Chloride dependence of glycine betaine transport in Halobacillus halophilus

Growth of Halobacillus halophilus is strictly chloride-dependent but the physiological basis for the chloride dependence remains to be elucidated. To address the function of Cl(-) in H. halophilus, a physiological study was performed. It was found that uptake of the compatible solute glycine betaine under isoosmotic conditions was stimulated by increasing salt concentrations. Uptake of glycine betaine required both, Na(+) and Cl(-). Cl(-) could be substituted by nitrate and bromide, but not by sulfate. Glycine betaine transport was optimal at around 0.7 M Cl(-). Cells responded to an osmotic upshock by accumulating glycine betaine, but only in the presence of chloride. These studies revealed the first chloride-dependent glycine betaine transporter in a prokaryote.     

Motility and flagellum synthesis in Halobacillus halophilus are chloride dependent

The motility of Halobacillus halophilus as observed on swarm agar plates was strictly dependent on the chloride concentration. Cl(-) was apparently not used as the coupling ion for flagellar rotation. Cells grown in the absence of chloride were devoid of flagella, but flagellation was restored upon the addition of chloride. These experiments indicate that chloride is involved in synthesis of flagella in H. halophilus.     

Flagellin gene (fliC) of Thermus thermophilus HB8: characterization of its product and involvement to flagella assembly and microbial motility

Thermus thermophilus HB8 flagellin protein (FliC) is encoded by the TTHC004 (fliC) gene, which is located in the pTT8 plasmid of the bacterium. Flagellin monomer and flagella fibres were isolated from a culture of T. thermophilus grown in rich medium, or in mineral salt medium with sodium gluconate as the carbon source. Western blot immunodetection with anti-FliC revealed a stable complex (FliC)(1)(FliS)(2) of flagellin (FliC, 27.7 kDa) with a homodimer of FliS (FliS, 18.2 kDa) that are encoded by TTHC004 and TTHC003 genes, respectively. The complex is dissociable at low pHs and/or by heat treatment. Glycan staining of purified flagella and treatment with N-glycosidase F suggested that flagellin of T. thermophilus is a glycosylated protein. Size exclusion chromatography revealed that flagellar filaments (FliC) have a molecular mass higher than 200 kDa. The formation of flagella is enhanced after prolonged cultivation time where phosphate and other nutrient were depleted, giving in the bacterium considerable swimming motility in low viscosity media.     

Flagellin polymerisation control by a cytosolic export chaperone

Assembly of the long helical filament of the bacterial flagellum requires polymerisation of ca 20,000 flagellin (FliC) monomeric subunits into the growing structure extending from the cell surface. Here, we show that export of Salmonella flagellin is facilitated specifically by a cytosolic protein, FliS, and that FliS binds to the FliC C-terminal helical domain, which contributes to stabilisation of flagellin subunit interactions during polymerisation. Stable complexes of FliS with flagellin were assembled efficiently in vitro, apparently by FliS homodimers binding to FliC monomers. The data suggest that FliS acts as a substrate-specific chaperone, preventing premature interaction of newly synthesised flagellin subunits in the cytosol. Compatible with this view, FliS was able to prevent in vitro polymerisation of FliC into filaments.     

Analysis of the role of the two flagella of Bradyrhizobium japonicum in competition for nodulation of soybean

Bradyrhizobium japonicum has two types of flagella. One has thin filaments consisting of the 33-kDa flagellins FliCI and FliCII (FliCI-II) and the other has thick filaments consisting of the 65-kDa flagellins FliC1, FliC2, FliC3, and FliC4 (FliC1-4). To investigate the roles of each flagellum in competition for nodulation, we obtained mutants deleted in fliCI-II and/or fliC1-4 in the genomic backgrounds of two derivatives from the reference strain USDA 110: the streptomycin-resistant derivative LP 3004 and its more motile derivative LP 3008. All mutations diminished swimming motility. When each mutant was co-inoculated with the parental strain on soybean plants cultivated in vermiculite either at field capacity or flooded, their competitiveness differed according to the flagellin altered. ΔfliCI-II mutants were more competitive, occupying 64-80% of the nodules, while ΔfliC1-4 mutants occupied 45-49% of the nodules. Occupation by the nonmotile double mutant decreased from 55% to 11% as the water content of the vermiculite increased from 85% to 95% field capacity to flooding. These results indicate that the influence of motility on competitiveness depended on the water status of the rooting substrate.     

Biochemical and molecular characterization of the biosynthesis of glutamine and glutamate, two major compatible solutes in the moderately halophilic bacterium Halobacillus halophilus

The moderately halophilic, chloride-dependent bacterium Halobacillus halophilus produces glutamate and glutamine as main compatible solutes at external salinities of 1.0 to 1.5 M NaCl. The routes for the biosynthesis of these solutes and their regulation were examined. The genome contains two genes potentially encoding glutamate dehydrogenases and two genes for the small subunit of a glutamate synthase, but only one gene for the large subunit. However, the expression of these genes was not salt dependent, nor were the corresponding enzymatic activities detectable in cell extracts of cells grown at different salinities. In contrast, glutamine synthetase activity was readily detectable in H. halophilus. Induction of glutamine synthetase activity was strictly salt dependent and reached a maximum at 3.0 M NaCl; chloride stimulated the production of active enzyme by about 300%. Two potential genes encoding a glutamine synthetase, glnA1 and glnA2, were identified. The expression of glnA2 but not of glnA1 was increased up to fourfold in cells adapted to high salt, indicating that GlnA2 is the glutamine synthetase involved in the synthesis of the solutes glutamate and glutamine. Furthermore, expression of glnA2 was stimulated twofold by the presence of chloride ions. Chloride exerted an even more pronounced effect on the enzymatic activity of preformed enzyme: in the absence of chloride in the assay buffer, glutamine synthetase activity was decreased by as much as 90%. These data demonstrate for the first time a regulatory role of a component of common salt, chloride, in the biosynthesis of compatible solutes.     

Pathoadaptive Mutations in Salmonella enterica Isolated after Serial Passage in Mice

How pathogenic bacteria adapt and evolve in the complex and variable environment of the host remains a largely unresolved question. Here we have used whole genome sequencing of Salmonella enterica serovar Typhimurium LT2 populations serially passaged in mice to identify mutations that adapt bacteria to systemic growth in mice. We found unique pathoadaptive mutations in two global regulators, phoQ and stpA, which increase the competitive indexes of the bacteria 3- to 5-fold. Also, all mouse-adapted lineages had changed the orientation of the hin invertable element, resulting in production of a FliC type of flagellum. Competition experiments in mice with locked flagellum mutants showed that strains expressing the FliC type of flagellum had a 5-fold increase in competitive index as compared to those expressing FljB type flagellum. Combination of the flagellum cassette inversion with the stpA mutation increased competitive indexes up to 20-fold. These experiments show that Salmonella can rapidly adapt to a mouse environment by acquiring a few mutations of moderate individual effect that when combined confer substantial increases in growth.     

Polyphasic taxonomy of a novel Halobacillus, Halobacillus thailandensis sp. nov. isolated from fish sauce.

In order to speed up fish sauce production, a more complete understanding of the microorganisms associated with the fermentation was needed. This study was undertaken to meet that need. A bacterium was isolated from a fish sauce production line containing 25% NaCl. It is a Gram-positive, rod-shaped bacillus with pointed ends, occurring as single cells, pairs, or short chains. Endospores are produced on a low nutrient medium and, in old cultures, the cells round up, even when undergoing division. The cell wall is relatively amorphous and similar to that of Gram-positive bacteria in structure and composition. Cells grown in a medium containing 10-20% salt possess thicker cell walls than those grown in a medium with 3% salt. Based on 16S rRNA sequence and DNA/DNA hybridization data, we conclude that the bacterium is a species of Halobacillus. This bacterium shares 99.2% and 97.2% 16S rRNA similarity with Halobacillus litoralis and Halobacillus halophilus respectively and DNA/DNA homology was lower than 70%, considered indicative of species similarity. Three highly expressed extra-cellular proteolytic enzymes with M(r) of approximately 100 kDa, 42 kDa and 17 kDa, respectively, were detected by gelatin-polyacrylamide gel electrophoresis. Activity of the 100 kDa and 17 kDa proteases was inhibited by phenylmethanesulphonyl fluoride (PMSF), without being affected by L-trans epoxysuccinyl-leucylamide 4-guanidino-butane (E-64), pepstatin, EDTA, or 1, 10-phenanthroline, leading to the conclusion that these enzymes are serine proteases. The 42-kDa protease was inhibited by EDTA and 1,10-phenanthroline, but not by PMSF, thus, being classified a metalloprotease. The strain has been successfully employed to improve fermentation in industrial production of fish sauce in Thailand.     

Growth phase-dependent switch in osmolyte strategy in a moderate halophile: ectoine is a minor osmolyte but major stationary phase solute in Halobacillus halophilus

The moderately halophilic, chloride-dependent bacterium Halobacillus halophilus switches its osmolyte strategy with the salinity in its environment by the production of different compatible solutes. Ectoine is produced predominantly at very high salinities, along with proline. Interestingly, ectoine production is growth phase dependent which led to a more than 1000-fold change in the ectoine : proline ratio from 0.04 in exponential to 27.4 in late stationary phase cultures. The genes encoding the ectoine biosynthesis pathway were identified on the chromosome in the order ectABC . They form an operon that is expressed in a salinity-dependent manner with low-level expression below 1.5 M NaCl but 10-fold and 23-fold increased expression at 2.5 and 3.0 M NaCl respectively. The temporal expression of genes involved in osmoresponse is different with gdh / gln and pro genes being first, followed by ect genes. Chloride had no effect on expression of ect genes, but stimulated cellular EctC synthesis as well as ectoine production. These data demonstrate, for the first time, a growth-phase dependent switch in osmolyte strategy in a moderate halophile and, additionally, represent another piece of the chloride regulon of H. halophilus .     

Involvement of SPI-2-encoded SpiC in flagellum synthesis in Salmonella enterica serovar Typhimurium

Background  

SpiC encoded within Salmonella pathogeniCity island 2 on the Salmonella enterica serovar Typhimurium chromosome is required for survival within macrophages and systemic infection in mice. Additionally, SpiC contributes to Salmonella-induced activation of the signal transduction pathways in macrophages by affecting the expression of FliC, a component of flagella filaments. Here, we show the contribution of SpiC in flagellum synthesis.     

In vivo and in vitro complementation study comparing the function of DnaK chaperone systems from halophilic lactic acid bacterium Tetragenococcus halophilus and Escherichia coli

In this study, we characterized the DnaK chaperone system from Tetragenococcus halophilus, a halophilic lactic acid bacterium. An in vivo complementation test showed that under heat stress conditions, T. halophilus DnaK did not rescue the growth of the Escherichia coli dnaK deletion mutant, whereas T. halophilus DnaJ and GrpE complemented the corresponding mutations of E. coli. Purified T. halophilus DnaK showed intrinsic weak ATPase activity and holding chaperone activity in vitro, but T. halophilus DnaK did not cooperate with the purified DnaJ and GrpE from either T. halophilus or E. coli in ATP hydrolysis or luciferase-refolding reactions under the conditions tested. E. coli DnaK, however, cross-reacted with those from both bacteria. This difference in the cooperation with DnaJ and GrpE appears to result in an inability of T. halophilus DnaK to replace the in vivo function of the DnaK chaperone of E. coli.     

Identification of a GntR family regulator BusRTha and its regulatory mechanism in the glycine betaine ABC transport system of Tetragenococcus halophilus

Extremophiles - Glycine betaine is one of the most effective compatible solutes of the halophilic lactic acid bacterium Tetragenococcus halophilus, the transportation of which is essential for its...     

Cell-cycle-dependent polar morphogenesis in Caulobacter crescentus: roles of phospholipid, DNA, and protein syntheses.

During swarmer cell differentiation in Caulobacter crescentus, morphogenesis at the swarmer pole is characterized by the loss of the flagellum, by the loss of phage receptor activity (PRA) (the ability of the cell to adsorb phage phi CbK), and finally by the initiation of stalk outgrowth at the site formerly occupied by the flagellum and the PRA. We show here that each of these events is a cell cycle-dependent event requiring continuous protein synthesis for its execution but occurring normally in the absence of DNA synthesis or phospholipid synthesis. During stalked-cell differentiation, the flagellum and PRA reappear and the stalk elongates considerably. We show here that these events are also cell cycle dependent, requiring not only de novo protein synthesis but also DNA and phospholipid syntheses. When synchronous cells dividing 160 min after collection were used, PRA reappearance occurred at 110 min. This PRA reappearance was dependent on a phospholipid synthesis-requiring event occurring at 70 min, a DNA synthesis-requiring event occurring at 95 min, and a protein synthesis-requiring event occurring at 108 min. In the absence of net phospholipid synthesis, stalk elongation appeared more or less normal, but the stalks eventually became fragile, and by 240 min, most of the stalks had broken off, leaving only stubs attached to the cell body.     

Simultaneous display of multiple foreign peptides in the FliD capping and FliC filament proteins of the Escherichia coli flagellum

The bacterial flagellum is composed of more than 20 different proteins. The filament, which constitutes the major extracellular part of the flagellum, is built up of approximately 20,000 FliC molecules that assemble at the growing distal end of the filament. A capping structure composed of five FliD molecules located at the tip of the filament promotes polymerization of FliC. Lack of FliD leads to release of the subunits into the growth medium. We show here that FliD can be successfully used in bacterial surface display. We tested various insertion sites in the capping protein, and the optimal region for display was at the variable region in FliD. Deletion and/or insertion at other sites resulted in decreased formation of flagella. We further developed the technique into a multihybrid display system in which three foreign peptides are simultaneously expressed within the same flagellum, i.e., D repeats of FnBPA from Staphylococcus aureus at the tip and fragments of YadA from Yersinia enterocolitica as well as SlpA from Lactobacillus crispatus along the filament. This technology can have biotechnological applications, e.g., in simultaneous delivery of several effector molecules.