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Antivirulence agents are considered a promising strategy to treat bacterial infections. Fluoropyrimidines possess antivirulence and antibiofilm activity against Gram-negative bacteria; however, their mechanism of action is yet unknown. Consistent with their known antibiofilm activity, fluoropyrimidines, particularly 5-fluorocytosine (5-FC), impair curli-dependent surface adhesion by Escherichia coli MG1655 via downregulation of curli fimbriae gene transcription. Curli inhibition requires fluoropyrimidine conversion into fluoronucleotides and is not mediated by c -di-GMP or the ymg-rcs envelope stress response axis, previously suggested as the target of fluorouracil antibiofilm activity in E. coli . In contrast, 5-FC hampered the transcription of curli activators RpoS and stimulated the expression of Fis, a curli repressor affected by nucleotide availability. This last observation suggested a possible perturbation of the de novo pyrimidine biosynthesis by 5-FC: indeed, exposure to 5-FC resulted in a ca. 2-fold reduction of UMP intracellular levels while not affecting ATP. Consistently, expression of the de novo pyrimidine biosynthesis genes carB and pyrB was upregulated in the presence of 5-FC. Our results suggest that the antibiofilm activity of fluoropyrimidines is mediated, at least in part, by perturbation of the pyrimidine nucleotide pool. We screened a genome library in search of additional determinants able to counteract the effects of 5-FC. We found that a DNA fragment encoding the unknown protein D8B36_18,480 and the N -terminal domain of the penicillin-binding protein 1b (PBP1b), involved in peptidoglycan synthesis, could restore curli production in the presence of 5-FC. Deletion of the PBP1b-encoding gene mrcB , induced csgBAC transcription, while overexpression of the gene encoding the D8B36_18,480 protein obliterated its expression, possibly as part of a coordinated response in curli regulation with PBP1b. While the two proteins do not appear to be direct targets of 5-FC, their involvement in curli regulation suggests a connection between peptidoglycan biosynthesis and curli production, which might become even more relevant upon pyrimidine starvation and reduced availability of UDP-sugars needed in cell wall biosynthesis. Overall, our findings link the antibiofilm activity of fluoropyrimidines to the redirection of at least two global regulators (RpoS, Fis) by induction of pyrimidine starvation. This highlights the importance of the de novo pyrimidines biosynthesis pathway in controlling virulence mechanisms in different bacteria and makes the pathway a potential target for antivirulence strategies., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

IFNγ-producing ex-Th17 cells ['Th1/17'] were shown to play a key pathogenic role in experimental colitis and are abundant in the intestine. Here, we identified and characterised a novel, potentially colitogenic subset of Th17 cells in the intestine of patients with Crohn's disease [CD]. Human Th17 cells expressing CCR5 ['pTh17'] co-expressed T-bet and RORC/γt and produced very high levels of IL-17, together with IFN-γ. They had a gene signature of Th17 effector cells and were distinct from established Th1/17 cells. pTh17 cells, but not Th1/17 cells, were associated with intestinal inflammation in CD, and decreased upon successful anti-TNF therapy with infliximab. Conventional CCR5[-]Th17 cells differentiated to pTh17 cells with IL-23 in vitro. Moreover, anti-IL-23 therapy with risankizumab strongly reduced pTh17 cells in the intestine. Importantly, intestinal pTh17 cells were selectively activated by adherent-invasive Escherichia coli [AIEC], but not by a commensal/probiotic E. coli strain. AIEC induced high levels of IL-23 and RANTES from dendritic cells [DC]. Intestinal CCR5+Th1/17 cells responded instead to cytomegalovirus and were reduced in ulcerative colitis [UC], suggesting an unexpected protective role. In conclusion, we identified an IL-23-inducible subset of human intestinal Th17 cells. pTh17 cells produced high levels of pro-inflammatory cytokines, were selectively associated with intestinal inflammation in CD, and responded to CD-associated AIEC, suggesting a key colitogenic role., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

In the search for structurally novel metabolites with antibacterial activity, innovative approaches must be implemented to increase the probability of discovering novel chemistry from microbial sources. Here we report on the application of metabolomic tools to the genus Actinoallomurus , a poorly explored member of the Actinobacteria. From examining extracts derived from 88 isolates belonging to this genus, we identified a family of cyclodepsipeptides acylated with a C 20 polyketide chain, which we named allopeptimicins. These molecules possess unusual structural features, including several double bonds in the amino-polyketide chain and four non-proteinogenic amino acids in the octapeptide. Remarkably, allopeptimicins are produced as a complex of active and inactive congeners, the latter carrying a sulfate group on the polyketide amine. This modification is also a mechanism of self-protection in the producer strain. The structural uniqueness of allopeptimicins is reflected in a biosynthetic gene cluster showing a mosaic structure, with dedicated gene cassettes devoted to formation of specialized precursors and modular assembly lines related to those from different pathways., Competing Interests: The authors declare the following competing financial interest(s): M. I., C. B., S. I. M., S. D. and M. S. are inventors of patent on allopeptimicins owned by NAICONS. M. I., A. G., C. B., A. T., S. I. M., S. D. and M. S. are employees and shareholders of NAICONS., (This journal is © The Royal Society of Chemistry.)

Staphylococcus epidermidis is an opportunistic pathogen and a frequent cause of nosocomial infections. In this work, we show that, among 51 S. epidermidis isolates from an Italian hospital, only a minority displayed biofilm formation, regardless of their isolation source (peripheral blood, catheter, or skin wounds); however, among the biofilm-producing isolates, those from catheters were the most efficient in biofilm formation. Interestingly, most isolates including strong biofilm producers displayed production levels of PIA (polysaccharide intercellular adhesin), the main S. epidermidis extracellular polysaccharide, similar to reference S. epidermidis strains classified as non-biofilm formers, and much lower than those classified as intermediate or high biofilm formers, possibly suggesting that high levels of PIA production do not confer a particular advantage for clinical isolates. Finally, while for the reference S. epidermidis strains the biofilm production clearly correlated with the decreased sensitivity to antibiotics, in particular, protein synthesis inhibitors, in our clinical isolates, such positive correlation was limited to tetracycline. In contrast, we observed an inverse correlation between biofilm formation and the minimal inhibitory concentrations for levofloxacin and teicoplanin. In addition, in growth conditions favoring PIA production, the biofilm-forming isolates showed increased sensitivity to daptomycin, clindamycin, and erythromycin, with increased tolerance to the trimethoprim/sulfamethoxazole association. The lack of direct correlation between the biofilm production and increased tolerance to antibiotics in S. epidermidis isolates from a clinical setting would suggest, at least for some antimicrobials, the possible existence of a trade-off between the production of biofilm determinants and antibiotic resistance.

In Crohn's disease (CD) patients, the adherent-invasive Escherichia coli (AIEC) pathovar contributes to the chronic inflammation typical of the disease via its ability to invade gut epithelial cells and to survive in macrophages. We show that, in the AIEC strain LF82, inactivation of the pyrD gene, encoding dihydroorotate dehydrogenase (DHOD), an enzyme of the de novo pyrimidine biosynthetic pathway, completely abolished its ability of to grow in a macrophage environment-mimicking culture medium. In addition, pyrD inactivation reduced flagellar motility and strongly affected biofilm formation by downregulating transcription of both type 1 fimbriae and curli subunit genes. Thus, the pyrD gene appears to be essential for several cellular processes involved in AIEC virulence. Interestingly, vidofludimus (VF), a DHOD inhibitor, has been proposed as an effective drug in CD treatment. Despite displaying a potentially similar binding mode for both human and E. coli DHOD in computational molecular docking experiments, VF showed no activity on either growth or virulence-related processes in LF82. Altogether, our results suggest that the crucial role played by the pyrD gene in AIEC virulence, and the presence of structural differences between E. coli and human DHOD allowing for the design of specific inhibitors, make E. coli DHOD a promising target for therapeutical strategies aiming at counteracting chronic inflammation in CD by acting selectively on its bacterial triggers.

Escherichia coli C is a strong biofilm producer in comparison to E. coli K-12 laboratory strains due to higher expression of the pgaABCD operon encoding the enzymes for the biosynthesis of the extracellular polysaccharide poly-β-1,6- N -acetylglucosamine (PNAG). The pgaABCD operon is negatively regulated at the post-transcriptional level by two factors, namely CsrA, a conserved RNA-binding protein controlling multiple pathways, and the RNA exonuclease polynucleotide phosphorylase (PNPase). In this work, we investigated the molecular bases of different PNAG production in C-1a and MG1655 strains taken as representative of E. coli C and K-12 strains, respectively. We found that pgaABCD operon expression is significantly lower in MG1655 than in C-1a; consistently, CsrA protein levels were much higher in MG1655. In contrast, we show that the negative effect exerted by PNPase on pgaABCD expression is much stronger in C-1a than in MG1655. The amount of CsrA and of the small RNAs CsrB, CsrC, and McaS sRNAs regulating CsrA activity is dramatically different in the two strains, whereas PNPase level is similar. Finally, the compensatory regulation acting between CsrB and CsrC in MG1655 does not occur in E. coli C. Our results suggest that PNPase preserves CsrA-dependent regulation by indirectly modulating csrA expression.

This article reports data on the occurrence and spread of three invasive mosquito species: Aedes japonicus, Aedes koreicus, and Aedes albopictus in two regions of Northeast Italy; resulting from larval and adult collections performed during the 2011-2020 period in the framework of different projects. Routine species identification was performed using morphological characters and complemented by molecular methods when required. For the years 2019 and 2020, detailed data are reported which update previous information on municipalities and sites where these species have been detected. Geo-referenced information on the presence of invasive mosquitoes is reported and demonstrated on maps. Additional data on the nature of breeding sites and the finding of native mosquito species in the same collections are also provided., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article., (© 2021 The Author(s). Published by Elsevier Inc.)

Collagen-based skin-like scaffolds (CBSS) are promising alternatives to skin grafts to repair wounds and injuries. In this work, we propose that the common marine invertebrate sea urchin represents a promising and eco-friendly source of native collagen to develop innovative CBSS for skin injury treatment. Sea urchin food waste after gonad removal was here used to extract fibrillar glycosaminoglycan (GAG)-rich collagen to produce bilayer (2D + 3D) CBSS. Microstructure, mechanical stability, permeability to water and proteins, ability to exclude bacteria and act as scaffolding for fibroblasts were evaluated. Our data show that the thin and dense 2D collagen membrane strongly reduces water evaporation (less than 5% of water passes through the membrane after 7 days) and protein diffusion (less than 2% of BSA passes after 7 days), and acts as a barrier against bacterial infiltration (more than 99% of the different tested bacterial species is retained by the 2D collagen membrane up to 48 h), thus functionally mimicking the epidermal layer. The thick sponge-like 3D collagen scaffold, structurally and functionally resembling the dermal layer, is mechanically stable in wet conditions, biocompatible in vitro (seeded fibroblasts are viable and proliferate), and efficiently acts as a scaffold for fibroblast infiltration. Thus, thanks to their chemical and biological properties, CBSS derived from sea urchins might represent a promising, eco-friendly, and economically sustainable biomaterial for tissue regenerative medicine.

Hypersecretion of proinflammatory cytokines and dysregulated activation of the IL-23/Th17 axis in response to intestinal microbiota dysbiosis are key factors in the pathogenesis of inflammatory bowel diseases (IBD). In this work, we studied how Lactobacillus and Bifidobacterium strains affect AIEC-LF82 virulence mechanisms and the consequent inflammatory response linked to the CCR6-CCL20 and IL-23/Th17 axes in Crohn's disease (CD) and ulcerative colitis (UC) patients. All Lactobacillus and Bifidobacterium strains significantly reduced the LF82 adhesion and persistence within HT29 intestinal epithelial cells, inhibiting IL-8 secretion while not affecting the CCR6-CCL20 axis. Moreover, they significantly reduced LF82 survival within macrophages and dendritic cells, reducing the secretion of polarizing cytokines related to the IL-23/Th17 axis, both in healthy donors (HD) and UC patients. In CD patients, however, only B. breve Bbr8 strain was able to slightly reduce the LF82 persistence within dendritic cells, thus hampering the IL-23/Th17 axis. In addition, probiotic strains were able to modulate the AIEC-induced inflammation in HD, reducing TNF-α and increasing IL-10 secretion by macrophages, but failed to do so in IBD patients. Interestingly, the probiotic strains studied in this work were all able to interfere with the IL-23/Th17 axis in UC patients, but not in CD patients. The different interaction mechanisms of probiotic strains with innate immune cells from UC and CD patients compared to HD suggest that testing on CD-derived immune cells may be pivotal for the identification of novel probiotic strains that could be effective also for CD patients.

The increasing incidence of infections caused by drug-resistant pathogens requires new efforts for the discovery of novel antibiotics. By screening microbial extracts in an assay aimed at identifying compounds interfering with cell wall biosynthesis, based on differential activity against a Staphylococcus aureus strain and its isogenic l-form, the potent enduracyclinones (1, 2), containing the uncommon amino acid enduracididine linked to a six-ring aromatic skeleton, were discovered from different Nonomuraea strains. The structures of 1 and 2 were established through a combination of derivatizations, oxidative cleavages, and NMR analyses of natural and 13 C- 15 N-labeled compounds. Analysis of the biosynthetic cluster provides the combination of genes for the synthesis of enduracididine and type II polyketide synthases. Enduracyclinones are active against Gram-positive pathogens (especially Staphylococcus spp.), including multi-drug-resistant strains, with minimal inhibitory concentrations in the range of 0.0005 to 4 μg mL -1 and with limited toxicity toward eukaryotic cells. The combined results from assays and macromolecular syntheses suggest a possible dual mechanism of action in which both peptidoglycan and DNA syntheses are inhibited by these molecules.

Aims: The aim of this study was to screen a chemical library consisting of over 1200 pharmacologically active, already approved off-patent compounds, to determine whether any of the compounds reduced or eliminated the invasion or intracellular replication phenotypes of Salmonella enterica serovar Typhimurium (S. Typhimurium)., Methods and Results: LacZ reporter and tissue culture-based infection assays were used to screen for compounds that significantly reduced expression of key virulence genes, and were required for the invasion or intracellular replication phenotypes of S. Typhimurium in host cells. The search lead to the discovery of four compounds that reduced invasion by between 90-100%, and two compounds that reduced intracellular replication by between 65-93% at concentrations of either 2, 10 or 50 μg ml -1 , relative to an untreated control strain., Conclusions: We identified six compounds that significantly reduced expression of S. Typhimurium virulence genes resulting in decreased in vitro virulence., Significance and Impact of the Study: The emergence of multidrug-resistant strains of Salmonella poses a considerable and growing worldwide threat to human and animal health. The screening of off-patent chemical libraries represents a potential discovery route for novel antimicrobials., (© 2018 The Society for Applied Microbiology.)

Most bacteria can switch between a planktonic, sometimes motile, form and a biofilm mode, in which bacterial cells can aggregate and attach to a solid surface. The transition between these two forms represents an example of bacterial adaptation to environmental signals and stresses. In 'environmental pathogens', namely, environmental bacteria that are also able to cause disease in animals and humans, signals associated either with the host or with the external environment, such as temperature, oxygen availability, nutrient concentrations etc., play a major role in triggering the switch between the motile and the biofilm mode, via complex regulatory mechanisms that control flagellar synthesis and motility, and production of adhesion factors. In this review article, we present examples of how environmental signals can impact biofilm formation and cell motility in the Gram negative bacteria Pseudomonas aeruginosa, Escherichia coli and in the Burkholderia genus, and how the switch between motile and biofilm mode can be an essential part of a more general process of adaptation either to the host or to the external environment., (© 2018 The Society for Applied Microbiology.)

Adherent-invasive Escherichia coli (AIEC) strains are overrepresented in the dysbiotic microbiota of Crohn's disease (CD) patients, and contribute to the onset of the chronic inflammation typical of the disease. However, the effects of anti-inflammatory drugs used for CD treatment on AIEC virulence have not yet been investigated. In this report, we show that exposure of AIEC LF82 strain to amino-6-mercaptopurine (6-MP) riboside, one of the most widely used anti-inflammatory drugs in CD, impairs its ability to adhere to, and consequently to invade, human epithelial cells. Notably, phagocytosis of LF82 treated with 6-MP by human macrophages is also reduced, suggesting that 6-MP affects AIEC cell surface determinants involved both in interaction with epithelial cells and in uptake by macrophages. Since a main target of 6-MP in bacterial cells is the inhibition of the important signal molecule c-di-GMP, we also tested whether perturbations in cAMP, another major signaling pathway in E. coli , might have similar effects on interactions with human cells. To this aim, we grew LF82 in the presence of glucose, which leads to inhibition of cAMP synthesis. Growth in glucose-supplemented medium resulted in a reduction in AIEC adhesion to epithelial cells and uptake by macrophages. Consistent with these results, both 6-MP and glucose can affect expression of cell adhesion-related genes, such as the csg genes, encoding thin aggregative fimbriae (curli). In addition, glucose strongly inhibits expression of the fim operon, encoding type 1 pili, a known AIEC determinant for adhesion to human cells. To further investigate whether 6-MP can indeed inhibit c-di-GMP signaling in AIEC, we performed biofilm and motility assays and determination of extracellular polysaccharides. 6-MP clearly affected biofilm formation and cellulose production, but also, unexpectedly, reduced cell motility, itself an important virulence factor for AIEC. Our results provide strong evidence that 6-MP can affect AIEC-host cell interaction by acting on the bacterial cell, thus strengthening the hypothesis that mercaptopurines might promote CD remission also by affecting gut microbiota composition and/or physiology, and suggesting that novel drugs targeting bacterial virulence and signaling might be effective in preventing chronic inflammation in CD.

Escherichia coli can commonly be found, either as a commensal, probiotic or a pathogen, in the human gastrointestinal (GI) tract. Biofilm formation and its regulation is surprisingly variable, although distinct regulatory pattern of red, dry and rough (rdar) biofilm formation arise in certain pathovars and even clones. In the GI tract, environmental conditions, signals from the host and from commensal bacteria contribute to shape E. coli biofilm formation within the multi-faceted multicellular communities in a complex and integrated fashion. Although some major regulatory networks, adhesion factors and extracellular matrix components constituting E. coli biofilms have been recognized, these processes have mainly been characterized in vitro and in the context of interaction of E. coli strains with intestinal epithelial cells. However, direct observation of E. coli cells in situ, and the vast number of genes encoding surface appendages on the core or accessory genome of E. coli suggests the complexity of the biofilm process to be far from being fully understood. In this review, we summarize biofilm formation mechanisms of commensal, probiotic and pathogenic E. coli in the context of the gastrointestinal tract.

Production of cellulose, a stress response-mediated process in enterobacteria, is modulated in Escherichia coli by the activity of the two pyrimidine nucleotide biosynthetic pathways, namely, the de novo biosynthetic pathway and the salvage pathway, which relies on the environmental availability of pyrimidine nitrogenous bases. We had previously reported that prevalence of the salvage over the de novo pathway triggers cellulose production via synthesis of the second messenger c-di-GMP by the DgcQ (YedQ) diguanylate cyclase. In this work, we show that DgcQ enzymatic activity is enhanced by UTP, whilst being inhibited by N-carbamoyl-aspartate, an intermediate of the de novo pathway. Thus, direct allosteric control by these ligands allows full DgcQ activity exclusively in cells actively synthesizing pyrimidine nucleotides via the salvage pathway. Inhibition of DgcQ activity by N-carbamoyl-aspartate appears to be favoured by protein-protein interaction between DgcQ and PyrB, a subunit of aspartate transcarbamylase, which synthesizes N-carbamoyl-aspartate. Our results suggest that availability of pyrimidine bases might be sensed, somehow paradoxically, as an environmental stress by E. coli. We hypothesize that this link might have evolved since stress events, leading to extensive DNA/RNA degradation or lysis of neighbouring cells, can result in increased pyrimidine concentrations and activation of the salvage pathway., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

In the bacterium Escherichia coli, some intermediates of the sulfate assimilation and cysteine biosynthesis pathway can act as signal molecules and modulate gene expression. In addition to sensing and utilization of sulphur sources, these signaling mechanisms also impact more global cell processes, such as resistance to antimicrobial agents and biofilm formation. In a recent work, we have shown that inactivation of the cysH gene, encoding phosphoadenosine-phosphosulfate (PAPS) reductase, and the consequent increase in intracellular PAPS concentration, strongly affect production of several cell surface-associated structures, enhancing surface adhesion and cell aggregation. In order to identify the molecular mechanism relaying intracellular PAPS concentration to regulation of cell surface-associated structures, we looked for mutations able to suppress the effects of cysH inactivation. We found that mutations in the adenylate cyclase-encoding cyaA gene abolished the effects of PAPS accumulation; consistent with this result, cyclic AMP (cAMP)-dependent gene expression appears to be increased in the cysH mutant. Experiments aimed at the direct identification of proteins interacting with either CysC or CysH, i.e. the PAPS-related proteins APS kinase and PAPS reductase, allowed us to identify several regulators, namely, CspC, CspE, HNS and HupA. Protein-protein interaction between HupA and CysH was confirmed by a bacterial two hybrid system, and inactivation of the hupA gene enhanced the effects of the cysH mutation in terms of production of cell surface-associated factors. Our results indicate that PAPS can modulate different regulatory systems, providing evidence that this molecule acts as a global signal molecule in E. coli., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Curli are bacterial appendages involved in the adhesion of cells to surfaces; their synthesis is regulated by many genes such as csgD and ompR. The expression of the two curli subunits (CsgA and CsgB) in Escherichia coli (E. coli) is regulated by CsgD; at the same time, csgD transcription is under the control of OmpR. Therefore, both genes are involved in the control of curli production. In this work, we elucidated the role of these genes in the nanomechanical and adhesive properties of E. coli MG1655 (a laboratory strain not expressing significant amount of curli) and its curli-producing mutants overexpressing OmpR and CsgD, employing atomic force microscopy (AFM). Nanomechanical analysis revealed that the expression of these genes gave origin to cells with a lower Young's modulus (E) and turgidity (P0), whereas the adhesion forces were unaffected when genes involved in curli formation were expressed. AFM was also employed to study the primary structure of the curli expressed through the freely jointed chain (FJC) model for polymers. CsgD increased the number of curli on the surface more than OmpR did, and the overexpression of both genes did not result in a greater number of curli. Neither of the genes had an impact on the structure (total length of the polymer and number and length of Kuhn segments) of the curli. Our results further suggest that, despite the widely assumed role of curli in cell adhesion, cell adhesion force is also dictated by surface properties because no relation between the number of curli expressed on the surface and cell adhesion was found.

DNA methylation damage can be induced by endogenous and exogenous chemical agents, which has led every living organism to develop suitable response strategies. We investigated protein expression profiles of Escherichia coli upon exposure to the alkylating agent methyl-methane sulfonate (MMS) by differential proteomics. Quantitative proteomic data showed a massive downregulation of enzymes belonging to the glycolytic pathway and fatty acids degradation, strongly suggesting a decrease of energy production. A strong reduction in the expression of the N-acetylneuraminate lyases (NanA) involved in the sialic acid metabolism was also observed. Using a null NanA mutant and DANA, a substrate analog acting as competitive inhibitor, we demonstrated that down regulation of NanA affects biofilm formation and adhesion properties of E. coli MV1161. Exposure to alkylating agents also decreased biofilm formation and bacterial adhesion to Caco-2 eukaryotic cell line by the adherent invasive E. coli (AIEC) strain LF82. Our data showed that methylation stress impairs E. coli adhesion properties and suggest a possible role of NanA in biofilm formation and bacteria host interactions.

Aim: Acinetobacter baumannii can cause sepsis with high mortality rates. We investigated whether glucose sensing might play a role in A. baumannii pathogenesis., Materials & Methods: We carried out transcriptome analysis and extracellular polysaccharide determination in an A. baumannii clinical isolate grown on complex medium with or without glucose supplementation, and assessed its ability to induce production of inflammatory cytokines in human macrophages., Results: Growth in glucose-supplemented medium strongly enhanced A. baumannii sugar anabolism, resulting in increasing lipopolysaccharide biosynthesis. In addition, glucose induced active shedding of lipopolysaccharide, in turn triggering a strong induction of inflammatory cytokines in human macrophages. Finally, hemolytic activity was strongly enhanced by growth in glucose-supplemented medium., Conclusion: We propose that sensing of exogenous glucose might trigger A. baumannii pathogenesis during sepsis.

In recent years, Escherichia coli has served as one of a few model bacterial species for studying cyclic di-GMP (c-di-GMP) signaling. The widely used E. coli K-12 laboratory strains possess 29 genes encoding proteins with GGDEF and/or EAL domains, which include 12 diguanylate cyclases (DGC), 13 c-di-GMP-specific phosphodiesterases (PDE), and 4 "degenerate" enzymatically inactive proteins. In addition, six new GGDEF and EAL (GGDEF/EAL) domain-encoding genes, which encode two DGCs and four PDEs, have recently been found in genomic analyses of commensal and pathogenic E. coli strains. As a group of researchers who have been studying the molecular mechanisms and the genomic basis of c-di-GMP signaling in E. coli, we now propose a general and systematic dgc and pde nomenclature for the enzymatically active GGDEF/EAL domain-encoding genes of this model species. This nomenclature is intuitive and easy to memorize, and it can also be applied to additional genes and proteins that might be discovered in various strains of E. coli in future studies., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

In bacteria, selective promoter recognition by RNA polymerase is achieved by its association with σ factors, accessory subunits able to direct RNA polymerase "core enzyme" (E) to different promoter sequences. Using Chromatin Immunoprecipitation-sequencing (ChIP-seq), we searched for promoters bound by the σ(S)-associated RNA polymerase form (Eσ(S)) during transition from exponential to stationary phase. We identified 63 binding sites for Eσ(S) overlapping known or putative promoters, often located upstream of genes (encoding either ORFs or non-coding RNAs) showing at least some degree of dependence on the σ(S)-encoding rpoS gene. Eσ(S) binding did not always correlate with an increase in transcription level, suggesting that, at some σ(S)-dependent promoters, Eσ(S) might remain poised in a pre-initiation state upon binding. A large fraction of Eσ(S)-binding sites corresponded to promoters recognized by RNA polymerase associated with σ(70) or other σ factors, suggesting a considerable overlap in promoter recognition between different forms of RNA polymerase. In particular, Eσ(S) appears to contribute significantly to transcription of genes encoding proteins involved in LPS biosynthesis and in cell surface composition. Finally, our results highlight a direct role of Eσ(S) in the regulation of non coding RNAs, such as OmrA/B, RyeA/B and SibC.

Aims: Due to their antimicrobial activity, silver nanoparticles (Ag-NPs) are being increasingly used in a number of industrial products. The accumulation of Ag-NPs in the soil might affect plant growth-promoting rhizobacteria and, in turn, the plants. We describe the effects of Ag-NPs on the soil bacteria Azotobacter vinelandii and Bacillus subtilis., Methods and Results: In growth-inhibition studies, A. vinelandii showed extreme sensitivity to Ag-NPs, compared to B. subtilis. We investigated the effects of Ag-NPs at subinhibitory concentrations, both on planktonic and sessile B. subtilis cells. As determined by 2,7-dichlorofluorescein-diacetate assays, Ag-NPs increase the formation of reactive oxygen species in planktonic cells, but not in sessile cells, suggesting the activation of scavenging systems in biofilms. Consistently, proteomic analysis in B. subtilis Ag-NPs-treated biofilms showed increased production of proteins related to quorum sensing and involved in stress responses and redox sensing. Extracellular polysaccharides production and inorganic phosphate solubilization were also increased, possibly as part of a coordinated response to stress., Conclusions: At low concentrations, Ag-NPs killed A. vinelandii and affected cellular processes in planktonic and sessile B. subtilis cells., Significance and Impact of the Study: Re-direction of gene expression, linked to selective toxicity, suggests a strong impact of Ag-NPs on soil bacterial communities., (© 2015 The Society for Applied Microbiology.)

The enterobacterium Escherichia coli can utilize a variety of molecules as sulfur sources, including cysteine, sulfate, thiosulfate and organosulfonates. An intermediate of the sulfate assimilation pathway, adenosine 59-phosphosulfate (APS), also acts as a signal molecule regulating the utilization of different sulfur sources. In this work, we show that inactivation of the cysH gene, leading to accumulation of phosphoadenosine 59-phosphosulfate (PAPS), also an intermediate of the sulfate assimilation pathway, results in increased surface adhesion and cell aggregation by activating the expression of the curli-encoding csgBAC operon. In contrast, curli production was unaffected by the inactivation of any other gene belonging to the sulfate assimilation pathway. Overexpression of the cysH gene downregulated csgBAC transcription, further suggesting a link between intracellular PAPS levels and curli gene expression. In addition to curli components, the Flu, OmpX and Slp proteins were also found in increased amounts in the outer membrane compartment of the cysH mutant; deletion of the corresponding genes suggested that these proteins also contribute to surface adhesion and cell surface properties in this strain. Our results indicate that, similar to APS, PAPS also acts as a signal molecule, albeit with a distinct mechanism and role: whilst APS regulates organosulfonate utilization, PAPS would couple availability of sulfur sources to remodulation of the cell surface, as part of a more global effect on cell physiology.

Burkholderia thailandensis, although normally avirulent for mammals, can infect macrophages in vitro and has occasionally been reported to cause pneumonia in humans. It is therefore used as a model organism for the human pathogen B. pseudomallei, to which it is closely related phylogenetically. We characterized the B. thailandensis clinical isolate CDC2721121 (BtCDC272) at the genome level and studied its response to environmental cues associated with human host colonization, namely, temperature and oxygen limitation. Effects of the different growth conditions on BtCDC272 were studied through whole genome transcription studies and analysis of proteins associated with the bacterial cell surface. We found that growth at 37°C, compared to 28°C, negatively affected cell motility and flagella production through a mechanism involving regulation of the flagellin-encoding fliC gene at the mRNA stability level. Growth in oxygen-limiting conditions, in contrast, stimulated various processes linked to virulence, such as lipopolysaccharide production and expression of genes encoding protein secretion systems. Consistent with these observations, BtCDC272 grown in oxygen limitation was more resistant to phagocytosis and strongly induced the production of inflammatory cytokines from murine macrophages. Our results suggest that, while temperature sensing is important for regulation of B. thailandensis cell motility, oxygen limitation has a deeper impact on its physiology and constitutes a crucial environmental signal for the production of virulence factors.

Bacterial cells often face hostile environmental conditions, to which they adapt by activation of stress responses. In Escherichia coli, environmental stresses resulting in significant reduction in growth rate stimulate the expression of the rpoS gene, encoding the alternative σ factor σ(S). The σ(S) protein associates with RNA polymerase, and through transcription of genes belonging to the rpoS regulon allows the activation of a 'general stress response', which protects the bacterial cell from harmful environmental conditions. Each step of this process is finely tuned in order to cater to the needs of the bacterial cell: in particular, selective promoter recognition by σ(S) is achieved through small deviations from a common consensus DNA sequence for both σ(S) and the housekeeping σ(70). Recognition of specific DNA elements by σ(S) is integrated with the effects of environmental signals and the interaction with regulatory proteins, in what represents a fascinating example of multifactorial regulation of gene expression. In this report, we discuss the function of the rpoS gene in the general stress response, and review the current knowledge on regulation of rpoS expression and on promoter recognition by σ(S)., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The ability of many pathogenic bacteria to grow as a biofilm results in lower susceptibility to antibiotic treatments and to the host immune response, thus leading to the development of chronic infections. The understanding that biofilms can play an important role in bacterial virulence has prompted the search for inhibitors of biofilm development and of biofilm-related cellular processes. In this report, we present two examples of target-based microbiological screenings for antimicrobials endowed with anti-biofilm activity, aimed respectively at the inhibition of the signal molecule cyclic di-GMP and of quorum sensing.

In Gram-negative bacteria, production of the signal molecule c-di-GMP by diguanylate cyclases (DGCs) is a key trigger for biofilm formation, which, in turn, is often required for the development of chronic bacterial infections. Thus, DGCs represent interesting targets for new chemotherapeutic drugs with anti-biofilm activity. We searched for inhibitors of the WspR protein, a Pseudomonas aeruginosa DGC involved in biofilm formation and production of virulence factors, using a set of microbiological assays developed in an Escherichia coli strain expressing the wspR gene. We found that azathioprine, an immunosuppressive drug used in the treatment of Crohn's disease, was able to inhibit WspR-dependent c-di-GMP biosynthesis in bacterial cells. However, in vitro enzymatic assays ruled out direct inhibition of WspR DGC activity either by azathioprine or by its metabolic derivative 2-amino-6-mercapto-purine riboside. Azathioprine is an inhibitor of 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase, an enzyme involved in purine biosynthesis, which suggests that inhibition of c-di-GMP biosynthesis by azathioprine may be due to perturbation of intracellular nucleotide pools. Consistent with this hypothesis, WspR activity is abolished in an E. coli purH mutant strain, unable to produce AICAR transformylase. Despite its effect on WspR, azathioprine failed to prevent biofilm formation by P. aeruginosa; however, it affected production of extracellular structures in E. coli clinical isolates, suggesting efficient inhibition of c-di-GMP biosynthesis in this bacterium. Our results indicate that azathioprine can prevent biofilm formation in E. coli through inhibition of c-di-GMP biosynthesis and suggest that such inhibition might contribute to its anti-inflammatory activity in Crohn's disease.

Background: Next generation sequencing (NGS) technologies have revolutionized gene expression studies and functional genomics analysis. However, further improvement of RNA sequencing protocols is still desirable, in order to reduce NGS costs and to increase its accuracy. In bacteria, a major problem in RNA sequencing is the abundance of ribosomal RNA (rRNA), which accounts for 95-98% of total RNA and can therefore hinder sufficient coverage of mRNA, the main focus of transcriptomic studies. Thus, efficient removal of rRNA is necessary to achieve optimal coverage, good detection sensitivity and reliable results. An additional challenge is presented by microorganisms with GC-rich genomes, in which rRNA removal is less efficient., Results: In this work, we tested two commercial kits for rRNA removal, either alone or in combination, on Burkholderia thailandensis. This bacterium, chosen as representative of the important Burkholderia genus, which includes both pathogenic and environmental bacteria, has a rather large (6.72 Mb) and GC-rich (67.7%) genome. Each enriched mRNA sample was sequenced through paired-end Illumina GAIIx run in duplicate, yielding between 10 and 40 million reads. We show that combined treatment with both kits allows an mRNA enrichment of more than 238-fold, enabling the sequencing of almost all (more than 90%) B. thailandensis transcripts from less than 10 million reads, without introducing any bias in mRNA relative abundance, thus preserving differential expression profile., Conclusions: The mRNA enrichment protocol presented in this work leads to an increase in detection sensitivity up to 770% compared to total RNA; such increased sensitivity allows for a corresponding reduction in the number of sequencing reads necessary for the complete analysis of whole transcriptome expression profiling. Thus we can conclude that the MICROBExpress/Ovation combined rRNA removal method could be suitable for RNA sequencing of whole transcriptomes of microorganisms with high GC content and complex genomes enabling at the same time an important scaling down of sequencing costs.

Background: Transition from planktonic cells to biofilm is mediated by production of adhesion factors, such as extracellular polysaccharides (EPS), and modulated by complex regulatory networks that, in addition to controlling production of adhesion factors, redirect bacterial cell metabolism to the biofilm mode., Results: Deletion of the pnp gene, encoding polynucleotide phosphorylase, an RNA processing enzyme and a component of the RNA degradosome, results in increased biofilm formation in Escherichia coli. This effect is particularly pronounced in the E. coli strain C-1a, in which deletion of the pnp gene leads to strong cell aggregation in liquid medium. Cell aggregation is dependent on the EPS poly-N-acetylglucosamine (PNAG), thus suggesting negative regulation of the PNAG biosynthetic operon pgaABCD by PNPase. Indeed, pgaABCD transcript levels are higher in the pnp mutant. Negative control of pgaABCD expression by PNPase takes place at mRNA stability level and involves the 5'-untranslated region of the pgaABCD transcript, which serves as a cis-element regulating pgaABCD transcript stability and translatability., Conclusions: Our results demonstrate that PNPase is necessary to maintain bacterial cells in the planktonic mode through down-regulation of pgaABCD expression and PNAG production.

This work showed that perturbations of the physiological steady-state level of reactive oxygen species (ROS) affected biofilm genesis and the characteristics of the model bacterium Azotobacter vinelandii. To get a continuous endogenous source of ROS, a strain exposed to chronic sub-lethal oxidative stress was deprived of the gene coding for the antioxidant rhodanese-like protein RhdA (MV474). In this study MV474 biofilm showed (i) a seven-fold higher growth rate, (ii) induction of catalase and alkyl-hydroxyl-peroxidase enzymes, (iii) higher average thicknesses due to increased production of a polysaccharide-rich extracellular matrix and (iv) less susceptibility to hydrogen peroxide than the wild-type strain (UW136). MV474 showed increased swimming and swarming activity and the swarming colonies experienced a higher level of oxidative stress compared to UW136. A continuous exogenous source of ROS increased biofilm formation in UW136. Overall, chronic sub-lethal oxidative events promoted sessile behavior in A. vinelandii.

Bacteria are often found in multicellular communities known as biofilms, which constitute a resistance form against environmental stresses. Extracellular adhesion and cell aggregation factors, responsible for bacterial biofilm formation and maintenance, are tightly regulated in response to physiological and environmental cues. We show that, in Escherichia coli, inactivation of genes belonging to the de novo uridine monophosphate (UMP) biosynthetic pathway impairs production of curli fibers and cellulose, important components of the bacterial biofilm matrix, by inhibiting transcription of the csgDEFG operon, thus preventing production of the biofilm master regulator CsgD protein. Supplementing growth media with exogenous uracil, which can be converted to UMP through the pyrimidine nucleotide salvage pathway, restores csgDEFG transcription and curli production. In addition, however, exogenous uracil triggers cellulose production, particularly in strains defective in either carB or pyrB genes, which encode enzymes catalyzing the first steps of de novo UMP biosynthesis. Our results indicate the existence of tight and complex links between pyrimidine metabolism and curli/cellulose production: transcription of the csgDEFG operon responds to pyrimidine nucleotide availability, while cellulose production is triggered by exogenous uracil in the absence of active de novo UMP biosynthesis. We speculate that perturbations in the UMP biosynthetic pathways allow the bacterial cell to sense signals such as starvation, nucleic acids degradation, and availability of exogenous pyrimidines, and to adapt the production of the extracellular matrix to the changing environmental conditions.

aidB is one of the four genes of E. coli that is induced by alkylating agents and regulated by Ada protein. Three genes (ada, alkA, and alkB) encode DNA repair proteins that remove or repair alkylated bases. However, the role of AidB remains unclear despite extensive efforts to determine its function in cells exposed to alkylating agents. The E. coli AidB protein was identified as a component of the protein complex that assembles at strong promoters. We demonstrate that AidB protein preferentially binds to UP elements, AT rich transcription enhancer sequences found upstream of many highly expressed genes, several DNA repair genes, and housekeeping genes. AidB allows efficient transcription from promoters containing an UP element upon exposure to a DNA methylating agent and protects downstream genes from DNA damage. The DNA binding domain is required to target AidB to specific genes preferentially protecting them from alkylation damage. However, deletion of AidB's DNA binding domain does not prevent its antimutagenic activity, instead this deletion appears to allow AidB to function as a cytoplasmic alkylation resistance protein. Our studies identify the role of AidB in alkylating agent exposed cells and suggest a new cellular strategy in which a subset of the genome is preferentially protected from damage by alkylating agents., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Specific promoter recognition by bacterial RNA polymerase is mediated by σ subunits, which assemble with RNA polymerase core enzyme (E) during transcription initiation. However, σ(70) (the housekeeping σ subunit) and σ(S) (an alternative σ subunit mostly active during slow growth) recognize almost identical promoter sequences, thus raising the question of how promoter selectivity is achieved in the bacterial cell. To identify novel sequence determinants for selective promoter recognition, we performed run-off/microarray (ROMA) experiments with RNA polymerase saturated either with σ(70) (Eσ(70)) or with σ(S) (Eσ(S)) using the whole Escherichia coli genome as DNA template. We found that Eσ(70), in the absence of any additional transcription factor, preferentially transcribes genes associated with fast growth (e.g. ribosomal operons). In contrast, Eσ(S) efficiently transcribes genes involved in stress responses, secondary metabolism as well as RNAs from intergenic regions with yet-unknown function. Promoter sequence comparison suggests that, in addition to different conservation of the -35 sequence and of the UP element, selective promoter recognition by either form of RNA polymerase can be affected by the A/T content in the -10/+1 region. Indeed, site-directed mutagenesis experiments confirmed that an A/T bias in the -10/+1 region could improve promoter recognition by Eσ(S).

Upon exposure to alkylating agents, Escherichia coli increases expression of aidB along with three genes (ada, alkA, and alkB) that encode DNA repair proteins. While the biological roles of the Ada, AlkA, and AlkB proteins have been defined, despite many efforts, the molecular functions of AidB remain largely unknown. In this study, we focused on the biological role of the AidB protein, and we demonstrated that AidB shows preferential binding to a DNA region that includes the upstream element of its own promoter, PaidB. The physiological significance of this specific interaction was investigated by in vivo gene expression assays, demonstrating that AidB can repress its own synthesis during normal cell growth. We also showed that the domain architecture of AidB is related to the different functions of the protein: the N-terminal region, comprising the first 439 amino acids (AidB "I-III"), possesses FAD-dependent dehydrogenase activity, while its C-terminal domain, corresponding to residues 440 to 541 (AidB "IV"), displays DNA binding activity and can negatively regulate the expression of its own gene in vivo. Our results define a novel role in gene regulation for the AidB protein and underline its multifunctional nature.

In Gram-negative bacteria, production of adhesion factors and extracellular polysaccharides (EPS) is promoted by the activity of diguanylate cyclases (DGCs), a class of enzymes able to catalyse the synthesis of the signal molecule bis-(3',5')-cyclic di-guanylic acid (c-di-GMP). In this report we show that in Escherichia coli, overexpression of the YddV protein, but not of other DGCs such as AdrA and YcdT, induces the production of the EPS poly-N-acetylglucosamine (PNAG) by stimulating expression of pgaABCD, the PNAG-biosynthetic operon. Stimulation of PNAG production and activation of pgaABCD expression by the YddV protein are abolished by inactivation of its GGDEF motif, responsible for DGC activity. Consistent with the effects of YddV overexpression, inactivation of the yddV gene negatively affects pgaABCD transcription and PNAG-mediated biofilm formation. pgaABCD regulation by the yddV gene also takes place in a mutant carrying a partial deletion of the csrA gene, which encodes the main regulator of pgaABCD expression, suggesting that YddV does not regulate pgaABCD through modulation of CsrA activity. Our results demonstrate that PNAG production does not simply respond to intracellular c-di-GMP concentration, but specifically requires the DGC activity of the YddV protein, thus supporting the notion that in E. coli, c-di-GMP biosynthesis by a given DGC protein triggers regulatory events that lead to activation of specific sets of EPS biosynthetic genes or proteins.

In bacteria, intracellular amounts of the signal molecule cyclic di-GMP (c-di-GMP) are determined by biosynthetic enzymes, or diguanylate cyclases (DGCs), and degradative enzymes, or c-di-GMP phosphodiesterases (c-PDEs). In Escherichia coli, the production of curli fibers, an important adhesion factor, responds to c-di-GMP. The yddV-dos operon, which encodes a DGC and a c-PDE acting as a protein complex, is highly expressed at a low growth temperature and in the stationary phase, i.e. conditions that also stimulate curli production. We show that perturbations in the balance between YddV and Dos, obtained either through inactivation of the yddV gene or through overproduction of either YddV or Dos, strongly affect curli production. Both YddV and Dos proteins regulate the transcription of the csgBAC operon, which encodes curli structural subunits, while not affecting the expression of the regulatory operon csgDEFG. Consistent with the role of both YddV and Dos proteins as oxygen sensors, their effects on csgBAC gene expression were dramatically reduced in cells grown under anoxic conditions. Our results show that the yddV-dos operon plays an important role in the expression of curli-encoding genes in aerobically growing E. coli, and suggest that YddV and Dos, through their opposite activities, might finely tune curli production in response to oxygen availability.

Bacteria can switch between planktonic forms (single cells) and biofilms, i.e., bacterial communities growing on solid surfaces and embedded in a matrix of extracellular polymeric substance. Biofilm formation by pathogenic bacteria often results in lower susceptibility to antibiotic treatments and in the development of chronic infections; thus, biofilm formation can be considered an important virulence factor. In recent years, much attention has been directed towards understanding the biology of biofilms and towards searching for inhibitors of biofilm development and of biofilm-related cellular processes. In this report, we review selected examples of target-based screening for anti-biofilm agents: We focus on inhibitors of quorum sensing, possibly the most characterized target for molecules with anti-biofilm activity, and on compounds interfering with the metabolism of the signal molecule cyclic di-GMP metabolism and on inhibitors of DNA and nucleotide biosynthesis, which represent a novel and promising class of biofilm inhibitors. Finally, we discuss the activation of biofilm dispersal as a novel mode of action for anti-biofilm compounds.

In Gram-negative bacteria, production of bis-(3',5')-cyclic diguanylic acid (c-di-GMP) by diguanylate cyclases (DGCs) is the main trigger for production of extracellular polysaccharides and for biofilm formation. Mutants affected in c-di-GMP biosynthesis are impaired in biofilm formation, thus making DGCs interesting targets for new antimicrobial agents with anti-biofilm activity. In this report, we describe a strategy for the screening for DGC inhibitors consisting of a combination of three microbiological assays. The primary assay utilizes an Escherichia coli strain overexpressing the adrA gene, encoding the DGC protein AdrA, and relies on detection of AdrA-dependent cellulose production as red colony phenotype on solid medium supplemented with the dye Congo red (CR). Presence of DGC inhibitors blocking AdrA activity would result in a white phenotype on CR medium. The CR assay can be performed in 96-well microtiter plates, making it suitable for high-throughput screenings. To confirm specific inhibition of c-di-GMP biosynthesis, chemical compounds positive in the CR assay are tested for their ability to inhibit biofilm formation and in a reporter gene assay which monitors expression of curli-encoding genes as a function of DGC activity. Screening of a chemical library using the described approach allowed us to identify sulfathiazole, an antimetabolite drug, as an inhibitor of c-di-GMP biosynthesis. Sulfathiazole probably affects c-di-GMP biosynthesis in an indirect fashion rather than by binding to DGCs; however, sulfathiazole represents the first example of drug able to affect biofilm formation by interfering with c-di-GMP metabolism.

Background: Acinetobacter baumannii is emerging as an important nosocomial pathogen. Multidrug resistance, as well as ability to withstand environmental stresses, makes eradication of A. baumannii difficult, particularly from hospital settings., Results: Over a six-year period, 73 isolates of A. baumannii were collected from infected patients in two hospitals in Italy. While 69 out of the 73 isolates displayed identical multidrug antibiotic resistance pattern, they were susceptible to carbapenems. Genetic profiles of these 69 isolates, determined by Pulsed Field Gel Electrophoresis (PFGE), indicated that they were genetically related and could be clustered in a specific clone, called SMAL. We tested the ability of the SMAL clone to form biofilm, an important determinant for bacterial colonization of the human host and for persistence in the hospital environment. Biofilm formation by A. baumannii SMAL, measured as surface adhesion to polystyrene, is strongly affected by growth conditions, being impaired in rich growth media such as LB, while being favoured in glucose-based medium. Surface adhesion in glucose-based media is inhibited by treatment with cellulase, suggesting that it depends on production of cellulose or of a chemically related extracellular polysaccharide. Exposure of A. baumannii SMAL to subinhibitory concentrations of imipenem resulted in biofilm stimulation and increased production of iron uptake proteins. Growth in iron-supplemented medium also stimulated surface adhesion, thus suggesting that increased intracellular iron concentrations might act as an environmental signal for biofilm formation in A. baumannii SMAL., Conclusions: Our results indicate that exposure to subinhibitory concentrations of imipenem can stimulate biofilm formation and induce iron uptake in a pathogenic strain of A. baumannii, with potential implications on antibiotic susceptibility and ability to persist in the human host.

Switching from single-cell (planktonic) to biofilm growth (and vice versa) is regulated by a variety of environmental and physiological cues. Signals leading to activation of stress responses often lead to biofilm formation which, in turn, can trigger induction of stress response mechanisms, suggesting direct cross-talk between the two cellular processes. Regulatory mechanisms of this process include two-component regulatory systems, master regulators such as the rpoS gene and signal molecules such as cyclic-di-GMP, in a tight and complex interplay.

In enterobacteria, the CsgD protein activates production of two extracellular structures: thin aggregative fimbriae (curli) and cellulose. While curli fibres promote biofilm formation and cell aggregation, the evidence for a direct role of cellulose as an additional determinant for biofilm formation is not as straightforward. The MG1655 laboratory strain of Escherichia coli only produces limited amounts of curli and cellulose; however, ectopic csgD expression results in strong stimulation of curli and cellulose production. We show that, in a csgD-overexpressing derivative of MG1655, cellulose production negatively affects curli-mediated surface adhesion and cell aggregation, thus acting as a negative determinant for biofilm formation. Consistent with this observation, deletion of the bcsA gene, necessary for cellulose production, resulted in a significant increase in curli-dependent adhesion. We found that cellulose production increased tolerance to desiccation, suggesting that the function of cellulose might be related to resistance to environmental stresses rather than to biofilm formation. Production of the curli/cellulose network in enterobacteria typically takes place at low growth temperature (<32 degrees C), but not at 37 degrees C. We show that CsgD overexpression can overcome temperature-dependent control of the curli-encoding csgBA operon, but not of the cellulose-related adrA gene, suggesting very tight temperature control of cellulose production in E. coli MG1655.

Bacteria can switch from a single-cell (planktonic) mode to a multicellular community (biofilm) mode via production of cell-cell aggregation and surface adhesion factors. In this report, we present evidence that the CsgD protein, a transcription regulator involved in biofilm formation in Escherichia coli, modulates the expression of the rpoS (sigma(S)) regulon. Protein pattern analysis of E. coli cells in stationary phase shows that CsgD affects the expression of several proteins encoded by sigma(S)-dependent genes. CsgD regulation of sigma(S)-dependent genes takes place at gene transcription level, does not bypass the need for rpoS, and is abolished in an rpoS-null mutant. Consistent with these results, we find that CsgD expression leads to an increase in sigma(S) intracellular concentration. Increase in sigma(S) cellular amount is mediated by CsgD-dependent transcription activation of iraP, encoding a factor involved in sigma(S) protein stabilization. Our results strongly suggest that the CsgD regulatory protein plays a major role as a relay between adhesion factors production and sigma(S)-dependent gene expression via sigma(S) protein stabilization. Direct coordination between biofilm formation and expression of the rpoS regulon could positively impact important biological processes, such as host colonization or response to environmental stresses.

Laboratory strains of Escherichia coli do not show significant ability to attach to solid surfaces and to form biofilms. We compared the adhesion properties of the E. coli PHL565 laboratory strain to eight environmental E. coli isolates: only four isolates displayed adhesion properties to glass significantly higher than PHL565. The ability of the adhesion-proficient isolates to attach to glass tubes strongly correlated with their ability to express curli (thin aggregative fimbriae), thus suggesting that curli are a common adhesion determinant in environmental strains. Despite its inability to attach to solid surfaces, growth of E. coli PHL565 in mixed cultures with Pseudomonas putida MT2 resulted in co-adhesion and in formation of a mixed E. coli/P. putida biofilm, which was able to colonize glass surfaces with dramatic efficiency compared to P. putida alone. E. coli/P. putida interactions stimulate initial adhesion to glass, and the presence of both bacterial species in the mature biofilm was confirmed by quantitative PCR. In contrast, no synergistic biofilm formation was observed in mixed cultures of E. coli with the Gram-positive bacterium Staphylococcus epidermidis. Interestingly, E. coli PHL565 also stimulated biofilm formation by bacterial communities isolated from drinking water distribution systems. Our results strongly suggest that co-adhesion and synergistic interaction with biofilm-forming species might represent an important mechanism, and a possible alternative strategy to production of adhesion determinants, for persistence and propagation of E. coli in the environment.

Curli fibers, encoded by the csgBAC genes, promote biofilm formation in Escherichia coli and other enterobacteria. Curli production is dependent on the CsgD transcription activator, which also promotes cellulose biosynthesis. In this study, we investigated the effects of CsgD expression from a weak constitutive promoter in the biofilm formation-deficient PHL565 strain of E. coli. We found that despite its function as a transcription activator, the CsgD protein is localized in the cytoplasmic membrane. Constitutive CsgD expression promotes biofilm formation by PHL565 and activates transcription from the csgBAC promoter; however, csgBAC expression remains dependent on temperature and the growth medium. Constitutive expression of the CsgD protein results in altered transcription patterns for at least 24 novel genes, in addition to the previously identified CsgD-dependent genes. The cspA and fecR genes, encoding regulatory proteins responding to cold shock and to iron, respectively, and yoaD, encoding a putative negative regulator of cellulose biosynthesis, were found to be some of the novel CsgD-regulated genes. Consistent with the predicted functional role, increased expression of the yoaD gene negatively affects cell aggregation, while yoaD inactivation results in stimulation of cell aggregation and leads to increased cellulose production. Inactivation of fecR results in significant increases in both cell aggregation and biofilm formation, while the effects of cspA are not as strong in the conditions tested. Our results indicate that CsgD can modulate cellulose biosynthesis through activation of the yoaD gene. In addition, the positive effect of CsgD on biofilm formation might be enhanced by repression of the fecR gene.

We investigated the influence of regulatory and pathogenicity island-associated factors (Hha, RpoS, LuxS, EvgA, RfaH, and tRNA5Leu) on biofilm formation by uropathogenic Escherichia coli (UPEC) strain 536. Only inactivation of rfaH, which encodes a transcriptional antiterminator, resulted in increased initial adhesion and biofilm formation by E. coli 536. rfaH inactivation in nonpathogenic E. coli K-12 isolate MG1655 resulted in the same phenotype. Transcriptome analysis of wild-type strain 536 and an rfaH mutant of this strain revealed that deletion of rfaH correlated with increased expression of flu orthologs. flu encodes antigen 43 (Ag43), which mediates autoaggregation and biofilm formation. We confirmed that deletion of rfaH leads to increased levels of flu and flu-like transcripts in E. coli K-12 and UPEC. Supporting the hypothesis that RfaH represses biofilm formation through reduction of the Ag43 level, the increased-biofilm phenotype of E. coli MG1655rfaH was reversed upon inactivation of flu. Deletion of the two flu orthologs, however, did not modify the behavior of mutant 536rfaH. Our results demonstrate that the strong initial adhesion and biofilm formation capacities of strain MG1655rfaH are mediated by both increased steady-state production of Ag43 and likely increased Ag43 presentation due to null rfaH-dependent lipopolysaccharide depletion. Although the roles of rfaH in the biofilm phenotype are different in UPEC strain 536 and K-12 strain MG1655, this study shows that RfaH, in addition to affecting the expression of bacterial virulence factors, also negatively controls expression and surface presentation of Ag43 and possibly another Ag43-independent factor(s) that mediates cell-cell interactions and biofilm formation.

Exposure of Escherichia coli MG1655 to environmental bacteriophages results in rapid selection for phage-tolerant subpopulations displaying increased biofilm formation. Analysis of one phage-tolerant strain revealed large amounts of the DNA-binding Dps protein in the outer membrane protein and production of fimbria-like structures. In dps and fimA mutant derivatives of MG1655, no selection of phage-tolerant bacteria upon exposure to bacteriophages occurred, suggesting a role for Dps and type I pili in bacteriophage tolerance.

The strict dependence of transcription from the aidB promoter (PaidB) on the Esigma(S) form of RNA polymerase is because of the presence of a C nucleotide as the first residue of the -10 promoter sequence (-12C), which does not allow an open complex formation by Esigma(70). In this report, sigma(70) mutants carrying either the Q437H or the T440I single amino acid substitutions, which allow -12C recognition by sigma(70), were tested for their ability to carry out transcription from PaidB. The Gln-437 and Thr-440 residues are located in region 2.4 of sigma(70) and correspond to Gln-152 and Glu-155 in sigma(S). Interestingly, the Q437H mutant of sigma(70), but not T440I, was able to promote an open complex formation and to initiate transcription at PaidB. In contrast to T440I, a T440E mutant was proficient in carrying out transcription from PaidB. No sigma(70) mutant displayed significantly increased interaction with a PaidB mutant in which the -12C was substituted by a T (PaidB((C12T))), which is also efficiently recognized by wild type sigma(70). The effect of the T440E mutation suggests that the corresponding Glu-155 residue in sigma(S) might be involved in -12C recognition. However, substitution to alanine of the Glu-155 residue, as well as of Gln-152, in the sigma(S) protein did not significantly affect Esigma(S) interaction with PaidB. Our results reiterate the importance of the -12C residue for sigma(S)-specific promoter recognition and strongly suggest that interaction with the -10 sequence and open complex formation are carried out by different determinants in the two sigma factors.

The sigma(S) subunit of RNA polymerase, the product of the rpoS gene, controls the expression of genes responding to starvation and cellular stresses. Using gene array technology, we investigated rpoS-dependent expression at the onset of stationary phase in Escherichia coli grown in rich medium. Forty-one genes were expressed at significantly lower levels in an rpoS mutant derived from the MG1655 strain; for 10 of these, we also confirmed rpoS and stationary-phase dependence by reverse transcription-PCR. Only seven genes (dps, osmE, osmY, sodC, rpsV, wrbA, and yahO) had previously been recognized as rpoS dependent. Several newly identified rpoS-dependent genes are involved in the uptake and metabolism of amino acids, sugars, and iron. Indeed, the rpoS mutant strain shows severely impaired growth on some sugars such as fructose and N-acetylglucosamine. The rpoS gene controls the production of indole, which acts as a signal molecule in stationary-phase cells, via regulation of the tnaA-encoded tryptophanase enzyme. Genes involved in protein biosynthesis, encoding the ribosome-associated protein RpsV (sra) and the initiation factor IF-1 (infA), were also induced in an rpoS-dependent fashion. Using primer extension, we determined the promoter sequences of a selection of rpoS-regulated genes representative of different functional classes. Significant fractions of these promoters carry sequence features specific for Esigma(S) recognition of the -10 region, such as cytosines at positions -13 (70%) and -12 (30%) as well as a TG motif located upstream of the -10 region (50%), thus supporting the TGN(0-2)C(C/T)ATA(C/A)T consensus sequence recently proposed for sigma(S).