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Pandemic Pseudomonas aeruginosa clone C strains encode two inner-membrane associated ATP-dependent FtsH proteases. PaftsH1 is located on the core genome and supports cell growth and intrinsic antibiotic resistance, whereas PaftsH2, a xenolog acquired through horizontal gene transfer from a distantly related species, is unable to functionally replace PaftsH1. We show that purified PaFtsH2 degrades fewer substrates than PaFtsH1. Replacing the 31-amino acid-extended linker region of PaFtsH2 spanning from the C-terminal end of the transmembrane helix-2 to the first seven highly divergent residues of the cytosolic AAA+ ATPase module with the corresponding region of PaFtsH1 improves hybrid-enzyme substrate processing in vitro and enables PaFtsH2 to substitute for PaFtsH1 in vivo. Electron microscopy indicates that the identity of this linker sequence influences FtsH flexibility. We find membrane-cytoplasmic (MC) linker regions of PaFtsH1 characteristically glycine-rich compared to those from FtsH2. Consequently, introducing three glycines into the membrane-proximal end of PaFtsH2's MC linker is sufficient to elevate its activity in vitro and in vivo. Our findings establish that the efficiency of substrate processing by the two PaFtsH isoforms depends on MC linker identity and suggest that greater linker flexibility and/or length allows FtsH to degrade a wider spectrum of substrates. As PaFtsH2 homologs occur across bacterial phyla, we hypothesize that FtsH2 is a latent enzyme but may recognize specific substrates or is activated in specific contexts or biological niches. The identity of such linkers might thus play a more determinative role in the functionality of and physiological impact by FtsH proteases than previously thought., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cyclic di-GMP signaling regulates sessile-to-motile lifestyle transition and associated physiological and metabolic features in bacteria. The presence of potential cyclic di-GMP turnover proteins in deepest branching bacteria indicates that cyclic di-GMP is an ancient signaling molecule. In this issue of The EMBO Journal, Cai et al (2023) describe light-induced activation of a thiosulfate oxidation pathway in the deep-sea cold-seep bacterium Qipengyuania flava, thus coupling cyclic di-GMP with the regulation of the global abiotic sulfur cycle., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Shewanella spp. play important ecological and biogeochemical roles, due in part to their versatile metabolism and swift integration of stimuli. While Shewanella spp. are primarily considered environmental microbes, Shewanella algae is increasingly recognized as an occasional human pathogen. S. algae shares the broad metabolic and respiratory repertoire of Shewanella spp. and thrives in similar ecological niches. In S. algae, nitrate and dimethyl sulfoxide (DMSO) respiration promote biofilm formation strain specifically, with potential implication of taxis and cyclic diguanosine monophosphate (c-di-GMP) signaling. Signal transduction systems in S. algae have not been investigated. To fill these knowledge gaps, we provide here an inventory of the c-di-GMP turnover proteome and chemosensory networks of the type strain S. algae CECT 5071 and compare them with those of 41 whole-genome-sequenced clinical and environmental S. algae isolates. Besides comparative analysis of genetic content and identification of laterally transferred genes, the occurrence and topology of c-di-GMP turnover proteins and chemoreceptors were analyzed. We found S. algae strains to encode 61 to 67 c-di-GMP turnover proteins and 28 to 31 chemoreceptors, placing S. algae near the top in terms of these signaling capacities per Mbp of genome. Most c-di-GMP turnover proteins were predicted to be catalytically active; we describe in them six novel N-terminal sensory domains that appear to control their catalytic activity. Overall, our work defines the c-di-GMP and chemosensory signal transduction pathways in S. algae, contributing to a better understanding of its ecophysiology and establishing S. algae as an auspicious model for the analysis of metabolic and signaling pathways within the genus Shewanella . IMPORTANCE Shewanella spp. are widespread aquatic bacteria that include the well-studied freshwater model strain Shewanella oneidensis MR-1. In contrast, the physiology of the marine and occasionally pathogenic species Shewanella algae is poorly understood. Chemosensory and c-di-GMP signal transduction systems integrate environmental stimuli to modulate gene expression, including the switch from a planktonic to sessile lifestyle and pathogenicity. Here, we systematically dissect the c-di-GMP proteome and chemosensory pathways of the type strain S. algae CECT 5071 and 41 additional S. algae isolates. We provide insights into the activity and function of these proteins, including a description of six novel sensory domains. Our work will enable future analyses of the complex, intertwined c-di-GMP metabolism and chemotaxis networks of S. algae and their ecophysiological role.

Bacillus thuringiensis is the most widely used eco-friendly biopesticide, containing two primary determinants of biocontrol, endospore and insecticidal crystal proteins (ICPs). The 2-methylcitrate cycle is a widespread carbon metabolic pathway playing a crucial role in channelling propionyl-CoA, but with poorly understood metabolic regulatory mechanisms. Here, we dissect the transcriptional regulation of the 2-methylcitrate cycle operon prpCDB and report its unprecedented role in controlling the sporulation process of B. thuringiensis. We found that the transcriptional activity of the prp operon encoding the three critical enzymes PrpC, PrpD, and PrpB in the 2-methylcitrate cycle was negatively regulated by the two global transcription factors CcpA and AbrB, while positively regulated by the LysR family regulator CcpC, which jointly account for the fact that the 2-methylcitrate cycle is specifically and highly active in the stationary phase of growth. We also found that the prpD mutant accumulated 2-methylcitrate, the intermediate metabolite of the 2-methylcitrate cycle, which delayed and inhibited sporulation at the early stage. Thus, our results not only revealed sophisticated transcriptional regulatory mechanisms for the metabolic 2-methylcitrate cycle but also identified 2-methylcitrate as a novel regulator of sporulation in B. thuringiensis., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Elevation of temperature within and above the physiological limit causes the unfolding and aggregation of cellular proteins, which can ultimately lead to cell death. Bacteria are therefore equipped with Hsp100 disaggregation machines that revert the aggregation process and reactivate proteins otherwise lost by aggregation. In Gram-negative bacteria, two disaggregation systems have been described: the widespread ClpB disaggregase, which requires cooperation with an Hsp70 chaperone, and the standalone ClpG disaggregase. ClpG co-exists with ClpB in selected bacteria and provides superior heat resistance. Here, we compared the activities of both disaggregases towards diverse model substrates aggregated in vitro and in vivo at different temperatures. We show that ClpG exhibits robust activity towards all disordered aggregates, whereas ClpB acts poorly on the protein aggregates formed at very high temperatures. Extreme temperatures are expected not only to cause extended protein unfolding, but also to result in an accelerated formation of protein aggregates with potentially altered chemical and physical parameters, including increased stability. We show that ClpG exerts higher threading forces as compared to ClpB, likely enabling ClpG to process "tight" aggregates formed during severe heat stress. This defines ClpG as a more powerful disaggregase and mechanistically explains how ClpG provides increased heat resistance., Competing Interests: The authors declare no conflict of interest.

A physical genome map of Pseudomonas aeruginosa PAO1 had been constructed in the late 1980s by combinatorial SpeI/DpnI long-range restriction mapping. Whereas SpeI was known to cleave within the infrequent 5'-ACTAGT hexanucleotide, the rare recognition site 5'-Gm6ATC for the methylation-sensitive 4-cutter DpnI in the PAO1 genome remained elusive. Resequencing the PAO1 genome with the methylation-sensitive Single Molecule Real-Time (SMRT) sequencing technology identified DpnI to cleave the PAO1 genome at 15 palindromic GAC(N)6Gm6ATC(N)6GTC recognition sites with the central Gm6ATC palindrome to be methylated on both strands by the clone PAO1-specific type I HsdM superfamily methyltransferase M.PaePAOI (PA2735)., (© FEMS 2019.)

AAA+ disaggregases solubilize aggregated proteins and confer heat tolerance to cells. Their disaggregation activities crucially depend on partner proteins, which target the AAA+ disaggregases to protein aggregates while concurrently stimulating their ATPase activities. Here, we report on two potent ClpG disaggregase homologs acquired through horizontal gene transfer by the species Pseudomonas aeruginosa and subsequently abundant P. aeruginosa clone C. ClpG exhibits high, stand-alone disaggregation potential without involving any partner cooperation. Specific molecular features, including high basal ATPase activity, a unique aggregate binding domain, and almost exclusive expression in stationary phase distinguish ClpG from other AAA+ disaggregases. Consequently, ClpG largely contributes to heat tolerance of P. aeruginosa primarily in stationary phase and boosts heat resistance 100-fold when expressed in Escherichia coli This qualifies ClpG as a potential persistence and virulence factor in P. aeruginosa ., Competing Interests: The authors declare no conflict of interest.

The invasion of epithelial cells by Salmonella enterica serovar Typhimurium is a very tightly regulated process. Signaling cascades triggered by different environmental and physiological signals converge to control HilD, an AraC regulator that coordinates the expression of several virulence factors. The expression of hilD is modulated at several steps of the expression process. Here, we report that the invasion of epithelial cells by S. Typhimurium strains lacking the Gre factors, GreA and GreB, is impaired. By interacting with the RNA polymerase secondary channel, the Gre factors prevent backtracking of paused complexes to avoid arrest during transcriptional elongation. Our results indicate that the Gre factors are required for the expression of the bacterial factors needed for epithelial cell invasion by modulating expression of HilD. This regulation does not occur at transcription initiation and depends on the capacity of the Gre factors to prevent backtracking of the RNA polymerase. Remarkably, genetic analyses indicate that the 3'-untranslated region (UTR) of hilD is required for Gre-mediated regulation of hilD expression. Our data provide new insight into the complex regulation of S. Typhimurium virulence and highlight the role of the hilD 3'-UTR as a regulatory motif.

Cyclic di-GMP was the first cyclic dinucleotide second messenger described, presaging the discovery of additional cyclic dinucleotide messengers in bacteria and eukaryotes. The GGDEF diguanylate cyclase (DGC) and EAL and HD-GYP phosphodiesterase (PDE) domains conduct the turnover of cyclic di-GMP. These three unrelated domains belong to superfamilies that exhibit significant variations in function, and they include both enzymatically active and inactive members, with a subset involved in synthesis and degradation of other cyclic dinucleotides. Here, we summarize current knowledge of sequence and structural variations that underpin the functional diversification of cyclic di-GMP turnover proteins. Moreover, we highlight that superfamily diversification is not restricted to cyclic di-GMP signaling domains, as particular DHH/DHHA1 domain and HD domain proteins have been shown to act as cyclic di-AMP phosphodiesterases. We conclude with a consideration of the current limitations that such diversity of action places on bioinformatic prediction of the roles of GGDEF, EAL, and HD-GYP domain proteins., (Copyright © 2017 American Society for Microbiology.)

Background: The secondary messenger cyclic di-GMP promotes biofilm formation by up regulating the expression of csgD, encoding the major regulator of rdar biofilm formation in Salmonella typhimurium. The GGDEF/EAL domain proteins regulate the c-di-GMP turnover. There are twenty- two GGDEF/EAL domain proteins in the genome of S. typhimurium. In this study, we dissect the role of individual GGDEF/EAL proteins for csgD expression and rdar biofilm development., Results: Among twelve GGDEF domains, two proteins upregulate and among fifteen EAL domains, four proteins down regulate csgD expression. We identified two additional GGDEF proteins required to promote optimal csgD expression. With the exception of the EAL domain of STM1703, solely, diguanylate cyclase and phosphodiesterase activities are required to regulate csgD mediated rdar biofilm formation. Identification of corresponding phosphodiesterases and diguanylate cyclases interacting in the csgD regulatory network indicates various levels of regulation by c-di-GMP. The phosphodiesterase STM1703 represses transcription of csgD via a distinct promoter upstream region., Conclusion: The enzymatic activity and the protein scaffold of GGDEF/EAL domain proteins regulate csgD expression. Thereby, c-di-GMP adjusts csgD expression at multiple levels presumably using a multitude of input signals.

Maintenance of protein homeostasis is essential for survival of all organisms. In bacteria, the protein quality control system has a broad physiological impact beyond heat shock resistance, being involved in virulence, antibiotic resistance, as well as protection against environmental stresses. Its contribution to rejuvenation and growth arrest suggests interference with protein quality control to be a novel antimicrobial strategy. Remarkably, a protein quality control module originating from environmental strains has been found to be horizontally transferred to predominant clonal groups of bacteria providing exquisite thermotolerance to recently emerged global pathogens suggesting that novel features related to protein homeostasis contribute to the transition to new environments., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In Salmonella enterica serovar Typhimurium (S. Typhimurium), biofilm-formation is controlled by the cytoplasmic intracellular small-molecular second messenger cyclic 3', 5'-di- guanosine monophosphate (c-di-GMP) through the activities of GGDEF and EAL domain proteins. Here we describe that deleting either dsbA or dsbB, respectively encoding a periplasmic protein disulfide oxidase and a cytoplasmic membrane disulfide oxidoreductase, resulted in increased biofilm-formation on solid medium. This increased biofilm-formation, defined as a red, dry and rough (rdar) colony morphotype, paralleled with enhanced expression of the biofilm master regulator CsgD and the biofilm-associated fimbrial subunit CsgA. Deleting csgD in either dsb mutant abrogated the enhanced biofilm-formation. Likewise, overexpression of the c-di-GMP phosphodiesterase YhjH, or mutationally inactivating the CsgD activator EAL-domain protein YdiV, reduced biofilm-formation in either of the dsb mutants. Intriguingly, deleting the GGDEF-EAL domain protein gene STM3615 (yhjK), previously not connected to rdar morphotype development, also abrogated the escalated rdar morphotype formation in dsb mutant backgrounds. Enhanced biofilm-formation in dsb mutants was furthermore annulled by exposure to the protein disulfide catalyst copper chloride. When analyzed for the effect of exogenous reducing stress on biofilm-formation, both dsb mutants initially showed an escalated rdar morphotype development that later dissolved to reveal a smooth mucoid colony morphotype. From these results we conclude that biofilm-development in S. Typhimurium is affected by periplasmic protein disulphide bond status through CsgD, and discuss the involvement of selected GGDEF/EAL domain protein(s) as signaling mediators.

In contrast to numerous enzymes involved in c-di-GMP synthesis and degradation in enterobacteria, only a handful of c-di-GMP receptors/effectors have been identified. In search of new c-di-GMP receptors, we screened the Escherichia coli ASKA overexpression gene library using the Differential Radial Capillary Action of Ligand Assay (DRaCALA) with fluorescently and radioisotope-labelled c-di-GMP. We uncovered three new candidate c-di-GMP receptors in E. coli and characterized one of them, BcsE. The bcsE gene is encoded in cellulose synthase operons in representatives of Gammaproteobacteria and Betaproteobacteria. The purified BcsE proteins from E. coli, Salmonella enterica and Klebsiella pneumoniae bind c-di-GMP via the domain of unknown function, DUF2819, which is hereby designated GIL, GGDEF I-site like domain. The RxGD motif of the GIL domain is required for c-di-GMP binding, similar to the c-di-GMP-binding I-site of the diguanylate cyclase GGDEF domain. Thus, GIL is the second protein domain, after PilZ, dedicated to c-di-GMP-binding. We show that in S. enterica, BcsE is not essential for cellulose synthesis but is required for maximal cellulose production, and that c-di-GMP binding is critical for BcsE function. It appears that cellulose production in enterobacteria is controlled by a two-tiered c-di-GMP-dependent system involving BcsE and the PilZ domain containing glycosyltransferase BcsA., (© 2014 John Wiley & Sons Ltd.)

The major sessility-motility lifestyle change and additional fundamental aspects of bacterial physiology, behaviour and morphology are regulated by the secondary messenger cyclic di-GMP (c-di-GMP). Although the c-di-GMP metabolizing enzymes and many receptors have been readily characterized upon discovery, the HD-GYP domain c-di-GMP phosphodiesterase family remained underinvestigated. In this issue of Molecular Microbiology, Bellini et al. provide an important step towards functional and structural characterization of the previously neglected HD-GYP domain family by resolving the crystal structure of PmGH, a catalytically active family member from the thermophilic bacterium Persephonella marina. The crystal structure revealed a novel tri-nuclear catalytic iron centre involved in c-di-GMP binding and catalysis and provides the structural basis to subsequently characterize in detail the catalytic mechanism of hydrolysis of c-di-GMP to GMP by HD-GYP domains., (© 2013 John Wiley & Sons Ltd.)

In animals, plants and the environment, Salmonella enterica serovar Typhimurium forms the red dry and rough (rdar) biofilm characterized by extracellular matrix components curli and cellulose. With complex expression control by at least ten transcription factors, the bistably expressed orphan response regulator CsgD directs rdar morphotype development. CsgD expression is an integral part of the Hfq regulon and the complex cyclic diguanosine monophosphate signaling network partially controlled by the global RNA-binding protein CsrA. Cell wall turnover and the periplasmic redox status regulate csgD expression on a post-transcriptional level by unknown mechanisms. Furthermore, phosphorylation of CsgD is a potential inactivation and degradation signal in biofilm dissolution. Including complex incoherent feed-forward loops, regulation of biofilm formation versus motility and virulence is of recognized complexity.

The ubiquitous second messenger c-di-GMP regulates the switching of bacterial lifestyles from motility to sessility and acute to chronic virulence to adjust bacterial fitness to altered environmental conditions. Conventionally, EAL proteins being c-di-GMP phosphodiesterases promote motility and acute virulence phenotypes such as invasion into epithelial cells and inhibit biofilm formation. We report here that in contradiction, the EAL-like protein STM1697 of Salmonella typhimurium suppresses motility, invasion into HT-29 epithelial cell line and secretion of the type three secretion system 1 effector protein SipA, whereas it promotes rdar biofilm formation and CsgD expression. STM1697 can, however, functionally replace the EAL-like protein STM1344 and vice versa, whereby both proteins neither degrade nor bind c-di-GMP. Like STM1344, STM1697 suppresses the transcription of class 2 and class 3 flagella regulon genes by binding to FlhD, a component of the master regulator of the flagella regulon FlhD4 C2 and act additively under numerous conditions. Interestingly, the interaction interface of STM1697 with FlhD2 is distinct from its paralogue STM1344. We predict that the stand alone EAL domain proteins STM1697 and STM1344 belong to a subclass of EAL domain proteins in S. typhimurium, which are all involved in motility, biofilm and virulence regulation through interaction with proteins that regulate flagella function., (© 2013 John Wiley & Sons Ltd.)

The RNA chaperone Hfq and its associated small RNAs (sRNAs) regulate a variety of phenotypes in bacteria. In this work, we show that Hfq is a master regulator of biofilm formation in Salmonella enterica serovar Typhimurium. Hfq and two Hfq-dependent sRNAs (ArcZ and SdsR) are required for rdar morphotype expression in S. typhimurium. Hfq controls rdar biofilm formation through the major biofilm regulator CsgD. While csgD mRNA steady-state levels are altered in a sdsR mutant, ArcZ seems to work mainly at the post-transcriptional level. Overexpression of ArcZ complemented rdar morphotype formation of an hfq mutant under plate-grown conditions. Although ArcZ activates rpoS expression, its effect on csgD expression is mainly independent of RpoS. ArcZ does not only regulate rdar morphotype expression, but also the transition between sessility and motility and the timing of type 1 fimbriae vs. curli fimbriae surface-attachment at ambient temperature. Consequently, ArcZ is a major regulator of rdar biofilm development.

In Salmonella enterica serovar Typhimurium, a biofilm mode of growth known as the rdar morphotype is regulated by several networks which sense multiple environmental signals. The transcriptional regulator CsgD is the major target for these regulatory pathways. In this study, we show that two lytic transglycosylases of family I, MltE and MltC, in combination increase CsgD expression and rdar morphotype. MltE and MltC, which share a highly similar transglycosylase SLT domain, work redundantly to regulate CsgD at the transcriptional and posttranscriptional levels. The effect of MltE and MltC on CsgD levels was independent of the known regulatory pathways that sense cell envelope stress. These findings reveal, for the first time, a specific function of lytic transglycosylases in S. Typhimurium and suggest the existence of a new signaling pathway that links cell wall turnover to biofilm formation.

CsgD and cyclic-3',5'-di-guanylate are key regulators of biofilm formation in Salmonella enterica serovar Typhimurium. Our results show that polynucleotide phosphorylase and NlpI oppositely altered expression of CsgD. Polynucleotide phosphorylase and NlpI also had opposite effects on the expression of yjcC, which codes for a cyclic-3',5'-di-guanylate phosphodiesterase affecting CsgD expression.

The transcriptional regulator CsgD of Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major regulator of biofilm formation required for the expression of csgBA, which encodes curli fimbriae, and adrA, coding for a diguanylate cyclase. CsgD is a response regulator with an N-terminal receiver domain with a conserved aspartate (D59) as a putative target site for phosphorylation and a C-terminal LuxR-like helix-turn-helix DNA binding motif, but the mechanisms of target gene activation remained unclear. To study the DNA-binding properties of CsgD we used electrophoretic mobility shift assays and DNase I footprint analysis to show that unphosphorylated CsgD-His(6) binds specifically to the csgBA and adrA promoter regions. In vitro transcription analysis revealed that CsgD-His(6) is crucial for the expression of csgBA and adrA. CsgD-His(6) is phosphorylated by acetyl phosphate in vitro, which decreases its DNA-binding properties. The functional impact of D59 in vivo was demonstrated as S. Typhimurium strains expressing modified CsgD protein (D59E and D59N) were dramatically reduced in biofilm formation due to decreased protein stability and DNA-binding properties in the case of D59E. In summary, our findings suggest that the response regulator CsgD functions in its unphosphorylated form under the conditions of biofilm formation investigated in this study.

Bacterial survival depends on the ability to switch between sessile and motile lifestyles in response to changing environmental conditions. In many species, this switch is governed by (3'-5')-cyclic-diguanosine monophosphate (c-di-GMP), a signalling molecule, which is metabolized by proteins containing GGDEF and/or EAL domains. Salmonella Typhimurium contains 20 such proteins. Here, we show that the RNA-binding protein CsrA regulates the expression of eight genes encoding GGDEF, GGDEF-EAL and EAL domain proteins. CsrA bound directly to the mRNA leaders of five of these genes, suggesting that it may regulate these genes post-transcriptionally. The c-di-GMP-specific phosphodiesterase STM3611, which reciprocally controls flagella function and production of biofilm matrix components, was regulated by CsrA binding to the mRNA, but was also indirectly regulated by CsrA through the FlhDC/FliA flagella cascade and STM1344. STM1344 is an unconventional (c-di-GMP-inactive) EAL domain protein, recently identified as a negative regulator of flagella gene expression. Here, we demonstrate that CsrA directly downregulates expression of STM1344, which in turn regulates STM3611 through fliA and thus reciprocally controls motility and biofilm factors. Altogether, our data reveal that the concerted and complex regulation of several genes encoding GGDEF/EAL domain proteins allows CsrA to control the motility-sessility switch in S. Typhimurium at multiple levels.

Bacterial persistence in the environment and in the infected host is often aided by the formation of exopolymer-enclosed communities known as biofilms. Heterogeneous gene expression takes place in microcompartments formed within the complex biofilm structure. This study describes cell differentiation within an isogenic bacterial cell population based on the example of biofilm formation by Salmonella enterica serovar Typhimurium. We analyzed the expression of the major biofilm regulator CsgD at the single-cell level with a chromosomal CsgD-green fluorescent protein (GFP) translational fusion. In individual cells, CsgD-GFP expression is mostly found in the cytoplasm. Quantitative expression analysis and results from three different models of S. Typhimurium biofilms demonstrated that CsgD is expressed in a bistable manner during biofilm development. CsgD expression is, however, monomodal when CsgD is expressed in larger amounts due to a promoter mutation or elevated levels of the secondary signaling molecule c-di-GMP. High levels of CsgD-GFP are associated with cellular aggregation in all three biofilm models. Furthermore, the subpopulation of cells expressing large amounts of CsgD is engaged in cellulose production during red, dry, and rough (rdar) morphotype development and in microcolony formation under conditions of continuous flow. Consequently, bistability at the level of CsgD expression leads to a corresponding pattern of task distribution in S. Typhimurium biofilms.

The bacterial second messenger cyclic di-GMP (c-di-GMP) regulates the transition between sessility and motility. In Salmonella enterica serovar Typhimurium, the expression of CsgD, the regulator of multicellular rdar morphotype behavior, is a major target of c-di-GMP signaling. CsgD expression is positively regulated by at least two diguanylate cyclases, GGDEF domain proteins, and negatively regulated by at least four phosphodiesterases, EAL domain proteins. Here, we show that in contrast to EAL domain proteins acting as phosphodiesterases, the EAL-like protein STM1344 regulated CsgD expression positively and motility negatively. STM1344, however, did not have a role in c-di-GMP turnover and also did not bind the nucleotide. STM1344 acted upstream of the phosphodiesterases STM1703 and STM3611, previously identified to participate in CsgD downregulation, where it repressed their expression. Consequently, although STM1344 has not retained a direct role in c-di-GMP metabolism, it still participates in the regulation of c-di-GMP turnover and has a role in the transition between sessility and motility.

Successful cell division in pro- and eukaryotes is ensured by checkpoints that regulate cell cycle progression. Structural and biochemical analyses of the DNA integrity scanning protein (DisA) have recently shown that its domain of unknown function, DUF147 [renamed DAC (for diadenylate cyclase)], has diadenylate cyclase activity. This diadenylate cyclase activity is abolished when DisA binds to branched DNA substrates, which arise during DNA double-strand breaks that can spontaneously occur during DNA replication. This finding identifies cyclic di(3'-->5')-adenylic acid (c-di-AMP) as a second messenger candidate that signals DNA integrity in Bacillus subtilis during sporulation, a specialized cell division process that leads to formation of a dormant cell called a spore. The DAC domain is widespread in Bacteria and Archaea; moreover, it is found in proteins containing diverse domains, suggesting that c-di-AMP acts as a second messenger molecule in response to various signals besides branched DNA. To elucidate the biological importance and molecular mechanisms of action for c-di-AMP and the recently recognized second messenger c-di-GMP will require a multidisciplinary approach.

Background: Curli, cellulose and the cell surface protein BapA are matrix components in Salmonella biofilms. In this study we have investigated the roles of these components for the morphology of bacteria grown as colonies on agar plates and within a biofilm on submerged mica surfaces by applying atomic force microscopy (AFM) and light microscopy., Results: AFM imaging was performed on colonies of Salmonella Typhimurium grown on agar plates for 24 h and on biofilms grown for 4, 8, 16 or 24 h on mica slides submerged in standing cultures. Our data show that in the wild type curli were visible as extracellular material on and between the cells and as fimbrial structures at the edges of biofilms grown for 16 h and 24 h. In contrast to the wild type, which formed a three-dimensional biofilm within 24 h, a curli mutant and a strain mutated in the global regulator CsgD were severely impaired in biofilm formation. A mutant in cellulose production retained some capability to form cell aggregates, but not a confluent biofilm. Extracellular matrix was observed in this mutant to almost the same extent as in the wild type. Overexpression of CsgD led to a much thicker and a more rapidly growing biofilm. Disruption of BapA altered neither colony and biofilm morphology nor the ability to form a biofilm within 24 h on the submerged surfaces. Besides curli, the expression of flagella and pili as well as changes in cell shape and cell size could be monitored in the growing biofilms., Conclusion: Our work demonstrates that atomic force microscopy can efficiently be used as a tool to monitor the morphology of bacteria grown as colonies on agar plates or within biofilms formed in a liquid at high resolution.

GGDEF and EAL domain proteins are involved in turnover of the novel secondary messenger cyclic di(3'-->5')-guanylic acid (c-di-GMP) in many bacteria. The rdar morphotype, a multicellular behavior of Salmonella enterica serovar Typhimurium characterized by the expression of the extracellular matrix components cellulose and curli fimbriae is controlled by c-di-GMP. In this work the roles of the EAL and GGDEF-EAL domain proteins on rdar morphotype development were investigated. Knockout of four of 15 EAL and GGDEF-EAL domain proteins upregulated rdar morphotype expression and expression of CsgD, the central regulator of the rdar morphotype, and partially downregulated c-di-GMP concentrations. More-detailed analysis showed that the EAL domain protein STM4264 and the GGDEF-EAL domain protein STM1703, which highly downregulated the rdar morphotype, have overlapping yet distinct functions. Another subset of EAL and GGDEF-EAL domain proteins influenced multicellular behavior in liquid culture and flagellum-mediated motility. Consequently, this work has shown that several EAL and GGDEF-EAL domain proteins, which act as phosphodiesterases, play a determinative role in the expression level of multicellular behavior of Salmonella enterica serovar Typhimurium.

CsgD is a master regulator of multicellular behaviour in Salmonella enterica serovar Typhimurium. Expression of CsgD is highly regulated on the transcriptional level. A nucleo-protein complex had been defined where the global regulators OmpR and integration host factor (IHF) bind up- and downstream of the csgD core promoter. In this study, the nucleo-protein complex of PcsgD was extended through characterization of additional OmpR and IHF binding sites that influence the transcriptional activity of the csgD promoter. Furthermore, the role of the 174 bp long 5'-untranslated region on transcriptional activity was defined.

Flagellin is the major cytokine-releasing factor when Salmonella enterica serovar Typhimurium (S. Typhimurium) infects intestinal epithelial cells. In this work it is shown that curli, an adhesive proteinaceous surface component of Enterobacteriaceae involved in biofilm formation of S. Typhimurium and Escherichia coli strains can bind flagellin and thus elicit an immune response by the intestinal epithelial cell line HT-29.

GGDEF and EAL domain proteins are involved in the turnover of the novel secondary messenger cyclic-di(3'-->5')-guanylic acid (c-di-GMP) in many bacteria. In this work the role of the 12 GGDEF domain proteins encoded by the Salmonella enterica serovar Typhimurium (S. Typhimurium) chromosome in rdar morphotype development was investigated. Previously, it was shown that the GGDEF domain protein AdrA activated the biosynthesis of cellulose by production of c-di-GMP. Enhancement of the c-di-GMP levels by overexpression of the GGDEF domain protein AdrA did lead to the activation of curli fimbriae biosynthesis through the elevated expression of CsgD and CsgA. Although knock-out of the chromosomal copy of adrA influenced CsgA expression, CsgD expression was not altered, although more than half of the total cellular c-di-GMP was produced by AdrA at 16 h of growth. On the other hand, chromosomally encoded GGDEF-EAL domain proteins STM2123 and STM3388 were required to additively activate CsgD expression on a transcriptional and post-transcriptional level. Enhanced c-di-GMP levels did overcome temperature regulation of rdar morphotype expression by activation of curli fimbriae as well as cellulose biosynthesis through CsgD expression. Thus in the regulatory cascade leading to rdar morphotype expression c-di-GMP activates several subsequent steps in the network.

GGDEF domain-containing proteins have been implicated in bacterial signal transduction and synthesis of the second messenger molecule cyclic-di-GMP. A number of GGDEF proteins are involved in controlling the formation of extracellular matrices. AdrA (Salmonella enterica serovar Typhimurium) and HmsT (Yersinia pestis) contain GGDEF domains and are required for extracellular cellulose production and biofilm formation, respectively. Here we show that hmsT is able to restore cellulose synthesis to a Salmonella serovar Typhimurium adrA mutant and that adrA can replace hmsT in Y. pestis Hms-dependent biofilm formation. Like Y. pestis HmsT overproducers, Y. pestis cells carrying adrA under the control of an arabinose-inducible promoter produced substantial biofilms in the presence of arabinose. Finally, we demonstrate that HmsT is involved in the synthesis of cyclic di-GMP.

The starvation-induced csgD gene of Salmonella typhimurium encodes for the positive transcriptional regulator of extracellular matrix components curli fimbriae and cellulose. To analyse regulatory elements of csgD promoter (PcsgD) response genetic studies combined with in vitro experiments were performed. Six binding sites (D1 to D6) for OmpR, a transcriptional regulator, were identified by gel shifts and DNase I footprints. While ompR is required for PcsgD expression, binding of OmpR-P to D2 centred immediately upstream of D1 at position -70.5 is proposed to repress PcsgD activity. The elevated expression of regulated and semiconstitutive PcsgD in response to microaerophilic conditions required integration host factor (IHF). Subsequently, two IHF-binding sites were identified up- and downstream of PcsgD. IHF competes with OmpR-P for binding at its upstream site IHF1, which overlaps with D3-D6 and thereby modulates the response to microaerophilic conditions. A complex regulatory network involving IHF, H-NS and OmpR is proposed whereby the nucleo-complex composition in the csgD-csgBA intergenic region is altered in response to oxygen tension.

Cellulose biosynthesis has recently been established for a variety of bacteria of diverse origin at the phenotypic and genetic levels. Novel regulatory pathways, which involve the second messenger bis-(3',5') cyclic diguanylic acid and several proteins with the GGDEF domain, participate in the regulation of cellulose biosynthesis. The biological significance of cellulose production in environmental, commensal and pathogenic bacteria is only punctually resolved. This review summarizes current knowledge on cellulose biosynthesis, its regulation and biological function.

Flagella-mediated swimming and swarming motility in Salmonella enterica serovar Typhimurium is intercalated with the cyclic di-guanylate monophosphate ( c-di-GMP) signalling network. In this study, we identified the GGDEF domain proteins STM2672, STM4551 and STM1987 as key di-guanylate cyclases involved in regulation of motility in a Δ yhjH phosphodiesterase gene deletion mutant with elevated c-di-GMP levels inhibiting motility. Surprisingly, these di-guanylate cyclases distinctively inhibited motility through the c-di-GMP receptors YcgR and the cellulose synthase BcsA, whereby STM2672 corresponded to YcgR, STM1987 to BcsA and STM4551 to both receptors. Although downregulation of motility is believed to prepare the bacterial cells for surface adhesion and biofilm formation, the major biofilm regulator CsgD of S. sv. Typhimurium was not involved in the regulation of swimming or swarming motility. Together with previously identified c-di-GMP networks regulating flagella-related phenotypes, flagella biosynthesis is a major target of c-di-GMP signalling in S. sv. Typhimurium. [ABSTRACT FROM AUTHOR]