The ancestral stringent response potentiator, DksA has been adapted throughout Salmonella evolution to orchestrate the expression of metabolic, motility, and virulence pathways.

DksA is a conserved RNA polymerase-binding protein known to play a key role in the stringent response of proteobacteria species, including many gastrointestinal pathogens. Here, we used RNA-sequencing of Escherichia coli, Salmonella bongori and Salmonella enterica serovar Typhimurium, together with phenotypic comparison to study changes in the DksA regulon, during Salmonella evolution. Comparative RNA-sequencing showed that under non-starved conditions, DksA controls the expression of 25%, 15%, and 20% of the E. coli, S. bongori, and S. enterica genes, respectively, indicating that DksA is a pleiotropic regulator, expanding its role beyond the canonical stringent response. We demonstrate that DksA is required for the growth of these three enteric bacteria species in minimal medium and controls the expression of the TCA cycle, glycolysis, pyrimidine biosynthesis, and quorum sensing. Interestingly, at multiple steps during Salmonella evolution, the type I fimbriae and various virulence genes encoded within SPIs 1, 2, 4, 5, and 11 have been transcriptionally integrated under the ancestral DksA regulon. Consequently, we show that DksA is necessary for host cells invasion by S. Typhimurium and S. bongori and for intracellular survival of S. Typhimurium in bone marrow-derived macrophages (BMDM). Moreover, we demonstrate regulatory inversion of the conserved motility-chemotaxis regulon by DksA, which acts as a negative regulator in E. coli, but activates this pathway in S. bongori and S. enterica. Overall, this study demonstrates the regulatory assimilation of multiple horizontally acquired virulence genes under the DksA regulon and provides new insights into the evolution of virulence genes regulation in Salmonella spp.

Global Transcriptional Repression of Diguanylate Cyclases by MucR1 Is Essential for Sinorhizobium-Soybean Symbiosis.

The ubiquitous bacterial second messenger c-di-GMP is intensively studied in pathogens but less so in mutualistic bacteria. Here, we report a genome-wide investigation of functional diguanylate cyclases (DGCs) synthesizing c-di-GMP from two molecules of GTP in Sinorhizobium fredii CCBAU45436, a facultative microsymbiont fixing nitrogen in nodules of diverse legumes, including soybean. Among 25 proteins harboring a putative GGDEF domain catalyzing the biosynthesis of c-di-GMP, eight functional DGCs were identified by heterogenous expression in Escherichia coli in a Congo red binding assay. This screening result was further verified by in vitro enzymatic assay with purified full proteins or the GGDEF domains from representative functional and nonfunctional DGCs. In the same in vitro assay, a functional EAL domain catalyzing the degradation of c-di-GMP into pGpG was identified in a protein that has an inactive GGDEF domain but with an active phosphodiesterase (PDE) function. The identified functional DGCs generally exhibited low transcription levels in soybean nodules compared to free-living cultures, as revealed in transcriptomes. An engineered upregulation of a functional DGC in nodules led to a significant increase of c-di-GMP level and symbiotic defects, which were not observed when a functional EAL domain was upregulated at the same level. Further transcriptional analysis and gel shift assay demonstrated that these functional DGCs were all transcriptionally repressed in nodules by a global pleiotropic regulator, MucR1, that is essential in Sinorhizobium-soybean symbiosis. These findings shed novel insights onto the systematic regulation of c-di-GMP biosynthesis in mutualistic symbiosis. IMPORTANCE The ubiquitous second messenger c-di-GMP is well-known for its role in biofilm formation and host adaptation of pathogens, whereas it is less investigated in mutualistic symbioses. Here, we reveal a cocktail of eight functional diguanylate cyclases (DGCs) catalyzing the biosynthesis of c-di-GMP in a broad-host-range Sinorhizobium that can establish nitrogen-fixing nodules on soybean and many other legumes. These functional DGCs are generally transcribed at low levels in soybean nodules compared to free-living conditions. The engineered nodule-specific upregulation of DGC can elevate the c-di-GMP level and cause symbiotic defects, while the upregulation of a phosphodiesterase that quenches c-di-GMP has no detectable symbiotic defects. Moreover, eight functional DGCs located on two different replicons are all directly repressed in nodules by a global silencer, MucR1, that is essential for Sinorhizobium-soybean symbiosis. These findings represent a novel mechanism of a strategic regulation of the c-di-GMP biosynthesis arsenal in prokaryote-eukaryote interactions.

Phylogeny Reveals Novel HipA-Homologous Kinase Families and Toxin-Antitoxin Gene Organizations.

Toxin-antitoxin modules function in the genetic stability of mobile genetic elements, bacteriophage defense, and antibiotic tolerance. A gain-of-function mutation of the Escherichia coli K-12 hipBA module can induce antibiotic tolerance in a subpopulation of bacterial cells, a phenomenon known as persistence. HipA is a Ser/Thr kinase that phosphorylates and inactivates glutamyl tRNA synthetase, inhibiting cellular translation and inducing the stringent response. Additional characterized HipA homologues include HipT from pathogenic E. coli O127 and YjjJ of E. coli K-12, which are encoded by tricistronic hipBST and monocistronic operons, respectively. The apparent diversity of HipA homologues in bacterial genomes inspired us to investigate overall phylogeny. Here, we present a comprehensive phylogenetic analysis of the Hip kinases in bacteria and archaea that expands on this diversity by revealing seven novel kinase families. Kinases of one family, encoded by monocistronic operons, consist of an N-terminal core kinase domain, a HipS-like domain, and a HIRAN (HIP116 Rad5p N-terminal) domain. HIRAN domains bind single- or double-stranded DNA ends. Moreover, five types of bicistronic kinase operons encode putative antitoxins with HipS-HIRAN, HipS, γδ-resolvase, or Stl repressor-like domains. Finally, our analysis indicates that reversion of hipBA gene order happened independently several times during evolution. IMPORTANCE Bacterial multidrug tolerance and persistence are problems of increasing scientific and medical significance. The first gene discovered to confer persistence was hipA, encoding the kinase toxin of the hipBA toxin-antitoxin (TA) module of E. coli. HipA-homologous kinases phosphorylate and thereby inactivate specific tRNA synthetases, thus inhibiting protein translation and cell proliferation. Here, we present a comprehensive phylogenetic analysis of bacterial Hip kinases and discover seven new families with novel operon structures and domains. Overall, Hip kinases are encoded by TA modules with at least 10 different genetic organizations, 7 of which have not been described before. These results open up exciting avenues for the experimental analysis of the superfamily of Hip kinases.

Investigating the amino acid sequences of membrane bound dihydroorotate:quinone oxidoreductases (DHOQOs): Structural and functional implications.

Dihydroorotate:quinone oxidoreductases (DHOQOs) are membrane bound enzymes responsible for oxidizing dihydroorotate (DHO) to orotate with concomitant reduction of quinone to quinol. They have FMN as prosthetic group and are part of the monotopic quinone reductase superfamily. These enzymes are also members of the dihydroorotate dehydrogenases (DHODHs) family, which besides membrane bound DHOQOs, class 2, includes soluble enzymes which reduce either NAD+ or fumarate, class 1. As key enzymes in both the de novo pyrimidine biosynthetic pathway as well as in the energetic metabolism, inhibitors of DHOQOs have been investigated as leads for therapeutics in cancer, immunological disorders and bacterial/viral infections. This work is a thorough bioinformatic approach on the structural conservation and taxonomic distribution of DHOQOs. We explored previously established structural/functional hallmarks of these enzymes, while searching for uncharacterized common elements. We also discuss the cellular role of DHOQOs and organize the identified protein sequences within six sub-classes 2A to 2F, according to their taxonomic origin and sequence traits. We concluded that DHOQOs are present in Archaea, Eukarya and Bacteria, including the first recognition in Gram-positive organisms. DHOQOs can be the single dihydroorotate dehydrogenase encoded in the genome of a species, or they can coexist with other DHODHs, as the NAD+ or fumarate reducing enzymes. Furthermore, we show that the type of catalytic base present in the active site is not an absolute criterium to distinguish between class 1 and class 2 enzymes. We propose the existence of a quinone binding motif ("ExAH") adjacent to a hydrophobic cavity present in the membrane interacting N-terminal domain.

Comparative Proteomics of Commensal and Pathogenic Strains of Escherichia coli.

BACKGROUND: Most of the proteomic studies in Escherichia coli have focussed on pathogenic strains, while very few studies have studied the commensal strains. It is important to study the commensal strains because under the selective pressure of their habitat, commensal strains might serve as reservoirs of virulent and pathogenic strains. OBJECTIVE: In this study, we have performed a comparative proteomic analysis of commensal and pathogenic strains of E. coli isolated from a major river flowing through northern India. METHODS: Proteins were resolved by two dimensional gel electrophoresis and the differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time of flight mass-spectrometry (MALDI-TOF MS). RESULTS: Many proteins of the commensal strain showed an increased expression compared to the pathogenic strain, of which seventeen proteins were identified by MALDI-TOF MS. Functional classification of these proteins revealed that they belonged to different functional pathways like energy metabolism, nucleotide and nucleoside conversions, translation, biosynthesis of amino acids and motility and energytaxis/chemotaxis. CONCLUSION: As per the best of our knowledge, this is the first report on comparative proteomic analysis of E. coli commensal and pathogenic strains of aquatic origin. Our results suggest that the increased production of these proteins might play an important role in adaptation of E. coli to a commensal/pathogenic lifestyle. However, further experiments are required to understand the precise role of these proteins in regulating the pathogenicity/commensalism of E. coli.

Escherichia coli Small Proteome.

Escherichia coli was one of the first species to have its genome sequenced and remains one of the best-characterized model organisms. Thus, it is perhaps surprising that recent studies have shown that a substantial number of genes have been overlooked. Genes encoding more than 140 small proteins, defined as those containing 50 or fewer amino acids, have been identified in E. coli in the past 10 years, and there is substantial evidence indicating that many more remain to be discovered. This review covers the methods that have been successful in identifying small proteins and the short open reading frames that encode them. The small proteins that have been functionally characterized to date in this model organism are also discussed. It is hoped that the review, along with the associated databases of known as well as predicted but undetected small proteins, will aid in and provide a roadmap for the continued identification and characterization of these proteins in E. coli as well as other bacteria.

Carriage of Distinct mcr-1-Harboring Plasmids by Unusual Serotypes of Salmonella.

Colistin acts as a last-resort antibiotic against lethal infections by carbapenem-resistant Enterobacterial pathogens. Enterobacteriaceae carrying mobile colistin resistance (MCR) genes are rapidly emerging and threatening human health and food safety. Despite mcr-1 being prevalent in Escherichia coli, its dissemination in Salmonella is not well characterized. Herein, two unusual serotypes of colistin-resistant Salmonella isolates are reported first, namely serotype Ngor (ST5399) and Goldcoast (ST2529). Using whole genome sequencing, it is shown that mcr-1 is located on the IncHI2-like plasmid pTB501 (188,527 bp) of strain SSDFZ54 and the IncX4-type plasmid pTB602 (33,303 bp) in strain SSDFZ69, respectively. Furthermore, the backbone, tra- and antimicrobial resistance genes relative variable regions in the mcr-1-bearing IncHI2 plasmids are systematically characterized. Phylogenetic analysis shows that all IncHI2-type plasmids from non-Chinese regions are clustered together, suggesting a possible Chinese origin. Taken together, these findings extend the understanding of Salmonella as a vehicle of mcr-1 carriage and distribution.

Diversity and sequence motifs of the bacterial SecA protein motor.

SecA is an essential component of the Sec protein secretion pathway in bacteria. Secretory proteins targeted to the Sec pathway by their N-terminal signal peptide bind to SecA, which couples binding and hydrolysis of adenosine triphosphate with movement of the secretory protein across the membrane-embedded SecYEG protein translocon. The phylogenetic diversity of bacteria raises the important question as to whether the region of SecA where the pre-protein binds has conserved sequence features that might impact the reaction mechanism of SecA. To address this question we established a large data set of SecA protein sequences and implemented a protocol to cluster and analyze these sequences according to features of two of the SecA functional domains, the protein binding domain and the nucleotide-binding domain 1. We identify remarkable sequence diversity of the protein binding domain, but also conserved motifs with potential role in protein binding. The N-terminus of SecA has sequence motifs that could help anchor SecA to the membrane. The overall sequence length and net estimated charge of SecA sequences depend on the organism.

Uropathogenic Escherichia coli strains harboring tosA gene were associated to high virulence genes and a multidrug-resistant profile.

TosA, a putative repeats-in-toxin protein that has recently gained importance as an antigenic molecule, has characteristics of nonfimbrial adhesins and can act as a virulence marker in uropathogenic Escherichia coli (UPEC) strains; however, little is known about the association of this protein with antibiotic resistance profiles in UPEC tosA+ clinical strains. The aim of this study was to evaluate UPEC tosA+ strains, including examining genetic diversity, associations with phylogenetic groups, resistance profiles, virulence genes, adherence assays, integrons, and extended-spectrum beta-lactamase phenotypes. Pulsed-field gel electrophoresis analysis grouped these strains into eight clusters with 62% genetic diversity. These strains were mainly associated with the multidrug-resistant profiles, together with an association with class 1 integron and the extended-spectrum beta-lactamase phenotype. Additionally, the strains exhibited a distribution of ≥96% for core-associated genes, while a variable distribution was identified for pathogenic islands-associated genes. Strong associations between UPEC tosA+ strains and two phylogenetic groups (B2 and D) were identified, including resistance to ß-lactam and non-ß-lactam antibiotics. The UPEC tosA+ clinical strains exhibited major adherence, which was related to the fitness and virulence genes. A recombinant TosA protein reacted with antibodies from the sera of urinary tract infection patients, and anti-recombinant TosA polyclonal antibodies also detected TosA expression in these strains. In conclusion, strains of UPEC tosA+ belonging to phylogenetic group B2 had a high frequency of fitness and virulence genes associated with class 1 integrons and the extended-spectrum beta-lactamase phenotype, which exhibited a high adherence profile. The TosA protein is expressed during infection with UPEC and is considered an immunogenic molecule.

Anti-inflammation effects of fucosylated chondroitin sulphate from Acaudina molpadioides by altering gut microbiota in obese mice.

This study evaluated the possible prebiotic effects of dietary fucosylated chondroitin sulfate from Acaudina molpadioides (Am-CHS) on the modulation of the gut microbiota and the improvement in the risk factors for chronic inflammation in high fat diet-fed mice. The results showed that the Am-CHS treatment greatly modified the gut microbiota, including the decrease in Bacteroidetes, increase in Firmicutes, elevation in Lactobacillus (intestinal barrier protector) and short chain fatty acid (SCFA)-producing bacteria (Lactobacillus, Bifidobacterium, and Lachnospiraceae NK4A136 group), and reduction in the lipopolysaccharide (LPS) producer (Escherichia coli). This modulation inhibited inflammatory response, manifesting the decreases in circulating proinflammatory cytokines and their mRNA expression, and the increases in interleukin-10. Dietary Am-CHS caused reductions in serum and fecal LPS concentrations and inhibition of transcription of toll-like receptor 4 (TLR4) and its downstream proteins. In addition, there were increases in the portal levels of fecal SCFAs, which probably contributed to an increase in the adenosine monophosphate-activated protein kinase (AMPK) protein in Am-CHS-treated mice. These results suggest that modulation of gut microbiota by Am-CHS can improve chronic inflammation by reducing LPS levels and TLR4 signaling. Modulation also appears to increase the levels of fecal SCFAs, which activates AMPK and finally leads to inflammation resistance.

Multitask Protein Function Prediction through Task Dissimilarity.

Automated protein function prediction is a challenging problem with distinctive features, such as the hierarchical organization of protein functions and the scarcity of annotated proteins for most biological functions. We propose a multitask learning algorithm addressing both issues. Unlike standard multitask algorithms, which use task (protein functions) similarity information as a bias to speed up learning, we show that dissimilarity information enforces separation of rare class labels from frequent class labels, and for this reason is better suited for solving unbalanced protein function prediction problems. We support our claim by showing that a multitask extension of the label propagation algorithm empirically works best when the task relatedness information is represented using a dissimilarity matrix as opposed to a similarity matrix. Moreover, the experimental comparison carried out on three model organism shows that our method has a more stable performance in both "protein-centric" and "function-centric" evaluation settings.

A Novel Arsenate-Resistant Determinant Associated with ICEpMERPH, a Member of the SXT/R391 Group of Mobile Genetic Elements.

ICEpMERPH, the first integrative conjugative element (ICE) of the SXT/R391 family isolated in the United Kingdom and Europe, was analyzed to determine the nature of its adaptive functions, its genetic structure, and its homology to related elements normally found in pathogenic Vibrio or Proteus species. Whole genome sequencing of Escherichia coli (E. coli) isolate K802 (which contains the ICEpMERPH) was carried out using Illumina sequencing technology. ICEpMERPH has a size of 110 Kb and 112 putative open reading frames (ORFs). The "hotspot regions" of the element were found to contain putative restriction digestion systems, insertion sequences, and heavy metal resistance genes that encoded resistance to mercury, as previously reported, but also surprisingly to arsenate. A novel arsenate resistance system was identified in hotspot 4 of the element, unrelated to other SXT/R391 elements. This arsenate resistance system was potentially linked to two genes: orf69, encoding an organoarsenical efflux major facilitator superfamily (MFS) transporter-like protein related to ArsJ, and orf70, encoding nicotinamide adenine dinucleotide (NAD)-dependent glyceraldehyde-3-phosphate dehydrogenase. Phenotypic analysis using isogenic strains of Escherichia coli strain AB1157 with and without the ICEpMERPH revealed resistance to low levels of arsenate in the range of 1-5 mM. This novel, low-level resistance may have an important adaptive function in polluted environments, which often contain low levels of arsenate contamination. A bioinformatic analysis on the novel determinant and the phylogeny of ICEpMERPH was presented.

Characterization of extended-spectrum ß-lactamase-producing Escherichia coli harboring mcr-1 and toxin genes from human fecal samples from China.

AIM: To characterize extended-spectrum ß-lactamase-producing Escherichia coli harboring the colistin resistance gene mcr-1 from human fecal samples collected in 2012 in a rural area of Shandong province, PR China. MATERIALS & METHODS: Whole-genome sequencing and antimicrobial susceptibility testing was performed on 25 mcr-1-positive isolates to determine carriage of antibiotic resistance and virulence genes, diversity and antibiotic resistance profiles. RESULTS: The isolates were highly genetically diverse and carried a large variety of different antibiotic resistance genes. The multidrug-resistance rate was high (96%). Virulence genes associated with intestinal pathogenic E. coli were carried by 32% of the isolates. CONCLUSION: Further monitoring of the epidemiological situation is necessary to ensure a preparedness for potential emergence of novel, difficult-to-treat strains and awareness of available treatment options.

Combining High-Resolution and Exact Calibration To Boost Statistical Power: A Well-Calibrated Score Function for High-Resolution MS2 Data.

To achieve accurate assignment of peptide sequences to observed fragmentation spectra, a shotgun proteomics database search tool must make good use of the very high-resolution information produced by state-of-the-art mass spectrometers. However, making use of this information while also ensuring that the search engine's scores are well calibrated, that is, that the score assigned to one spectrum can be meaningfully compared to the score assigned to a different spectrum, has proven to be challenging. Here we describe a database search score function, the "residue evidence" (res-ev) score, that achieves both of these goals simultaneously. We also demonstrate how to combine calibrated res-ev scores with calibrated XCorr scores to produce a "combined p value" score function. We provide a benchmark consisting of four mass spectrometry data sets, which we use to compare the combined p value to the score functions used by several existing search engines. Our results suggest that the combined p value achieves state-of-the-art performance, generally outperforming MS Amanda and Morpheus and performing comparably to MS-GF+. The res-ev and combined p-value score functions are freely available as part of the Tide search engine in the Crux mass spectrometry toolkit ( http://crux.ms ).

Single-target regulators form a minor group of transcription factors in Escherichia coli K-12.

The identification of regulatory targets of all TFs is critical for understanding the entire network of the genome regulation. The lac regulon of Escherichia coli K-12 W3110 is composed of the lacZYA operon and its repressor lacI gene, and has long been recognized as the seminal model of transcription regulation in bacteria with only one highly preferred target. After the Genomic SELEX screening in vitro of more than 200 transcription factors (TFs) from E. coli K-12, however, we found that most TFs regulate multiple target genes. With respect to the number of regulatory targets, a total of these 200 E. coli TFs form a hierarchy ranging from a single target to as many as 1000 targets. Here we focus a total of 13 single-target TFs, 9 known TFs (BetI, KdpE, LacI, MarR, NanR, RpiR, TorR, UlaR and UxuR) and 4 uncharacterized TFs (YagI, YbaO, YbiH and YeaM), altogether forming only a minor group of TFs in E. coli. These single-target TFs were classified into three groups based on their functional regulation.

Three Hcp homologs with divergent extended loop regions exhibit different functions in avian pathogenic Escherichia coli.

Type VI secretion systems (T6SSs) contribute to the pathogenicity of avian pathogenic Escherichia coli (APEC), one of the leading causative agents of sepsis and meningitis in poultry. The Hcp protein is a core component of the T6SS tail tube and acts as an exported receptor and a chaperone of effectors. In this study, four distinct Hcp types (Ia, Ib, IIa, and IIb) were designated in Gram-negative bacteria, three of which were widely distributed in APEC. We detected divergence in transcription levels among three hcp clusters in 50% duck serum and demonstrated that hcp1 was upregulated by relieving Fur repression. Further analyses revealed that the host serum could activate the hcp2B operon by H-NS derepression to transcribe the downstream xmtU/xmtV pair for inter-bacterial antagonism. Notably, in a structural analysis based on the genetic classification, Hcp proteins exhibited significant differences in the extended loop regions, suggesting that these regions were related to their functional properties. Indeed, the variant region Vs2 (Loop L2, 3) in Hcp1 and Hcp2B was essential for the delivery of antibacterial effectors and the inhibition of macrophage phagocytosis. Further analyses using a duck model indicated that these Hcps play different roles in the pathogenic processes of APEC and immunoprotection. These results indicated that the functional differentiation of Hcp homologs was driven by differences in transcriptional regulation, extended loop regions, and effector delivery.

Relevant Patented Biotechnological Applications of Ecotin: An Update.

BACKGROUND: Ecotins are serine protease inhibitors which are generally found in the periplasmic compartment. These inhibitors act on a wide range of serine proteases with different efficiencies. Actually, only few Ecotins were studied, and the main characterized proteins were derived from Escherichia coli. Functional studies of this latter protein allowed the development of numerous patents related to Ecotin relevant biotechnological applications. OBJECTIVE: This review aims to give an update on the relevant Ecotins already described and to provide a concise overview concerning the relevant patented applications of these serine protease inhibitors. METHOD: In this review, we focus on the analysis of Ecotin diversity and their distribution using Pfam protein data base. Moreover, we report a detailed overview regarding the biotechnological applications of the Ecotins based on all patents associated to Ecotins and their biotechnological applications searched in European Patent Office (Espacenet), United States Patent and National Patent Collections (WIPO) patents databases. RESULTS: On the basis of this analysis, we demonstrate that Ecotins are mostly present in bacteria. Study of Ecotin sequences and their biochemical properties reveals that they are a small serine protease inhibitor group. The high stability and specificity of Ecotins promote their biotechnological uses in several fields. The original structural organization of Ecotin-protease complexes and their flexibility lead to several patented applications. CONCLUSION: This review showed that Ecotins have many attractive biotechnological applications. Potential of Ecotins needs to be more investigated seeing the limited available data related to this protein family. Thus, further functional analyses will promote the use of Ecotins.

Contribution of AcrAB-TolC to multidrug resistance in an Escherichia coli sequence type 131 isolate.

Drug efflux by resistance-nodulation-cell division (RND)-type transporters, such as AcrAB-TolC of Escherichia coli, is an important resistance mechanism in Gram-negative bacteria; however, its contribution to multidrug resistance (MDR) in clinical isolates is poorly defined. We inactivated acrB of a sequence type 131 E. coli human isolate that showed high-level MDR, but had no mutations within the known efflux-associated local or global regulators. The resistance profile of the acrB deletion mutant revealed significantly increased susceptibility to drugs from seven antibiotic classes, including agents usually inactive against Gram-negative bacteria, notably the new oxazolidinone, tedizolid (512-fold enhanced susceptibility). AcrB deficiency reduced, but did not abolish, the efflux of dyes, which indicates the activity of at least one more efflux transporter. The findings demonstrate the efficacy of AcrAB-TolC-mediated broad-spectrum drug efflux, including agents primarily developed for Gram-positive pathogens, in a clinical isolate representative of a globally-emerging lineage. The results illustrate the need to develop molecules modified to impede their transport by AcrAB-TolC and its homologues and new efflux inhibitors.

Role of Cyclic Di-GMP and Exopolysaccharide in Type IV Pilus Dynamics.

For Pseudomonas aeruginosa, levels of cyclic di-GMP (c-di-GMP) govern the transition from the planktonic state to biofilm formation. Type IV pili (T4P) are crucial determinants of biofilm structure and dynamics, but it is unknown how levels of c-di-GMP affect pilus dynamics. Here, we scrutinized how c-di-GMP affects molecular motor properties and adhesive behavior of T4P. By means of retraction, T4P generated forces of ∼30 pN. Deletion mutants in the proteins with known roles in biofilm formation, swarming motility, and exopolysaccharide (EPS) production (specifically, the diguanylate cyclases sadC and roeA or the c-di-GMP phosphodiesterase bifA) showed only modest effects on velocity or force of T4P retraction. At high levels of c-di-GMP, the production of exopolysaccharides, particularly of Pel, is upregulated. We found that Pel production strongly enhances T4P-mediated surface adhesion of P. aeruginosa, suggesting that T4P-matrix interactions may be involved in biofilm formation by P. aeruginosa Finally, our data support the previously proposed model of slingshot-like "twitching" motility of P. aeruginosaIMPORTANCE Type IV pili (T4P) play various important roles in the transition of bacteria from the planktonic state to the biofilm state, including surface attachment and surface sensing. Here, we investigate adhesion, dynamics, and force generation of T4P after bacteria engage a surface. Our studies showed that two critical components of biofilm formation by Pseudomonas aeruginosa, T4P and exopolysaccharides, contribute to enhanced T4P-mediated force generation by attached bacteria. These data indicate a crucial role for the coordinated impact of multiple biofilm-promoting factors during the early stages of attachment to a surface. Our data are also consistent with a previous model explaining why pilus-mediated motility in P. aeruginosa results in characteristic "twitching" behavior.