Intermediate filament-like proteins in bacteria and a cytoskeletal function in Streptomyces

Actin and tubulin cytoskeletons are conserved and widespread in bacteria. A strikingly intermediate filament (IF)-like cytoskeleton, composed of crescentin, is also present in Caulobacter crescentus and determines its specific cell shape. However, the broader significance of this finding remained obscure, because crescentin appeared to be unique to Caulobacter. Here we demonstrate that IF-like function is probably a more widespread phenomenon in bacteria. First, we show that 21 genomes of 26 phylogenetically diverse species encoded uncharacterized proteins with a central segmented coiled coil rod domain, which we regarded as a key structural feature of IF proteins and crescentin. Experimental studies of three in silico predicted candidates from Mycobacterium and other actinomycetes revealed a common IF-like property to spontaneously assemble into filaments in vitro. Furthermore, the IF-like protein FilP formed cytoskeletal structures in the model actinomycete Streptomyces coelicolor and was needed for normal growth and morphogenesis. Atomic force microscopy of living cells revealed that the FilP cytoskeleton contributed to mechanical fitness of the hyphae, thus closely resembling the function of metazoan IF. Together, the bioinformatic and experimental data suggest that an IF-like protein architecture is a versatile design that is generally present in bacteria and utilized to perform diverse cytoskeletal tasks

Roles of curli, cellulose and BapA in Salmonella biofilm morphology studied by atomic force microscopy

<p>Abstract</p> <p>Background</p> <p>Curli, cellulose and the cell surface protein BapA are matrix components in <it>Salmonella </it>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.</p> <p>Results</p> <p>AFM imaging was performed on colonies of <it>Salmonella </it>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.</p> <p>Conclusion</p> <p>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.</p

The Escherichia coli BarA-UvrY two-component system is a virulence determinant in the urinary tract

BACKGROUND: The Salmonella enterica BarA-SirA, the Erwinia carotovora ExpS-ExpA, the Vibrio cholerae BarA-VarA and the Pseudomonas spp GacS-GacA all belong to the same orthologous family of two-component systems as the Escherichia coli BarA-UvrY. In the first four species it has been demonstrated that disruption of this two-component system leads to a clear reduction in virulence of the bacteria. Our aim was to determine if the Escherichia coli BarA-UvrY two-component system is connected with virulence using a monkey cystitis model. RESULTS: Cystitis was generated in Macaque fascularis monkeys by infecting the bladder with a 1:1 mixture of the uropathogenic Escherichia coli isolate DS17 and a derivative where the uvrY gene had been disrupted with a kanamycin resistance gene. Urine was collected through bladder punctuation at subsequent time intervals and the relative amount of uvrY mutant was determined. This showed that inactivation of the UvrY response regulator leads to a reduced fitness. In similar competitions in culture flasks with Luria Broth (LB) the uvrY mutant rather had a higher fitness than the wild type. When the competitions were done in flasks with human urine the uvrY mutant initially had a lower fitness. This was followed by a fluctuation in the level of mutant in the long-term culture, with a pattern that was specific for the individual urines that were tested. Addition of LB to the different urine competition cultures however clearly led to a consistently higher fitness of the uvrY mutant. CONCLUSION: This paper demonstrates that the BarA-UvrY two-component system is a determinant for virulence in a monkey cystitis model. The observed competition profiles strengthen our previous hypothesis that disruption of the BarA-UvrY two-component system impairs the ability of the bacteria to switch between different carbon sources. The urine in the bladder contains several different carbon sources and its composition changes over time. Inability to efficiently switch between the carbon sources may thus provide an explanation to the reduced fitness of the uvrY mutant in the cystitis model

Bacterial virulence and adaptation mediated by two-compnent system signalling

One of the most fundamental ways of signal perception and propagation is mediated by the bacterial two-component system (TCS). These types of systems mediate bacterial adaptation that may cause disease in a host due to expression of virulence factors. Understanding the principles behind the bacterial adaptation process mediated by TCSs may thus aid in the development of novel types of antibiotics. In this work we first characterized the impact of the BarA-UvrY TCS when it comes to adaptation of E. coli to varying carbon sources both in vitro and in a monkey cystitis infection model. Growth and efficient utilization of limited nutrients is central for the bacteria when it comes to the infection process. A bacterium mutated in the BarA-UvrY TCS is deficient in the ability to switch between different carbon sources and thus severely attenuated when nutrients are limited and vary over time. However, when gluconeogenic carbon sources are present in excess our results indicate that lack of the BarA-UvrY TCS leads to a higher fitness, probably due to an increased amount of free CsrA protein promoting gluconeogensis. To get more insight regarding the molecular function of the BarA-UvrY TCS we benefited from the ability of UvrY to be activated by acetyl phosphate. This enabled us to characterize BarA sensor proteins with mutations in domains believed to be important for sensor signal propagation and phosphatase activity. The results indicate that the HAMP domain (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase) in BarA is vital for the kinase and phosphatase switching ability. of the sensor and that the two N-terminal domains of BarA are both involved in dephosphorylating UvrY. The dephosphorylating process was also shown to be mediated by a dimer of two BarA sensor proteins. The BarA-UvrY TCSs controls the carbon storage regulator (Csr) system that has earlier been implicated in biofilm formation in both E. coli and Salmonella. We decided to establish a model for studying the kinetics and the impact of these and other regulatory systems on early Salmonella biofilm formation, using a combination of atomic force microscopy and light microscopy. Following the initial proliferation phase, we observed a dispersal of the bacteria and formation of microcolonies that subsequently merged into a confluent biofilm. The dispersal was clearly distinct from the detachment phase that occurs after formation of the mature biofilm. Mutations in different global regulatory genes and genes controlling the production of extracellular polymeric substances (EPS) had a moderate to severe impact on the ability of Salmonella to form a biofilm. Loss of the CsrA protein had a drastic effect on cell morphology and caused a loss of EPS and flagella, unlike loss of the CsrB and CsrC ncRNAs, which caused an increase in EPS and flagella production. In the last study the yhdA gene was identified, via a transposon screening approach, as a factor affecting BarA-UvrY TCS signaling. The yhdA gene encodes a 646 amino acid protein containing both a GGDEF-like and an EAL-like domain. These domains are involved in formation and breakdown of 3',5'-cyclic diguanylic acid (c-di-GMP), a second messenger important for the switch between the vegetative and sessile phase of the bacteria. Additional complementation studies using the yhdA gene expressed in trans, and the ability of UvrY to be activated by acetyl phosphate in the absence of BarA, suggested that YhdA affects the ability of the BarA sensor to switch between phosphatase and kinase activity

The Escherichia coli BarA-UvrY Two-Component System Is Needed for Efficient Switching between Glycolytic and Gluconeogenic Carbon Sources

The Escherichia coli BarA and UvrY proteins were recently demonstrated to constitute a novel two-component system, although its function has remained largely elusive. Here we show that mutations in the sensor kinase gene, barA, or the response regulator gene, uvrY, in uropathogenic E. coli drastically affect survival in long-term competition cultures. Using media with gluconeogenic carbon sources, the mutants have a clear growth advantage when competing with the wild type, but using media with carbon sources feeding into the glycolysis leads to a clear growth advantage for the wild type. Results from competitions with mutants in the carbon storage regulation system, CsrA/B, known to be a master switch between glycolysis and gluconeogenesis, led us to propose that the BarA-UvrY two-component system controls the Csr system. Taking these results together, we propose the BarA-UvrY two-component system is crucial for efficient adaptation between different metabolic pathways, an essential function for adaptation to a new environment

Molecular Characterization of the Acid-Inducible asr Gene of Escherichia coli and Its Role in Acid Stress Response

Enterobacteria have developed numerous constitutive and inducible strategies to sense and adapt to an external acidity. These molecular responses require dozens of specific acid shock proteins (ASPs), as shown by genomic and proteomic analysis. Most of the ASPs remain poorly characterized, and their role in the acid response and survival is unknown. We recently identified an Escherichia coli gene, asr (acid shock RNA), encoding a protein of unknown function, which is strongly induced by high environmental acidity (pH < 5.0). We show here that Asr is required for growth at moderate acidity (pH 4.5) as well as for the induction of acid tolerance at moderate acidity, as shown by its ability to survive subsequent transfer to extreme acidity (pH 2.0). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of acid-shocked E. coli cells harboring a plasmid-borne asr gene demonstrated that the Asr protein is synthesized as a precursor with an apparent molecular mass of 18 kDa. Mutational studies of the asr gene also demonstrated the Asr preprotein contains 102 amino acids. This protein is subjected to an N-terminal cleavage of the signal peptide and a second processing event, yielding 15- and 8-kDa products, respectively. Only the 8-kDa polypeptide was detected in acid-shocked cells containing only the chromosomal copy of the asr gene. N-terminal sequencing and site-directed mutagenesis revealed the two processing sites in the Asr protein precursor. Deletion of amino acids encompassing the processing site required for release of the 8-kDa protein resulted in an acid-sensitive phenotype similar to that observed for the asr null mutant, suggesting that the 8-kDa product plays an important role in the adaptation to acid shock. Analysis of Asr:PhoA fusions demonstrated a periplasmic location for the Asr protein after removal of the signal peptide. Homologues of the asr gene from other Enterobacteriaceae were cloned and shown to be induced in E. coli under acid shock conditions

Genetic and Functional Characterization of the Escherichia coli BarA-UvrY Two-Component System: Point Mutations in the HAMP Linker of the BarA Sensor Give a Dominant-Negative Phenotype

The BarA-UvrY two-component system family is strongly associated with virulence but is poorly understood at the molecular level. During our attempts to complement a barA deletion mutant, we consistently generated various mutated BarA proteins. We reasoned that characterization of the mutants would help us to better understand the signal transduction mechanism in tripartite sensors. This was aided by the demonstrated ability to activate the UvrY regulator with acetyl phosphate independently of the BarA sensor. Many of the mutated BarA proteins had poor complementation activity but could counteract the activity of the wild-type sensor in a dominant-negative fashion. These proteins carried point mutations in or near the recently identified HAMP linker, previously implicated in signal transduction between the periplasm and cytoplasm. This created sensor proteins with an impaired kinase activity and a net dephosphorylating activity. Using further site-directed mutagenesis of a HAMP linker-mutated protein, we could demonstrate that the phosphoaccepting aspartate 718 and histidine 861 are crucial for the dephosphorylating activity. Additional analysis of the HAMP linker-mutated BarA sensors demonstrated that a dephosphorylating activity can operate via phosphotransfer within a tripartite sensor dimer in vivo. This also means that a tripartite sensor can be arranged as a dimer even in the dephosphorylating mode

High resolution AFM images of pili-like fimbrial structures and flagella in the curli mutant MAE14 after 4 h at two different scan sizes

<p><b>Copyright information:</b></p><p>Taken from "Roles of curli, cellulose and BapA in biofilm morphology studied by atomic force microscopy"</p><p>http://www.biomedcentral.com/1471-2180/7/70</p><p>BMC Microbiology 2007;7():70-70.</p><p>Published online 24 Jul 2007</p><p>PMCID:PMC1949822.</p><p></p

The BarA-UvrY two-component system is a virulence determinant in the urinary tract-0

<p><b>Copyright information:</b></p><p>Taken from "The BarA-UvrY two-component system is a virulence determinant in the urinary tract"</p><p>BMC Microbiology 2006;6():27-27.</p><p>Published online 10 Mar 2006</p><p>PMCID:PMC1421404.</p><p>Copyright © 2006 Tomenius et al; licensee BioMed Central Ltd.</p> (110-7 and 40-6) at day 0. Urine samples and vaginal smears were collected (day 2, 5 & 7). The percentage of the mutant was determined in A. urine and B. vaginal smears