaeobacter phage MD18, a novel species of bacteriophage isolated in Woods Hole, MA, exhibits potent lytic ability against P. inhibens and appears to be of the Siphoviridae morphotype. Consistent with this finding, the sequence of the MD18 revealed significant similarity to another siphophage, the recently discovered Roseobacter phage DSS3P8. We incubated MD18 with a library of barcoded P. inhibens transposon insertion mutants and identified 22 genes that appear to be required for phage predation of this host. Network analysis of these genes using genomic position, GO term enrichment, and protein associations reveals that these genes are enriched for roles in assembly of a type IV pilus (T4P) and regulators of cellular morphology. Our results suggest that T4P serve as receptors for a novel marine virus that targets P. inhibens.ImportanceBacteriophages are useful non-antibiotic therapeutics for bacterial infections as well as threats to industries utilizing bacterial agents. This study identifies Phaeobacter phage MD18, the first documented phage of Phaeobacter inhibens, a bacterium with promising use as a probiotic for aquatic farming industries. Genomic analysis suggests that the Phaeobacter phage MD18 has evolved to enhance its replication in P. inhibens by adopting favorable tRNA genes as well as through genomic sequence adaptation to resemble host codon usage. Lastly, a high-throughput analysis of P. inhibens transposon insertion mutants identifies genes that modulate host susceptibility to phage MD18 and implicates the type IV pilus as the likely receptor recognized for adsorption. This study marks the first characterization of the relationship between P. inhibens and an environmentally sampled phage, which informs our understanding of natural threats to the bacterium and may promote the development of novel phage technologies for genetic manipulation of this host.

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Identification and initial characterization of a new pair of sibling sRNAs of Neisseria gonorrhoeae involved in type IV pilus biogenesis

Microbiology ◽

10.1099/mic.0.001080 ◽

2021 ◽

Vol 167 (9) ◽

Author(s):

Marie Zachary ◽

Susanne Bauer ◽

Maximilian Klepsch ◽

Katharina Wagler ◽

Bruno Hüttel ◽

...

Keyword(s):

Target Genes ◽

Target Prediction ◽

Type Iv Pilus ◽

Content Type ◽

Type Iv ◽

Gene Expression Control ◽

Initial Characterization ◽

Pilus Biogenesis ◽

Regulatory Rnas

Non-coding regulatory RNAs mediate post-transcriptional gene expression control by a variety of mechanisms relying mostly on base-pairing interactions with a target mRNA. Though a plethora of putative non-coding regulatory RNAs have been identified by global transcriptome analysis, knowledge about riboregulation in the pathogenic Neisseriae is still limited. Here we report the initial characterization of a pair of sRNAs of N. gonorrhoeae , TfpR1 and TfpR2, which exhibit a similar secondary structure and identical single-stranded seed regions, and therefore might be considered as sibling sRNAs. By combination of in silico target prediction and sRNA pulse expression followed by differential RNA sequencing we identified target genes of TfpR1 which are involved in type IV pilus biogenesis and DNA damage repair. We provide evidence that members of the TfpR1 regulon can also be targeted by the sibling TfpR2.

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Discovery of a New Neisseria gonorrhoeae Type IV Pilus Assembly Factor, TfpC

mBio ◽

10.1128/mbio.02528-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Linda I. Hu ◽

Shaohui Yin ◽

Egon A. Ozer ◽

Lee Sewell ◽

Saima Rehman ◽

...

Keyword(s):

Cell Surface ◽

Neisseria Gonorrhoeae ◽

Bacterial Cell ◽

Bacterial Species ◽

Type Iv Pili ◽

Type Iv Pilus ◽

Transmitted Infection ◽

Content Type ◽

Sexually Transmitted ◽

Type Iv

ABSTRACT Neisseria gonorrhoeae relies on type IV pili (T4p) to promote colonization of their human host and to cause the sexually transmitted infection gonorrhea. This organelle cycles through a process of extension and retraction back into the bacterial cell. Through a genetic screen, we identified the NGO0783 locus of N. gonorrhoeae strain FA1090 as containing a gene encoding a protein required to stabilize the type IV pilus in its extended, nonretracted conformation. We have named the gene tfpC and the protein TfpC. Deletion of tfpC produces a nonpiliated colony morphology, and immuno-transmission electron microscopy confirms that the pili are lost in the ΔtfpC mutant, although there is some pilin detected near the bacterial cell surface. A copy of the tfpC gene expressed from a lac promoter restores pilus expression and related phenotypes. A ΔtfpC mutant shows reduced levels of pilin protein, but complementation with a tfpC gene restored pilin to normal levels. Bioinformatic searches show that there are orthologues in numerous bacterial species, but not all type IV pilin-expressing bacteria contain orthologous genes. Coevolution and nuclear magnetic resonance (NMR) analysis indicates that TfpC contains an N-terminal transmembrane helix, a substantial extended/unstructured region, and a highly charged C-terminal coiled-coil domain. IMPORTANCE Most bacterial species express one or more extracellular organelles called pili/fimbriae that are required for many properties of each bacterial cell. The Neisseria gonorrhoeae type IV pilus is a major virulence and colonization factor for the sexually transmitted infection gonorrhea. We have discovered a new protein of Neisseria gonorrhoeae called TfpC that is required to maintain type IV pili on the bacterial cell surface. There are similar proteins found in other members of the Neisseria genus and many other bacterial species important for human health.

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ThePseudomonas aeruginosaPilSR Two-Component System Regulates Both Twitching and Swimming Motilities

mBio ◽

10.1128/mbio.01310-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 18

Author(s):

Sara L. N. Kilmury ◽

Lori L. Burrows

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iv Pili ◽

Geobacter Sulfurreducens ◽

Type Iv Pilus ◽

Two Component System ◽

Component System ◽

Content Type ◽

Swimming Motility ◽

Type Iv ◽

Two Component

ABSTRACTMotility is an important virulence trait for many bacterial pathogens, allowing them to position themselves in appropriate locations at appropriate times. The motility structures type IV pili and flagella are also involved in sensing surface contact, which modulates pathogenicity. InPseudomonas aeruginosa, the PilS-PilR two-component system (TCS) regulates expression of the type IV pilus (T4P) major subunit PilA, while biosynthesis of the single polar flagellum is regulated by a hierarchical system that includes the FleSR TCS. Previous studies ofGeobacter sulfurreducensandDichelobacter nodosusimplicated PilR in regulation of non-T4P-related genes, including some involved in flagellar biosynthesis. Here we used transcriptome sequencing (RNA-seq) analysis to identify genes in addition topilAwith changes in expression in the absence ofpilR. Among the genes identified were 10 genes whose transcription increased in thepilAmutant but decreased in thepilRmutant, despite both mutants lacking T4P and pilus-related phenotypes. The products of these inversely dysregulated genes, many of which were hypothetical, may be important for virulence and surface-associated behaviors, as mutants had altered swarming motility, biofilm formation, type VI secretion system expression, and pathogenicity in a nematode model. Further, the PilSR TCS positively regulated transcription offleSR, and thus many genes in the FleSR regulon. As a result,pilSRdeletion mutants had defects in swimming motility that were independent of the loss of PilA. Together, these data suggest that in addition to controlling T4P expression, PilSR could have a broader role in the regulation ofP. aeruginosamotility and surface sensing behaviors.IMPORTANCESurface appendages such as type IV pili and flagella are important for establishing surface attachment and infection in a host in response to appropriate cues. The PilSR regulatory system that controls type IV pilus expression inPseudomonas aeruginosahas an established role in expression of the major pilin PilA. Here we provide evidence supporting a new role for PilSR in regulating flagellum-dependent swimming motility in addition to pilus-dependent twitching motility. Further, even though bothpilAandpilRmutants lack PilA and pili, we identified sets of genes downregulated in thepilRmutant and upregulated in apilAmutant as well as genes downregulated only in apilRmutant, independent of pilus expression. This finding suggests that change in the inner membrane levels of PilA is only one of the cues to which PilR responds to modulate gene expression. Identification of PilR as a regulator of multiple motility pathways may make it an interesting therapeutic target for antivirulence compounds.

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Cyclic Di-GMP Regulates Type IV Pilus-Dependent Motility in Myxococcus xanthus

Journal of Bacteriology ◽

10.1128/jb.00281-15 ◽

2015 ◽

Vol 198 (1) ◽

pp. 77-90 ◽

Cited By ~ 26

Author(s):

Dorota Skotnicka ◽

Tobias Petters ◽

Jan Heering ◽

Michael Hoppert ◽

Volkhard Kaever ◽

...

Keyword(s):

Myxococcus Xanthus ◽

Lifestyle Changes ◽

Gliding Motility ◽

Cyclase Activity ◽

Diguanylate Cyclase ◽

Type Iv Pilus ◽

Cell Agglutination ◽

Content Type ◽

Type Iv ◽

Altered Cell

ABSTRACTThe nucleotide-based second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is involved in regulating a plethora of processes in bacteria that are typically associated with lifestyle changes.Myxococcus xanthusundergoes major lifestyle changes in response to nutrient availability, with the formation of spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. Here, we investigated the function of c-di-GMP inM. xanthusand show that this bacterium synthesizes c-di-GMP during growth. Manipulation of the c-di-GMP level by expression of either an active, heterologous diguanylate cyclase or an active, heterologous phosphodiesterase correlated with defects in type IV pilus (T4P)-dependent motility, whereas gliding motility was unaffected. An increased level of c-di-GMP correlated with reduced transcription of thepilAgene (which encodes the major pilin of T4P), reduced the assembly of T4P, and altered cell agglutination, whereas a decreased c-di-GMP level correlated with altered cell agglutination. The systematic inactivation of the 24 genes inM. xanthusencoding proteins containing GGDEF, EAL, or HD-GYP domains, which are associated with c-di-GMP synthesis, degradation, or binding, identified three genes encoding proteins important for T4P-dependent motility, whereas all mutants had normal gliding motility. Purified DmxA had diguanylate cyclase activity, whereas the hybrid histidine protein kinases TmoK and SgmT, each of which contains a GGDEF domain, did not have diguanylate cyclase activity. These results demonstrate that c-di-GMP is important for T4P-dependent motility inM. xanthus.IMPORTANCEWe provide the first direct evidence thatM. xanthussynthesizes c-di-GMP and demonstrate that c-di-GMP is important for T4P-dependent motility, whereas we did not obtain evidence that c-di-GMP regulates gliding motility. The data presented uncovered a novel mechanism for regulation of T4P-dependent motility, in which increased levels of c-di-GMP inhibit transcription of thepilAgene (which encodes the major pilin of T4P), ultimately resulting in the reduced assembly of T4P. Moreover, we identified an enzymatically active diguanylate cyclase that is important for T4P-dependent motility.

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Type IV Pilus Glycosylation Mediates Resistance of Pseudomonas aeruginosa to Opsonic Activities of the Pulmonary Surfactant Protein A

Infection and Immunity ◽

10.1128/iai.02874-14 ◽

2015 ◽

Vol 83 (4) ◽

pp. 1339-1346 ◽

Cited By ~ 18

Author(s):

Rommel M. Tan ◽

Zhizhou Kuang ◽

Yonghua Hao ◽

Francis Lee ◽

Timothy Lee ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Pulmonary Surfactant ◽

Protein A ◽

Lung Infection ◽

Surfactant Protein ◽

Surfactant Protein A ◽

Type Iv Pilus ◽

Content Type ◽

Type Iv ◽

Pulmonary Surfactant Protein

Pseudomonas aeruginosais a major bacterial pathogen commonly associated with chronic lung infections in cystic fibrosis (CF). Previously, we have demonstrated that the type IV pilus (Tfp) ofP. aeruginosamediates resistance to antibacterial effects of pulmonary surfactant protein A (SP-A). Interestingly,P. aeruginosastrains with group I pilins areO-glycosylated through the TfpO glycosyltransferase with a single subunit of O-antigen (O-ag). Importantly, TfpO-mediatedO-glycosylation is important for virulence in mouse lungs, exemplified by more frequent lung infection in CF with TfpO-expressingP. aeruginosastrains. However, the mechanism underlying the importance of Tfp glycosylation inP. aeruginosapathogenesis is not fully understood. Here, we demonstrated one mechanism of increased fitness mediated byO-glycosylation of group 1 pilins on Tfp in theP. aeruginosaclinical isolate 1244. Using an acute pneumonia model in SP-A+/+versus SP-A−/−mice, theO-glycosylation-deficient ΔtfpOmutant was found to be attenuated in lung infection. Both 1244 and ΔtfpOstrains showed equal levels of susceptibility to SP-A-mediated membrane permeability. In contrast, the ΔtfpOmutant was more susceptible to opsonization by SP-A and by other pulmonary and circulating opsonins, SP-D and mannose binding lectin 2, respectively. Importantly, the increased susceptibility to phagocytosis was abrogated in the absence of opsonins. These results indicate thatO-glycosylation of Tfp with O-ag specifically confers resistance to opsonization during host-mediated phagocytosis.

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Type IV Pilus Biogenesis, Twitching Motility, and DNA Uptake in Thermus thermophilus: Discrete Roles of Antagonistic ATPases PilF, PilT1, and PilT2

Applied and Environmental Microbiology ◽

10.1128/aem.03218-13 ◽

2013 ◽

Vol 80 (2) ◽

pp. 644-652 ◽

Cited By ~ 18

Author(s):

Ralf Salzer ◽

Friederike Joos ◽

Beate Averhoff

Keyword(s):

Natural Transformation ◽

Thermophilic Bacteria ◽

Bacterial Species ◽

Thermus Thermophilus ◽

Dna Uptake ◽

Twitching Motility ◽

Type Iv Pilus ◽

Content Type ◽

Ecological Diversification ◽

Type Iv

ABSTRACTNatural transformation has a large impact on lateral gene flow and has contributed significantly to the ecological diversification and adaptation of bacterial species.Thermus thermophilusHB27 has emerged as the leading model organism for studies of DNA transporters in thermophilic bacteria. Recently, we identified a zinc-binding polymerization nucleoside triphosphatase (NTPase), PilF, which is essential for the transport of DNA through the outer membrane. Here, we present genetic evidence that PilF is also essential for the biogenesis of pili. One of the most challenging questions was whetherT. thermophilushas any depolymerization NTPase acting as a counterplayer of PilF. We identified two depolymerization NTPases, PilT1 (TTC1621) and PilT2 (TTC1415), both of which are required for type IV pilus (T4P)-mediated twitching motility and adhesion but dispensable for natural transformation. This suggests that T4P dynamics are not required for natural transformation. The latter finding is consistent with our suggestion that inT. thermophilus, T4P and natural transformation are linked but distinct systems.

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Lysobacter PilR, the Regulator of Type IV Pilus Synthesis, Controls Antifungal Antibiotic Production via a Cyclic di-GMP Pathway

Applied and Environmental Microbiology ◽

10.1128/aem.03397-16 ◽

2017 ◽

Vol 83 (7) ◽

Cited By ~ 17

Author(s):

Yuan Chen ◽

Jing Xia ◽

Zhenhe Su ◽

Gaoge Xu ◽

Mark Gomelsky ◽

...

Keyword(s):

Response Regulator ◽

Antibiotic Production ◽

Second Messenger ◽

Twitching Motility ◽

Type Iv Pilus ◽

Content Type ◽

Lysobacter Enzymogenes ◽

Antifungal Antibiotic ◽

Type Iv ◽

Two Component

ABSTRACT Lysobacter enzymogenes is a ubiquitous soil gammaproteobacterium that produces a broad-spectrum antifungal antibiotic, known as heat-stable antifungal factor (HSAF). To increase HSAF production for use against fungal crop diseases, it is important to understand how HSAF synthesis is regulated. To gain insights into transcriptional regulation of the HSAF synthesis gene cluster, we generated a library with deletion mutations in the genes predicted to encode response regulators of the two-component signaling systems in L. enzymogenes strain OH11. By quantifying HSAF production levels in the 45 constructed mutants, we identified two strains that produced significantly smaller amounts of HSAF. One of the mutations affected a gene encoding a conserved bacterial response regulator, PilR, which is commonly associated with type IV pilus synthesis. We determined that L. enzymogenes PilR regulates pilus synthesis and twitching motility via a traditional pathway, by binding to the pilA promoter and upregulating pilA expression. Regulation of HSAF production by PilR was found to be independent of pilus formation. We discovered that the pilR mutant contained significantly higher intracellular levels of the second messenger cyclic di-GMP (c-di-GMP) and that this was the inhibitory signal for HSAF production. Therefore, the type IV pilus regulator PilR in L. enzymogenes activates twitching motility while downregulating antibiotic HSAF production by increasing intracellular c-di-GMP levels. This study identifies a new role of a common pilus regulator in proteobacteria and provides guidance for increasing antifungal antibiotic production in L. enzymogenes. IMPORTANCE PilR is a widespread response regulator of the two-component system known for regulating type IV pilus synthesis in proteobacteria. Here we report that, in the soil bacterium Lysobacter enzymogenes, PilR regulates pilus synthesis and twitching motility, as expected. Unexpectedly, PilR was also found to control intracellular levels of the second messenger c-di-GMP, which in turn inhibits production of the antifungal antibiotic HSAF. The coordinated production of type IV pili and antifungal antibiotics has not been observed previously.

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Type IV Pilus Secretins Have Extracellular C Termini

mBio ◽

10.1128/mbio.00322-15 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 3

Author(s):

Joshua A. Lieberman ◽

Courtney D. Petro ◽

Stefani Thomas ◽

Austin Yang ◽

Michael S. Donnenberg

Keyword(s):

Outer Membrane ◽

Secondary Structure Prediction ◽

Outer Membrane Proteins ◽

Gram Negative Bacteria ◽

Structural Constraints ◽

Type Iv Pilus ◽

Gram Negative ◽

Content Type ◽

Atomic Structures ◽

Type Iv

ABSTRACTType IV pili (T4Ps) are surface appendages used by Gram-negative and Gram-positive pathogens for motility and attachment to epithelial surfaces. In Gram-negative bacteria, such as the important pediatric pathogen enteropathogenicEscherichia coli(EPEC), during extension and retraction, the pilus passes through an outer membrane (OM) pore formed by the multimeric secretin complex. The secretin is common to Gram-negative assemblies, including the related type 2 secretion (T2S) system and the type 3 secretion (T3S) system. The N termini of the secretin monomers are periplasmic and in some systems have been shown to mediate substrate specificity. In this study, we mapped the topology of BfpB, the T4P secretin from EPEC, using a combination of biochemical and biophysical techniques that allowed selective identification of periplasmic and extracellular residues. We applied rules based on solved atomic structures of outer membrane proteins (OMPs) to generate our topology model, combining the experimental results with secondary structure prediction algorithms and direct inspection of the primary sequence. Surprisingly, the C terminus of BfpB is extracellular, a result confirmed by flow cytometry for BfpB and a distantly related T4P secretin, PilQ, fromPseudomonas aeruginosa. Keeping with prior evidence, the C termini of two T2S secretins and one T3S secretin were not detected on the extracellular surface. On the basis of our data and structural constraints, we propose that BfpB forms a beta barrel with 16 transmembrane beta strands. We propose that the T4P secretins have a C-terminal segment that passes through the center of each monomer.IMPORTANCESecretins are multimeric proteins that allow the passage of secreted toxins and surface structures through the outer membranes (OMs) of Gram-negative bacteria. To date, there have been no atomic structures of the C-terminal region of a secretin, although electron microscopy (EM) structures of the complex are available. This work provides a detailed topology prediction of the membrane-spanning domain of a type IV pilus (T4P) secretin. Our study used innovative techniques to provide new and comprehensive information on secretin topology, highlighting similarities and differences among secretin subfamilies. Additionally, the techniques used in this study may prove useful for the study of other OM proteins.

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Characterization of the Exopolysaccharide Biosynthesis Pathway in Myxococcus xanthus

Journal of Bacteriology ◽

10.1128/jb.00335-20 ◽

2020 ◽

Vol 202 (19) ◽

Author(s):

María Pérez-Burgos ◽

Inmaculada García-Romero ◽

Jana Jung ◽

Eugenia Schander ◽

Miguel A. Valvano ◽

...

Keyword(s):

Repeat Unit ◽

Myxococcus Xanthus ◽

Type Iv Pilus ◽

Fruiting Body Formation ◽

Content Type ◽

Body Formation ◽

Polysaccharide Biosynthesis ◽

Type Iv ◽

Biosynthetic Machinery ◽

Dependent Pathway

ABSTRACT Myxococcus xanthus arranges into two morphologically distinct biofilms depending on its nutritional status, i.e., coordinately spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. A secreted polysaccharide, referred to as exopolysaccharide (EPS), is a structural component of both biofilms and is also important for type IV pilus-dependent motility and fruiting body formation. Here, we characterize the biosynthetic machinery responsible for EPS biosynthesis using bioinformatics, genetics, heterologous expression, and biochemical experiments. We show that this machinery constitutes a Wzx/Wzy-dependent pathway dedicated to EPS biosynthesis. Our data support that EpsZ (MXAN_7415) is the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for the initiation of the repeat unit synthesis. Heterologous expression experiments support that EpsZ has galactose-1-P transferase activity. Moreover, MXAN_7416, renamed WzxEPS, and MXAN_7442, renamed WzyEPS, are the Wzx flippase and Wzy polymerase responsible for translocation and polymerization of the EPS repeat unit, respectively. In this pathway, EpsV (MXAN_7421) also is the polysaccharide copolymerase and EpsY (MXAN_7417) the outer membrane polysaccharide export (OPX) protein. Mutants with single in-frame deletions in the five corresponding genes had defects in type IV pilus-dependent motility and a conditional defect in fruiting body formation. Furthermore, all five mutants were deficient in type IV pilus formation, and genetic analyses suggest that EPS and/or the EPS biosynthetic machinery stimulates type IV pilus extension. Additionally, we identify a polysaccharide biosynthesis gene cluster, which together with an orphan gene encoding an OPX protein make up a complete Wzx/Wzy-dependent pathway for synthesis of an unknown polysaccharide. IMPORTANCE The secreted polysaccharide referred to as exopolysaccharide (EPS) has important functions in the social life cycle of M. xanthus; however, little is known about how EPS is synthesized. Here, we characterized the EPS biosynthetic machinery and showed that it makes up a Wzx/Wzy-dependent pathway for polysaccharide biosynthesis. Mutants lacking a component of this pathway had reduced type IV pilus-dependent motility and a conditional defect in development. These analyses also suggest that EPS and/or the EPS biosynthetic machinery is important for type IV pilus formation.

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