Probable Role of Type IV Pili of Aeromonas hydrophila in Human Pathogenicity.

BACKGROUND: Aeromonas hydrophila is a widely recognized broad-spectrum pathogen that primarily targets the gastrointestinal tract. Type IV pili (T4P) are proteinaceous nano-machines located on the bacterial cell surface, playing a crucial role in host colonization and infection. Regrettably, the T4P systems of A. hydrophila remain largely underexplored. METHODS: A. hydrophila genomes with complete genome assembly and annotation reports up to 31 March 2023, were obtained from the NCBI Genome database or KEGG genome database, followed by a global search for T4P secretion system genes. Protein sequences of these manually curetted genes were used as secondary quarry for Synteny analysis. Protein-protein interaction analysis was performed by string analysis and in silico study of genomic islands. RESULTS: We identified 27 orthologs of type IV pili (T4P) nano-machine components in A. hydrophila. These orthologs are primarily distributed across three operons: pilABCD, pilMNOPQ, and pilVWXY. While the first two operons are commonly found in all experimental genomes, the presence of the pilVWXY operon, coding for 11 orthologs, is reported here for the first time in A. hydrophila. Notably, the complete pilVWXY operon is absent in nonvirulent strains. A genomic islands study between a nonvirulent and hypervirulent strain also confirms absence of most of the genes coded by pilVWXY in nonvirulent strain. Interestingly, among the 51 experimental genomes analyzed, the pilVWXY operon was completely absent in 10 strains, most of which are categorized as nonvirulent; Conclusions: The distribution of two major type IV pili (T4P) nano-machines, PilABCDMNOPQ and PilVWXY, is reported here for the first time in A. hydrophila. Additionally, this study suggests a potential role for the PilVWXY nano-machine in establishing human disease.

From isotopically labeled DNA to fluorescently labeled dynamic pili: building a mechanistic model of DNA transport to the cytoplasmic membrane.

SUMMARYNatural competence, the physiological state wherein bacteria produce proteins that mediate extracellular DNA transport into the cytosol and the subsequent recombination of DNA into the genome, is conserved across the bacterial domain. DNA must successfully translocate across formidable permeability barriers during import, including the cell membrane(s) and the cell wall, that are normally impermeable to large DNA polymers. This review will examine the mechanisms underlying DNA transport from the extracellular space to the cytoplasmic membrane. First, the challenges inherent to DNA movement through the cell periphery will be discussed to provide context for DNA transport during natural competence. The following sections will trace the development of a comprehensive model for DNA translocation to the cytoplasmic membrane, highlighting the crucial studies performed over the last century that have contributed to building contemporary DNA import models. Finally, this review will conclude by reflecting on what is still unknown about the process and the possible solutions to overcome these limitations.

Type IV pili of Enterobacteriaceae species.

Type IV pili (T4Ps) are surface filaments widely distributed among bacteria and archaea. T4Ps are involved in many cellular functions and contribute to virulence in some species of bacteria. Due to the diversity of T4Ps, different properties have been observed for homologous proteins that make up T4Ps in various organisms. In this review, we highlight the essential components of T4Ps, their functions, and similarities to related systems. We emphasize the unique T4Ps of enteric pathogens within the Enterobacteriaceae family, which includes pathogenic strains of Escherichia coli and Salmonella. These include the bundle-forming pilus (BFP) of enteropathogenic E. coli (EPEC), longus (Lng) and colonization factor III (CFA/III) of enterotoxigenic E. coli (ETEC), T4P of Salmonella enterica serovar Typhi, Colonization Factor Citrobacter (CFC) of Citrobacter rodentium, T4P of Yersinia pseudotuberculosis, a ubiquitous T4P that was characterized in enterohemorrhagic E. coli (EHEC), and the R64 plasmid thin pilus. Finally, we highlight areas for further study.

The phage-encoded PIT4 protein affects multiple two-component systems of Pseudomonas aeruginosa.

IMPORTANCE: More and more Pseudomonas aeruginosa isolates have become resistant to antibiotics like carbapenem. As a consequence, P. aeruginosa ranks in the top three of pathogens for which the development of novel antibiotics is the most crucial. The pathogen causes both acute and chronic infections, especially in patients who are the most vulnerable. Therefore, efforts are urgently needed to develop alternative therapies. One path explored in this article is the use of bacteriophages and, more specifically, phage-derived proteins. In this study, a phage-derived protein was studied that impacts key virulence factors of the pathogen via interaction with multiple histidine kinases of TCSs. The fundamental insights gained for this protein can therefore serve as inspiration for the development of an anti-virulence compound that targets the bacterial TCS.

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.

Discovery of Two Inhibitors of the Type IV Pilus Assembly ATPase PilB as Potential Antivirulence Compounds.

With the pressing antibiotic resistance pandemic, antivirulence has been increasingly explored as an alternative strategy against bacterial infections. The bacterial type IV pilus (T4P) is a well-documented virulence factor and an attractive target for small molecules for antivirulence purposes. The PilB ATPase is essential for T4P biogenesis because it catalyzes the assembly of monomeric pilins into the polymeric pilus filament. Here, we describe the identification of two PilB inhibitors by a high-throughput screen (HTS) in vitro and their validation as effective inhibitors of T4P assembly in vivo. We used Chloracidobacterium thermophilum PilB as a model enzyme to optimize an ATPase assay for the HTS. From a library of 2,320 compounds, benserazide and levodopa, two approved drugs for Parkinson's disease, were identified and confirmed biochemically to be PilB inhibitors. We demonstrate that both compounds inhibited the T4P-dependent motility of the bacteria Myxoccocus xanthus and Acinetobacter nosocomialis. Additionally, benserazide and levodopa were shown to inhibit A. nosocomialis biofilm formation, a T4P-dependent process. Using M. xanthus as a model, we showed that both compounds inhibited T4P assembly in a dose-dependent manner. These results suggest that these two compounds are effective against the PilB protein in vivo. The potency of benserazide and levodopa as PilB inhibitors both in vitro and in vivo demonstrate potentials of the HTS and its two hits here for the development of anti-T4P chemotherapeutics. IMPORTANCE Many bacterial pathogens use their type IV pilus (T4P) to facilitate and maintain an infection in a human host. Small-molecule inhibitors of the production or assembly of the T4P are promising for the treatment and prevention of infections by these bacteria, especially in our fight against antibiotic-resistant pathogens. Here, we report the development and implementation of a method to identify anti-T4P chemicals from compound libraries by high-throughput screen. This led to the identification and validation of two T4P inhibitors both in the test tubes and in bacteria. The discovery and validation pipeline reported here as well as the confirmation of two anti-T4P inhibitors provide new venues and leads for the development of chemotherapeutics against antibiotic-resistant infections.

Analysis of Myxococcus xanthus Vegetative Biofilms With Microtiter Plates.

The bacterium Myxococcus xanthus forms both developmental and vegetative types of biofilms. While the former has been studied on both agar plates and submerged surfaces, the latter has been investigated predominantly on agar surfaces as swarming colonies. Here we describe the development of a microplate-based assay for the submerged biofilms of M. xanthus under vegetative conditions. We examined the impacts of inoculation, aeration, and temperature to optimize the conditions for the assay. Aeration was observed to be critical for the effective development of submerged biofilms by M. xanthus, an obligate aerobic bacterium. In addition, temperature plays an important role in the development of M. xanthus submerged biofilms. It is well established that the formation of submerged biofilms by many bacteria requires both exopolysaccharide (EPS) and the type IV pilus (T4P). EPS constitutes part of the biofilm matrix that maintains and organizes bacterial biofilms while the T4P facilitates surface attachment as adhesins. For validation, we used our biofilm assay to examine a multitude of M. xanthus strains with various EPS and T4P phenotypes. The results indicate that the levels of EPS, but not of piliation, positively correlate with submerged biofilm formation in M. xanthus.

High-Throughput Screen for Inhibitors of the Type IV Pilus Assembly ATPase PilB.

The bacterial type IV pilus (T4P) is a prominent virulence factor in many significant human pathogens, some of which have become increasingly antibiotic resistant. Antivirulence chemotherapeutics are considered a promising alternative to antibiotics because they target the disease process instead of bacterial viability. However, a roadblock to the discovery of anti-T4P compounds is the lack of a high-throughput screen (HTS) that can be implemented relatively easily and economically. Here, we describe the first HTS for the identification of inhibitors specifically against the T4P assembly ATPase PilB in vitroChloracidobacterium thermophilum PilB (CtPilB) had been demonstrated to have robust ATPase activity and the ability to bind its expected ligands in vitro. We utilized CtPilB and MANT-ATP, a fluorescent ATP analog, to develop a binding assay and adapted it for an HTS. As a proof of principle, we performed a pilot screen with a small compound library of kinase inhibitors and identified quercetin as a PilB inhibitor in vitro Using Myxococcus xanthus as a model bacterium, we found quercetin to reduce its T4P-dependent motility and T4P assembly in vivo. These results validated our HTS as effective in identifying PilB inhibitors. This assay may prove valuable in seeking leads for the development of antivirulence chemotherapeutics against PilB, an essential and universal component of all bacterial T4P systems.IMPORTANCE Many bacterial pathogens use their type IV pili (T4P) to facilitate and maintain infection of a human host. Small chemical compounds that inhibit the production or assembly of T4P hold promise in the treatment and prevention of infections, especially in the era of increasing threats from antibiotic-resistant bacteria. However, few chemicals are known to have inhibitory or anti-T4P activity. Their identification has not been easy due to the lack of a method for the screening of compound collections or libraries on a large scale. Here, we report the development of an assay that can be scaled up to screen compound libraries for inhibitors of a critical T4P assembly protein. We further demonstrate that it is feasible to use whole cells to examine potential inhibitors for their activity against T4P assembly in a bacterium.

The Role of Minor Pilins in Assembly and Function of the Competence Pilus of Streptococcus pneumoniae.

The remarkable genomic plasticity of Streptococcus pneumoniae largely depends on its ability to undergo natural genetic transformation. To take up extracellular DNA, S. pneumoniae assembles competence pili composed of the major pilin ComGC. In addition to ComGC, four minor pilins ComGD, E, F, and G are expressed during bacterial competence, but their role in pilus biogenesis and transformation is unknown. Here, using a combination of protein-protein interaction assays we show that all four proteins can directly interact with each other. Pneumococcal ComGG stabilizes the minor pilin ComGD and ComGF and can interact with and stabilize the major pilin ComGC, thus, deletion of ComGG abolishes competence pilus assembly. We further demonstrate that minor pilins are present in sheared pili fractions and find ComGF to be incorporated along the competence pilus by immunofluorescence and electron microscopy. Finally, mutants of the invariant Glu5 residue (E5), ComGDE5A or ComGEE5A, but not ComGFE5A, were severely impaired in pilus formation and function. Together, our results suggest that ComGG, lacking E5, is essential for competence pilus assembly and function, and plays a central role in connecting the pneumococcal minor pilins to ComGC.

Function and Benefits of Natural Competence in Cyanobacteria: From Ecology to Targeted Manipulation.

Natural competence is the ability of a cell to actively take up and incorporate foreign DNA in its own genome. This trait is widespread and ecologically significant within the prokaryotic kingdom. Here we look at natural competence in cyanobacteria, a group of globally distributed oxygenic photosynthetic bacteria. Many cyanobacterial species appear to have the genetic potential to be naturally competent, however, this ability has only been demonstrated in a few species. Reasons for this might be due to a high variety of largely uncharacterised competence inducers and a lack of understanding the ecological context of natural competence in cyanobacteria. To shed light on these questions, we describe what is known about the molecular mechanisms of natural competence in cyanobacteria and analyse how widespread this trait might be based on available genomic datasets. Potential regulators of natural competence and what benefits or drawbacks may derive from taking up foreign DNA are discussed. Overall, many unknowns about natural competence in cyanobacteria remain to be unravelled. A better understanding of underlying mechanisms and how to manipulate these, can aid the implementation of cyanobacteria as sustainable production chassis.

Pseudomonas aeruginosa is capable of natural transformation in biofilms.

Natural transformation is a mechanism that enables competent bacteria to acquire naked, exogenous DNA from the environment. It is a key process that facilitates the dissemination of antibiotic resistance and virulence determinants throughout bacterial populations. Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that produces large quantities of extracellular DNA (eDNA) that is required for biofilm formation. P. aeruginosa has a remarkable level of genome plasticity and diversity that suggests a high degree of horizontal gene transfer and recombination but is thought to be incapable of natural transformation. Here we show that P. aeruginosa possesses homologues of all proteins known to be involved in natural transformation in other bacterial species. We found that P. aeruginosa in biofilms is competent for natural transformation of both genomic and plasmid DNA. Furthermore, we demonstrate that type-IV pili (T4P) facilitate but are not absolutely essential for natural transformation in P. aeruginosa.

ChpC controls twitching motility-mediated expansion of Pseudomonas aeruginosa biofilms in response to serum albumin, mucin and oligopeptides.

Twitching motility-mediated biofilm expansion occurs via coordinated, multi-cellular collective behaviour to allow bacteria to actively expand across surfaces. Type-IV pili (T4P) are cell-associated virulence factors which mediate twitching motility via rounds of extension, surface attachment and retraction. The Chp chemosensory system is thought to respond to environmental signals to regulate the biogenesis, assembly and twitching motility function of T4P. In other well characterised chemosensory systems, methyl-accepting chemotaxis proteins (MCPs) feed environmental signals through a CheW adapter protein to the histidine kinase CheA to modulate motility. The Pseudomonas aeruginosa Chp system has an MCP PilJ and two CheW adapter proteins, PilI and ChpC, that likely interact with the histidine kinase ChpA to feed environmental signals into the system. In the current study we show that ChpC is involved in the response to host-derived signals serum albumin, mucin and oligopeptides. We demonstrate that these signals stimulate an increase in twitching motility, as well as in levels of 3'-5'-cyclic adenosine monophosphate (cAMP) and surface-assembled T4P. Interestingly, our data shows that changes in cAMP and surface piliation levels are independent of ChpC but that the twitching motility response to these environmental signals requires ChpC. Furthermore, we show that protease activity is required for the twitching motility response of P. aeruginosa to environmental signals. Based upon our data we propose a model whereby ChpC feeds these environmental signals into the Chp system, potentially via PilJ or another MCP, to control twitching motility. PilJ and PilI then modulate T4P surface levels to allow the cell to continue to undergo twitching motility. Our study is the first to link environmental signals to the Chp chemosensory system and refines our understanding of how this system controls twitching motility-mediated biofilm expansion in P. aeruginosa.

Biological synthesis of high-conductive pili in aerobic bacterium Pseudomonas aeruginosa.

Bioelectrical nanowires as ecomaterials have great potential on environmental applications. A wide range of bacteria can express type IV pili (T4P), which are long protein fibers assembled from PilA. The T4P of Geobacter sulfurreducens are well known as "microbial nanowires," yet T4P of Pseudomonas aeruginosa (PaT4P) was believed to be poorly conductive. P. aeruginosa is an aerobic and electrochemically active bacterium. Its T4P have been known to be responsible for surface attachment, twitching motility and biofilm formation. Here, we show that PaT4P can be highly conductive while assembled by a truncated P. aeruginosa PilA (PaPilA) containing only N-terminus 61 amino acids. Furthermore, increasing the number of aromatic amino acids in the PaPilA1-61 significantly enhances the conductivity of pili and the bioelectricity output of P. aeruginosa in microbial fuel cell system, suggesting a potential application of PaT4P as a conductive nanomaterial. The N-terminal region of PilA from diverse eubacteria is highly conserved, implying a general way to synthesize highly conductive microbial nanowires and to increase the bioelectricity output of microbial fuel cell.

A global genomic approach uncovers novel components for twitching motility-mediated biofilm expansion in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed twitching motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa twitching motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in twitching motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggest that the flagellum-associated gene products have a differential effect on twitching motility, based on whether components are intra- or extracellular. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens.

Mangrovicella endophytica gen. nov., sp. nov., a new member of the family Aurantimonadaceae isolated from Aegiceras corniculatum.

A Gram-negative, aerobic, motile and short-rod-shaped bacterium, designated strain 5T4P-12-1T, was isolated from a piece of surface-sterilized bark of Aegiceras corniculatum collected from Cotai Ecological Zones in Macao, China and tested by a polyphasic approach to clarify its taxonomic position. Strain 5T4P-12-1T grew optimally with 0-1 % (w/v) NaCl at 30 °C and at pH 7.0-8.0. The 16S rRNA gene sequence of strain 5T4P-12-1T had the highest similarity (96.7 %) to Aureimonas altamirensis DSM 21988T. Phylogenic analysis based on 16S rRNA gene sequences and coding sequences of 98 protein clusters showed that the strain represented a novel genus of the family Aurantimonadaceae. The predominant quinone system of strain 5T4P-12-1T was ubiquinone 10. The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylmethylethanolamine, an unidentified aminophospholipid, three unidentified aminolipids, three unidentified phospholipids and three unidentified lipids. The major fatty acids (>10 % of total fatty acids) were C18 : 1ω7c (55.4 %) and C18 : 1 2-OH (15.6 %). The DNA G+C content of strain 5T4P-12-1T was 66.5 mol%. Based on the phylogenic, phenotypic and chemotaxonomic features, strain 5T4P-12-1T is considered to represent a novel species of a new genus in the family Aurantimonadaceae, for which the name Mangrovicella endophytica gen. nov., sp. nov. is proposed. The type strain is 5T4P-12-1T (=KCTC 62053T=CGMCC 1.16279 T).

Shearing and Enrichment of Extracellular Type IV Pili.

Pili are widespread among bacteria. Type IVa pili (T4aP) are associated with a variety of bacterial functions, including adhesion, motility, natural transformation, biofilm formation, and force-dependent signaling. In pathogenic bacteria, T4aP play a crucial role during infection and have been the subject of hundreds of studies. Methods for the isolation and purification of T4aP were first described in the 1970s. Purified pili have been used for studies of filament protein content, morphology, immunogenicity, post-translational modifications, and X-ray crystallography. We detail a tried-and-true method of isolating large amounts of native T4aP from bacterial surfaces. The method requires supplies and equipment that are available in most microbiology labs.

ChpA Controls Twitching Motility and Broadly Affects Gene Expression in the Biological Control Agent Lysobacter enzymogenes.

Lysobacter enzymogenes (L. enzymogenes) is an agriculturally important Gram-negative bacterium that employs T4P (type IV pili)-driven twitching motility to exhibit its antifungal function. Yet, it is still unclear how this bacterium regulates its twitching motility. Here, by using strain OH11 as the working model organism, we showed that a hybrid two-component system ChpA acts as a positive regulator in controlling twitching motility in L. enzymogenes. ChpA is a hybrid TCS (two-component transduction system) contains 7 domains including those for auto-phosphorylation and phosphate group transfer, as well as a phosphate receiver (REC) domain. Mutation of chpA completely abolished the wild-type twitching motility, as evidenced by the absence of mobile cells at the margin of the mutant colonies. Further studies of domain-deletion and phenotypic characterization reveal that domains responsible for phosphorylation and phosphotransfer, but not the REC domain, were indispensable for ChpA in regulating twitching motility. Transcriptome analyses of the chpA knockout strain indicated that ChpA was extensively involved in controlling expression of a wide variety of genes (totaling 243). The products of these differentially expressed genes were involved in multiple physiological and biological functions in L. enzymogenes. Thus, we have not only identified a new regulator controlling twitching motility in L. enzymogenes, but also provided the first report demonstrating the broad impact of the conserved ChpA in gene regulation in Gram-negative bacteria.

Micro-Patterned Surfaces That Exploit Stigmergy to Inhibit Biofilm Expansion.

Twitching motility is a mode of surface translocation that is mediated by the extension and retraction of type IV pili and which, depending on the conditions, enables migration of individual cells or can manifest as a complex multicellular collective behavior that leads to biofilm expansion. When twitching motility occurs at the interface of an abiotic surface and solidified nutrient media, it can lead to the emergence of extensive self-organized patterns of interconnected trails that form as a consequence of the actively migrating bacteria forging a furrow network in the substratum beneath the expanding biofilm. These furrows appear to direct bacterial movements much in the same way that roads and footpaths coordinate motor vehicle and human pedestrian traffic. Self-organizing systems such as these can be accounted for by the concept of stigmergy which describes self-organization that emerges through indirect communication via persistent signals within the environment. Many bacterial communities are able to actively migrate across solid and semi-solid surfaces through complex multicellular collective behaviors such as twitching motility and flagella-mediated swarming motility. Here, we have examined the potential of exploiting the stigmergic behavior of furrow-mediated trail following as a means of controlling bacterial biofilm expansion along abiotic surfaces. We found that incorporation of a series of parallel micro-fabricated furrows significantly impeded active biofilm expansion by Pseudomonas aeruginosa and Proteus vulgaris. We observed that in both cases bacterial movements tended to be directed along the furrows. We also observed that narrow furrows were most effective at disrupting biofilm expansion as they impeded the ability of cells to self-organize into multicellular assemblies required for escape from the furrows and migration into new territory. Our results suggest that the implementation of micro-fabricated furrows that exploit stigmergy may be a novel approach to impeding active biofilm expansion across abiotic surfaces such as those used in medical and industrial settings.

The Hsp70-like StkA functions between T4P and Dif signaling proteins as a negative regulator of exopolysaccharide in Myxococcus xanthus.

Myxococcus xanthus displays a form of surface motility known as social (S) gliding. It is mediated by the type IV pilus (T4P) and requires the exopolysaccharide (EPS) to function. It is clear that T4P retraction powers S motility. EPS on a neighboring cell or deposited on a gliding surface is proposed to anchor the distal end of a pilus and trigger T4P retraction at its proximal end. Inversely, T4P has been shown to regulate EPS production upstream of the Dif signaling pathway. Here we describe the isolation of two Tn insertions at the stk locus which had been known to play roles in cellular cohesion and formation of cell groups. An insertion in stkA (MXAN_3474) was identified based on its ability to restore EPS to a pilA deletion mutant. The stkA encodes a DnaK or Hsp70 homolog and it is upstream of stkB (MXAN_3475) and stkC (MXAN_3476). A stkB insertion was identified in a separate genetic screen because it eliminated EPS production of an EPS(+) parental strain. Our results with in-frame deletions of these three stk genes indicated that the stkA mutant produced increased level of EPS while stkB and stkC mutants produced less EPS relative to the wild type. S motility and developmental aggregation were affected by deletions of stkA and stkB but only minimally by the deletion of stkC. Genetic epistasis indicated that StkA functions downstream of T4P but upstream of the Dif proteins as a negative regulator of EPS production in M. xanthus.

Self-organization of bacterial biofilms is facilitated by extracellular DNA.

Twitching motility-mediated biofilm expansion is a complex, multicellular behavior that enables the active colonization of surfaces by many species of bacteria. In this study we have explored the emergence of intricate network patterns of interconnected trails that form in actively expanding biofilms of Pseudomonas aeruginosa. We have used high-resolution, phase-contrast time-lapse microscopy and developed sophisticated computer vision algorithms to track and analyze individual cell movements during expansion of P. aeruginosa biofilms. We have also used atomic force microscopy to examine the topography of the substrate underneath the expanding biofilm. Our analyses reveal that at the leading edge of the biofilm, highly coherent groups of bacteria migrate across the surface of the semisolid media and in doing so create furrows along which following cells preferentially migrate. This leads to the emergence of a network of trails that guide mass transit toward the leading edges of the biofilm. We have also determined that extracellular DNA (eDNA) facilitates efficient traffic flow throughout the furrow network by maintaining coherent cell alignments, thereby avoiding traffic jams and ensuring an efficient supply of cells to the migrating front. Our analyses reveal that eDNA also coordinates the movements of cells in the leading edge vanguard rafts and is required for the assembly of cells into the "bulldozer" aggregates that forge the interconnecting furrows. Our observations have revealed that large-scale self-organization of cells in actively expanding biofilms of P. aeruginosa occurs through construction of an intricate network of furrows that is facilitated by eDNA.