Functional Analysis of the Major Pilin Proteins of Type IV Pili in Streptococcus sanguinis CGMH010.

The pil gene cluster for Type IV pilus (Tfp) biosynthesis is commonly present and highly conserved in Streptococcus sanguinis. Nevertheless, Tfp-mediated twitching motility is less common among strains, and the factors determining twitching activity are not fully understood. Here, we analyzed the functions of three major pilin proteins (PilA1, PilA2, and PilA3) in the assembly and activity of Tfp in motile S. sanguinis CGMH010. Using various recombinant pilA deletion strains, we found that Tfp composed of different PilA proteins varied morphologically and functionally. Among the three PilA proteins, PilA1 was most critical in the assembly of twitching-active Tfp, and recombinant strains expressing motility generated more structured biofilms under constant shearing forces compared to the non-motile recombinant strains. Although PilA1 and PilA3 shared 94% identity, PilA3 could not compensate for the loss of PilA1, suggesting that the nature of PilA proteins plays an essential role in twitching activity. The single deletion of individual pilA genes had little effect on the invasion of host endothelia by S. sanguinis CGMH010. In contrast, the deletion of all three pilA genes or pilT, encoding the retraction ATPase, abolished Tfp-mediated invasion. Tfp- and PilT-dependent invasion were also detected in the non-motile S. sanguinis SK36, and thus, the retraction of Tfp, but not active twitching, was found to be essential for invasion.

Myxococcus xanthus PilB interacts with c-di-GMP and modulates motility and biofilm formation.

The regulation of biofilm and motile states as alternate bacterial lifestyles has been studied extensively in flagellated bacteria, where the second messenger cyclic-di-GMP (cdG) plays a crucial role. However, much less is known about the mechanisms of such regulation in motile bacteria without flagella. The bacterial type IV pilus (T4P) serves as a motility apparatus that enables Myxococcus xanthus to move on solid surfaces. PilB, the T4P assembly ATPase, is, therefore, required for T4P-dependent motility in M. xanthus. Interestingly, T4P is also involved in the regulation of exopolysaccharide as the biofilm matrix material in this bacterium. A newly discovered cdG-binding domain, MshEN, is conserved in the N-terminus of PilB (PilBN) in M. xanthus and other bacteria. This suggests that cdG may bind to PilB to control the respective outputs that regulate biofilm development and T4P-powered motility. In this study, we aimed to validate M. xanthus PilB as a cdG effector protein. We performed a systematic mutational analysis of its cdG-binding domain to investigate its relationship with motility, piliation, and biofilm formation. Excluding those resulting in low levels of PilB protein, all other substitution mutations in PilBN resulted in pilB mutants with distinct and differential phenotypes in piliation and biofilm levels in M. xanthus. This suggests that the PilBN domain plays dual roles in modulating motility and biofilm levels, and these two functions of PilB can be dependent on and independent of each other in M. xanthus. IMPORTANCE The regulation of motility and biofilm by cyclic-di-GMP in flagellated bacteria has been extensively investigated. However, our knowledge regarding this regulation in motile bacteria without flagella remains limited. Here, we aimed to address this gap by investigating a non-flagellated bacterium with motility powered by bacterial type-IV pilus (T4P). Previous studies hinted at the possibility of Myxococcus xanthus PilB, the T4P assembly ATPase, serving as a cyclic-di-GMP effector involved in regulating both motility and biofilm. Our findings strongly support the hypothesis that PilB directly interacts with cyclic-di-GMP to act as a potential switch to promote biofilm formation or T4P-dependent motility. These results shed light on the bifurcation of PilB functions and its pivotal role in coordinating biofilm formation and T4P-mediated motility.

Deep mutational scanning of the Neisseria meningitidis major pilin reveals the importance of pilus tip-mediated adhesion.

Type IV pili (TFP) are multifunctional micrometer-long filaments expressed at the surface of many prokaryotes. In Neisseria meningitidis, TFP are crucial for virulence. Indeed, these homopolymers of the major pilin PilE mediate interbacterial aggregation and adhesion to host cells. However, the mechanisms behind these functions remain unclear. Here, we simultaneously determined regions of PilE involved in pilus display, auto-aggregation, and adhesion by using deep mutational scanning and started mining this extensive functional map. For auto-aggregation, pili must reach a minimum length to allow pilus-pilus interactions through an electropositive cluster of residues centered around Lys140. For adhesion, results point to a key role for the tip of the pilus. Accordingly, purified pili interacting with host cells initially bind via their tip-located major pilin and then along their length. Overall, these results identify functional domains of PilE and support a direct role of the major pilin in TFP-dependent aggregation and adhesion.

Expression of a Phage-Encoded Gp21 Protein Protects Pseudomonas aeruginosa against Phage Infection.

There is a continuously expanding gap between predicted phage gene sequences and their corresponding functions, which has largely hampered the development of phage therapy. Previous studies reported several phage proteins that could interfere with the intracellular processes of the host to obtain efficient infection. But few phage proteins that protect host against phage infection have been identified and characterized in detail. Here, we isolate a phage, vB_Pae_QDWS, capable of infecting Pseudomonas aeruginosa PAO1 and report that its encoded Gp21 protein protects PAO1 against phage infection. Expression of Gp21 regulates bacterial quorum sensing with an inhibitory effect in low cell density and an activation effect in high cell density. By testing the type IV pilus (TFP)-mediated twitching motility and transmission electron microscopy analysis, Gp21 was found to decrease the pilus synthesis. Further, by constructing the TFP synthesis gene pilB mutant and performing adsorption and phage resistance assay, we demonstrated that the Gp21 protein could block phage infection via decreasing the TFP-mediated phage adsorption. Gp21 is a novel protein that inhibits phage efficacy against bacteria. The study deepens our understanding of phage-host interactions. IMPORTANCE The majority of the annotated phage genes are currently deposited as "hypothetical protein" with unknown function. Research has revealed that some phage proteins serve to inhibit or redirect the host intracellular processes for phage infection. Conversely, we report a phage encoded protein Gp21 that protects the host against phage infection. The pathways that Gp21 involved in antiphage defense in Pseudomonas aeruginosa PAO1 interfere with quorum sensing and decrease type IV pilus-mediated phage adsorption. Gp21 is a novel protein with a low sequence homology with other reported twitching inhibitory proteins. As a lytic phage-derived protein, Gp21 expression protects P. aeruginosa PAO1 from reinfection by phage vB_Pae_QDWS, which may explain the well-known pseudolysogeny caused by virulent phages. Our discoveries provide valuable new insight into phage-host evolutionary dynamics.

The polar Ras-like GTPase MglA activates type IV pilus via SgmX to enable twitching motility in Myxococcus xanthus.

Type IV pili (Tfp) are highly conserved macromolecular structures that fulfill diverse cellular functions, such as adhesion to host cells, the import of extracellular DNA, kin recognition, and cell motility (twitching). Outstandingly, twitching motility enables a poorly understood process by which highly coordinated groups of hundreds of cells move in cooperative manner, providing a basis for multicellular behaviors, such as biofilm formation. In the social bacteria Myxococcus xanthus, we know that twitching motility is under the dependence of the small GTPase MglA, but the underlying molecular mechanisms remain elusive. Here we show that MglA complexed to GTP recruits a newly characterized Tfp regulator, termed SgmX, to activate Tfp machines at the bacterial cell pole. This mechanism also ensures spatial regulation of Tfp, explaining how MglA switching provokes directional reversals. This discovery paves the way to elucidate how polar Tfp machines are regulated to coordinate multicellular movements, a conserved feature in twitching bacteria.

Burkholderia pseudomallei JW270 Is Lethal in the Madagascar Hissing Cockroach Infection Model and Can Be Utilized at Biosafety Level 2 to Identify Putative Virulence Factors.

Burkholderia pseudomallei, the causative agent of melioidosis, is classified by the CDC as a tier 1 select agent, and work involving it must be performed in a biosafety level 3 (BSL-3) laboratory. Three BSL-2 surrogate strains derived from B. pseudomallei 1026b, a virulent clinical isolate, have been removed from the CDC select agent list. These strains, Bp82, B0011, and JW270, are highly attenuated in rodent models of melioidosis and cannot be utilized to identify virulence determinants because of their high 50% lethal dose (LD50). We previously demonstrated that the Madagascar hissing cockroach (MHC) is a tractable surrogate host to study the innate immune response against Burkholderia. In this study, we found that JW270 maintains its virulence in MHCs. This surprising result indicates that it may be possible to identify potential virulence genes in JW270 by using MHCs at BSL-2. We tested this hypothesis by constructing JW270 mutations in genes that are required (hcp1) or dispensable (hcp2) for B. pseudomallei virulence in rodents. JW270 Δhcp1 was avirulent in MHCs and JW270 Δhcp2 was virulent, suggesting that MHCs can be used at BSL-2 for the discovery of important virulence factors. JW270 ΔBPSS2185, a strain harboring a mutation in a type IV pilin locus (TFP8) required for full virulence in BALB/c mice, was also found to be attenuated in MHCs. Finally, we demonstrate that the hmqA-G locus, which encodes the production of a family of secondary metabolites called 4-hydroxy-3-methyl-2-alkylquinolines, is important for JW270 virulence in MHCs and may represent a novel virulence determinant.

Bacterial glycocalyx integrity drives multicellular swarm biofilm dynamism.

Exopolysaccharide (EPS) layers on the bacterial cell surface are key determinants of biofilm establishment and maintenance, leading to the formation of higher-order 3D structures that confer numerous survival benefits to a cell community. In addition to a specific cell-associated EPS glycocalyx, we recently revealed that the social δ-proteobacterium Myxococcus xanthus secretes a novel biosurfactant polysaccharide (BPS) to the extracellular milieu. Together, secretion of the two polymers (EPS and BPS) is required for type IV pilus (T4P)-dependent swarm expansion via spatio-specific biofilm expression profiles. Thus the synergy between EPS and BPS secretion somehow modulates the multicellular lifecycle of M. xanthus. Herein, we demonstrate that BPS secretion functionally alters the EPS glycocalyx via destabilization of the latter, fundamentally changing the characteristics of the cell surface. This impacts motility behaviors at the single-cell level and the aggregative capacity of cells in groups via cell-surface EPS fibril formation as well as T4P production, stability, and positioning. These changes modulate the structure of swarm biofilms via cell layering, likely contributing to the formation of internal swarm polysaccharide architecture. Together, these data reveal the manner by which the combined secretion of two distinct polymers induces single-cell changes that modulate swarm biofilm communities.

The structure and mechanism of the bacterial type II secretion system.

The type II secretion system (T2SS) is a multi-protein complex used by many bacteria to move substrates across their cell membrane. Substrates released into the environment serve as local and long-range effectors that promote nutrient acquisition, biofilm formation, and pathogenicity. In both animals and plants, the T2SS is increasingly recognized as a key driver of virulence. The T2SS spans the bacterial cell envelope and extrudes substrates through an outer membrane secretin channel using a pseudopilus. An inner membrane assembly platform and a cytoplasmic motor controls pseudopilus assembly. This microreview focuses on the structure and mechanism of the T2SS. Advances in cryo-electron microscopy are enabling increasingly elaborate sub-complexes to be resolved. However, key questions remain regarding the mechanism of pseudopilus extension and retraction, and how this is coupled with the choreography of the substrate moving through the secretion system. The T2SS is part of an ancient type IV filament superfamily that may have been present within the last universal common ancestor (LUCA). Overall, mechanistic principles that underlie T2SS function have implication for other closely related systems such as the type IV and tight adherence pilus systems.

Prevalence of Type IV Pili-Mediated Twitching Motility in Streptococcus sanguinis Strains and Its Impact on Biofilm Formation and Host Adherence.

Type IV pili (Tfp) are known to mediate several biological activities, including surface-dependent twitching motility. Although a pil gene cluster for Tfp biosynthesis is found in all sequenced Streptococcus sanguinis strains, Tfp-mediated twitching motility is less commonly detected. Upon examining 81 clinical strains, 39 strains generated twitching zones on blood agar plates (BAP), while 27 strains displayed twitching on Todd-Hewitt (TH) agar. Although BAP appears to be more suitable for the development of twitching zones, 5 strains exhibited twitching motility only on TH agar, indicating that twitching motility is not only strain specific but also sensitive to growth media. Furthermore, different twitching phenotypes were observed in strains expressing comparable levels of pilT, encoding the retraction ATPase, suggesting that the twitching phenotype on agar plates is regulated by multiple factors. By using a PilT-null and a pilin protein-null derivative (CHW02) of twitching-active S. sanguinis CGMH010, we found that Tfp retraction was essential for biofilm stability. Further, biofilm growth was amplified in CHW02 in the absence of shearing force, indicating that S. sanguinis may utilize other ligands for biofilm formation in the absence of Tfp. Similar to SK36, Tfp from CGMH010 were required for colonization of host cells, but PilT only marginally affected adherence and only in the twitching-active strain. Taken together, the results suggest that Tfp participates in host cell adherence and that Tfp retraction facilitates biofilm stability. IMPORTANCE Although the gene clusters encoding Tfp are commonly present in Streptococcus sanguinis, not all strains express surface-dependent twitching motility on agar surfaces. Regardless of whether the Tfp could drive motility, Tfp can serve as a ligand for the colonization of host cells. Though many S. sanguinis strains lack twitching activity, motility can enhance biofilm stability in a twitching-active strain; thus, perhaps motility provides little or no advantage to the survival of bacteria within dental plaque. Rather, Tfp retraction could provide additional advantages for the bacteria to establish infections outside the oral cavity.

Inhibitors of the Neisseria meningitidis PilF ATPase provoke type IV pilus disassembly.

Despite the availability of antibiotics and vaccines, Neisseria meningitidis remains a major cause of meningitis and sepsis in humans. Due to its extracellular lifestyle, bacterial adhesion to host cells constitutes an attractive therapeutic target. Here, we present a high-throughput microscopy-based approach that allowed the identification of compounds able to decrease type IV pilus-mediated interaction of bacteria with endothelial cells in the absence of bacterial or host cell toxicity. Compounds specifically inhibit the PilF ATPase enzymatic activity that powers type IV pilus extension but remain inefficient on the ATPase that promotes pilus retraction, thus leading to rapid pilus disappearance from the bacterial surface and loss of pili-mediated functions. Structure activity relationship of the most active compound identifies specific moieties required for the activity of this compound and highlights its specificity. This study therefore provides compounds targeting pilus biogenesis, thereby inhibiting bacterial adhesion, and paves the way for a novel therapeutic option for meningococcal infections.

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

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.

Amuc_1102 from Akkermansia muciniphila adopts an immunoglobulin-like fold related to archaeal type IV pilus.

Akkermansia muciniphila is a kind of beneficial microorganism colonized in the human gut. A. muciniphila is closely related to human intestinal health and has a good effect on diseases related to intestinal metabolism. The proteins encoded by the Amuc_1098-Amuc_1102 gene cluster, which are related to the formation and assembly of the pilus, are highly expressed in the membrane protein components of A. muciniphila. In this paper, we report the crystal structure of Amuc_1102 at a resolution of 1.75 Å, which adopts an immunoglobulin (Ig)-like fold. Amuc_1102 shares a similar fold to three archaeal proteins related to type IV pilus (T4P)-like structure, Pilin, FlaF, and FlaG, indicating a similar function. Amuc_1102 exists as a trimer both in the crystal structure and in solution, which differs from the assemblies of Pilin, FlaF, and FlaG. This study provides a structural basis for the elucidation of the T4P formation of A. muciniphila.

Caldicellulosiruptor bescii Adheres to Polysaccharides via a Type IV Pilin-Dependent Mechanism.

Biological hydrolysis of cellulose above 70°C involves microorganisms that secrete free enzymes and deploy separate protein systems to adhere to their substrate. Strongly cellulolytic Caldicellulosiruptor bescii is one such extreme thermophile, which deploys modular, multifunctional carbohydrate-acting enzymes to deconstruct plant biomass. Additionally, C. bescii also encodes noncatalytic carbohydrate binding proteins, which likely evolved as a mechanism to compete against other heterotrophs in carbon-limited biotopes that these bacteria inhabit. Analysis of the Caldicellulosiruptor pangenome identified a type IV pilus (T4P) locus encoded upstream of the tapirins, that is encoded by all Caldicellulosiruptor species. In this study, we sought to determine if the C. bescii T4P plays a role in attachment to plant polysaccharides. The major C. bescii pilin (CbPilA) was identified by the presence of pilin-like protein domains, paired with transcriptomics and proteomics data. Using immuno-dot blots, we determined that the plant polysaccharide xylan induced production of CbPilA 10- to 14-fold higher than glucomannan or xylose. Furthermore, we are able to demonstrate that recombinant CbPilA directly interacts with xylan and cellulose at elevated temperatures. Localization of CbPilA at the cell surface was confirmed by immunofluorescence microscopy. Lastly, a direct role for CbPilA in cell adhesion was demonstrated using recombinant CbPilA or anti-CbPilA antibodies to reduce C. bescii cell adhesion to xylan and crystalline cellulose up to 4.5- and 2-fold, respectively. Based on these observations, we propose that CbPilA and, by extension, the T4P play a role in Caldicellulosiruptor cell attachment to plant biomass.IMPORTANCE Most microorganisms are capable of attaching to surfaces in order to persist in their environment. Type IV (T4) pili produced by certain mesophilic Firmicutes promote adherence; however, a role for T4 pili encoded by thermophilic members of this phylum has yet to be demonstrated. Prior comparative genomics analyses identified a T4 pilus locus possessed by an extremely thermophilic genus within the Firmicutes Here, we demonstrate that attachment to plant biomass-related carbohydrates by strongly cellulolytic Caldicellulosiruptor bescii is mediated by T4 pilins. Surprisingly, xylan but not cellulose induced expression of the major T4 pilin. Regardless, the C. bescii T4 pilin interacts with both polysaccharides at high temperatures and is located to the cell surface, where it is directly involved in C. bescii attachment. Adherence to polysaccharides is likely key to survival in environments where carbon sources are limiting, allowing C. bescii to compete against other plant-degrading microorganisms.

Molecular and Functional Analysis of the Type IV Pilus Gene Cluster in Streptococcus sanguinis SK36.

Streptococcus sanguinis, dominant in the oral microbiome, is the only known streptococcal species possessing a pil gene cluster for the biosynthesis of type IV pili (Tfp). Although this cluster is commonly present in the genome of S. sanguinis, most of the strains do not express Tfp-mediated twitching motility. Thus, this study was designed to investigate the biological functions encoded by the cluster in the twitching-negative strain S. sanguinis SK36. We found that the cluster was transcribed as an operon, with three promoters located 5' to the cluster and one in the intergenic region between SSA_2307 and SSA_2305. Studies using promoter-cat fusion strains revealed that the transcription of the cluster was mainly driven by the distal 5' promoter, which is located more than 800 bases 5' to the first gene of the cluster, SSA_2318. Optimal expression of the cluster occurred at the early stationary growth phase in a CcpA-dependent manner, although a CcpA-binding consensus is absent in the promoter region. Expression of the cluster resulted in a short hairlike surface structure under transmission electron microscopy. Deletion of the putative pilin genes (SSA_2313 to SSA_2315) abolished the biosynthesis of this structure and significantly reduced the adherence of SK36 to HeLa and SCC-4 cells. Mutations in the pil genes downregulated biofilm formation by S. sanguinis SK36. Taken together, the results demonstrate that Tfp of SK36 are important for host cell adherence, but not for motility, and that expression of the pil cluster is subject to complex regulation.IMPORTANCE The proteins and assembly machinery of the type IV pili (Tfp) are conserved throughout bacteria and archaea, and yet the function of this surface structure differs from species to species and even from strain to strain. As seen in Streptococcus sanguinis SK36, the expression of the Tfp gene cluster results in a hairlike surface structure that is much shorter than the typical Tfp. This pilus is essential for the adherence of SK36 but is not involved in motility. Being a member of the highly diverse dental biofilm, perhaps S. sanguinis could more effectively utilize this structure to adhere to host cells and to interact with other microbes within the same niche.

The type IV pilus assembly motor PilB is a robust hexameric ATPase with complex kinetics.

The bacterial type IV pilus (T4P) is a versatile nanomachine that functions in pathogenesis, biofilm formation, motility, and horizontal gene transfer. T4P assembly is powered by the motor ATPase PilB which is proposed to hydrolyze ATP by a symmetrical rotary mechanism. This mechanism, which is deduced from the structure of PilB, is untested. Here, we report the first kinetic studies of the PilB ATPase, supporting co-ordination among the protomers of this hexameric enzyme. Analysis of the genome sequence of Chloracidobacterium thermophilum identified a pilB gene whose protein we then heterologously expressed. This PilB formed a hexamer in solution and exhibited highly robust ATPase activity. It displays complex steady-state kinetics with an incline followed by a decline over an ATP concentration range of physiological relevance. The incline is multiphasic and the decline signifies substrate inhibition. These observations suggest that variations in intracellular ATP concentrations may regulate T4P assembly and T4P-mediated functions in vivo in accordance with the physiological state of bacteria with unanticipated complexity. We also identified a mutant pilB gene in the genomic DNA of C. thermophilum from an enrichment culture. The mutant PilB variant, which is significantly less active, exhibited similar inhibition of its ATPase activity by high concentrations of ATP. Our findings here with the PilB ATPase from C. thermophilum provide the first line of biochemical evidence for the co-ordination among PilB protomers consistent with the symmetrical rotary model of catalysis based on structural studies.

Genome-wide screen identifies host colonization determinants in a bacterial gut symbiont.

Animal guts are often colonized by host-specialized bacterial species to the exclusion of other transient microorganisms, but the genetic basis of colonization ability is largely unknown. The bacterium Snodgrassella alvi is a dominant gut symbiont in honey bees, specialized in colonizing the hindgut epithelium. We developed methods for transposon-based mutagenesis in S. alvi and, using high-throughput DNA sequencing, screened genome-wide transposon insertion (Tn-seq) and transcriptome (RNA-seq) libraries to characterize both the essential genome and the genes facilitating host colonization. Comparison of Tn-seq results from laboratory cultures and from monoinoculated worker bees reveal that 519 of 2,226 protein-coding genes in S. alvi are essential in culture, whereas 399 are not essential but are beneficial for gut colonization. Genes facilitating colonization fall into three broad functional categories: extracellular interactions, metabolism, and stress responses. Extracellular components with strong fitness benefits in vivo include trimeric autotransporter adhesins, O antigens, and type IV pili (T4P). Experiments with T4P mutants establish that T4P in S. alvi likely function in attachment and biofilm formation, with knockouts experiencing a competitive disadvantage in vivo. Metabolic processes promoting colonization include essential amino acid biosynthesis and iron acquisition pathways, implying nutrient scarcity within the hindgut environment. Mechanisms to deal with various stressors, such as for the repair of double-stranded DNA breaks and protein quality control, are also critical in vivo. This genome-wide study identifies numerous genetic networks underlying colonization by a gut commensal in its native host environment, including some known from more targeted studies in other host-microbe symbioses.

A Natural Mouse Model for Neisseria Colonization.

Commensals are important for the proper functioning of multicellular organisms. How a commensal establishes persistent colonization of its host is little understood. Studies of this aspect of microbe-host interactions are impeded by the absence of an animal model. We have developed a natural small animal model for identifying host and commensal determinants of colonization and of the elusive process of persistence. Our system couples a commensal bacterium of wild mice, Neisseria musculi, with the laboratory mouse. The pairing of a mouse commensal with its natural host circumvents issues of host restriction. Studies are performed in the absence of antibiotics, hormones, invasive procedures, or genetic manipulation of the host. A single dose of N. musculi, administered orally, leads to long-term colonization of the oral cavity and gut. All mice are healthy. Susceptibility to colonization is determined by host genetics and innate immunity. For N. musculi, colonization requires the type IV pilus. Reagents and powerful tools are readily available for manipulating the laboratory mouse, allowing easy dissection of host determinants controlling colonization resistance. N. musculi is genetically related to human-dwelling commensal and pathogenic Neisseria and encodes host interaction factors and vaccine antigens of pathogenic Neisseria Our system provides a natural approach for studying Neisseria-host interactions and is potentially useful for vaccine efficacy studies.

Sigma factor RpoN employs a dual transcriptional regulation for controlling twitching motility and biofilm formation in Lysobacter enzymogenes OH11.

Lysobacter is a Gram-negative genus comprising a group of environmental bacteria with abilities to produce abundant novel antibiotics, as well as adopting a unique type IV pilus (T4P)-mediated twitching motility (TM) that remains poorly understood. Here, we employ L. enzymogenes OH11 exhibiting significant antifungal activity as a working model to address this issue. Via mutating the 28 potential sigma factors in strain OH11, we have identified one protein RpoNOH11 (sigma 54) that is indispensable for T4P formation and TM. We further showed that RpoNOH11 not only regulates the transcription of pilA, but also another crucial gene chpA that encodes a hybrid two-component transduction system. The L. enzymogenes RpoNOH11 was found to directly bind to the promoter of chpA to control its transcription, which is found to be essential for the T4P-mediated TM. To our knowledge, such a transcriptional regulation performed by RpoN in control of bacterial TM has never been reported. Finally, we showed that L. enzymogenes OH11 could also produce biofilm that is likely employed by this strain to infect fungal pathogens. Mutation of rpoN OH11, pilA and chpA all led to a significant decrease in biofilm formation, suggesting that the dual transcriptional regulation of pilA and chpA by RpoNOH11 plays a key role for RpoNOH11 to modulate the biofilm formation in L. enzymogenes. Overall, this study identified chpA as a new target of RpoN for controlling the T4P-mediated twitching motility and biofilm formation in L. enzymogenes OH11.

Two direct gene targets contribute to Clp-dependent regulation of type IV pilus-mediated twitching motility in Lysobacter enzymogenes OH11.

Lysobacter enzymogenes is an agriculturally important Gram-negative bacterium that employs a multitude of antifungal mechanisms to inhibit and infect filamentous fungal pathogens, through secretion of antifungal antibiotic HSAF (heat-stable antifungal factor), formation of T4P (type IV pilus)-mediated twitching motility, and production of extracellular chitinase. Interestingly, all such key antifungal factors seem to be controlled by Clp, a master regulator in L. enzymogenes; however, the underlying mechanisms are poorly understood. Here, employing strain OH11 as a working model, we show that Clp plays a dual role in controlling OH11 twitching motility. It controls transcription of pilA, a major T4P structure pilin gene, via directly binding to its promoter region, as well as regulates the gene transcription of pilMONOPQ operon, whose products were essential for T4P assembly, by directly binding to a similar promoter sequence. We also truncated the Clp-binding region of the pilA promoter fragment down to 41 bp to identify the potential Clp-binding sequence. In addition, the Clp-recognized pilM promoter motif of the L. enzymogenes strains is similarly conserved as the pilA promoter, both with a conserved 5'-GTG and a conserved CAC-3', spaced by ten highly variable nucleotides. Thus, this study identified two direct and previously uncharacterized gene targets of Clp contributing to its regulation in the L. enzymogenes twitching motility. Overall, our findings further elucidate the molecular genetics of Clp-dependent twitching motility in Lysobacter.

Type IV pilus biogenesis genes and their roles in biofilm formation in the biological control agent Lysobacter enzymogenes OH11.

Type IV pilus (T4P) is widespread in bacteria, yet its biogenesis mechanism and functionality is only partially elucidated in a limited number of bacterial species. Here, by using strain OH11 as the model organism, we reported the identification of 26 T4P structural or functional component (SFC) proteins in the Gram-negative Lysobacter enzymogenes, which is a biocontrol agent potentially exploiting T4P-mediated twitching motility for antifungal activity. Twenty such SFC coding genes were individually knocked-out in-frame to create a T4P SFC deletion library. By using combined phenotypic and genetic approaches, we found that 14 such SFCs, which were expressed from four operons, were essential for twitching motility. These SFCs included the minor pilins (PilEi, PilXi, PilVi, and FimTi), the anti-retraction protein PilY1i, the platform protein PilC, the extension/extraction ATPases (PilB, PilT, and PilU), and the PilMNOPQ complex. Among these, mutation of pilT or pilU caused a hyper piliation, while the remaining 12 SFCs were indispensable for pilus formation. Ten (FimTi, PilY1i, PilB, PilT, PilU, and the PilMNOPQ complex) of the 14 SFC proteins, as well as PilA, were further shown to play a key role in L. enzymogenes biofilm formation. Overall, our results provide the first report to dissect the genetic basis of T4P biogenesis and its role in biofilm formation in L. enzymogenes in detail, which can serve as an alternative platform for studying T4P biogenesis and its antifungal function.