Adaptive response of Amphibacillus xylanus to normal aerobic and forced oxidative stress conditions.

Amphibacillus xylanus grows at the same rate and with the same cell yield under aerobic and anaerobic conditions. Under aerobic conditions, it exhibits vigorous oxygen consumption in spite of lacking a respiratory system and haem catalase. To understand the adaptive response of A. xylanus to oxidative stresses, a genomic analysis of A. xylanus was conducted. The analysis showed that A. xylanus has the genes of four metabolic systems: two pyruvate metabolic pathways, a glycolytic metabolic pathway and an NADH oxidase (Nox)-AhpC (Prx) system. A transcriptional study confirmed that A. xylanus has these metabolic systems. Moreover, genomic analysis revealed the presence of two genes for NADH oxidase (nox1 and nox2), both of which were identified in the transcriptional analysis. The nox1 gene in A. xylanus was highly expressed under normal aerobic conditions but that of nox2 was not. A purification study of NADH oxidases indicated that the gene product of nox1 is a primary metabolic enzyme responsible for metabolism of both oxygen and reactive oxygen species. A. xylanus was successfully grown under forced oxidative stress conditions such as 0.1 mM H2O2, 0.3 mM paraquat and 80 % oxygen. Proteomic analysis revealed that manganese SOD, Prx, pyruvate dehydrogenase complex E1 and E3 components, and riboflavin synthase ß-chain are induced under normal aerobic conditions, and the other proteins except the five aerobically induced proteins were not induced under forced oxidative stress conditions. Taken together, the present findings indicate that A. xylanus has a unique defence system against forced oxidative stress.

The relationship between a coiled morphology and Mbl in alkaliphilic Bacillus halodurans C-125 at neutral pH values.

The facultative alkaliphilic Bacillus halodurans C-125 can grow in a pH range from 6.8 to 10.8. The morphology of the cells grown at pH values above 7.5 is rod shaped, whereas, that gown at pH values less than 7.5 is coiled. Cytoplasmic membrane staining revealed that this coiled morphology was formed not by one filamentous cell, but by many chained bent/non-bent cells. Prokaryotic actin and tubulin homologs (MreB, Mbl MreBH, and FtsZ, respectively) are known to function as bacterial cytoskeleton proteins. The transcription levels of ftsZ, mreB, and mreBH genes were hardly affected by growth pH. However, the level of the mbl gene was significantly decreased at neutral pH values. Moreover, the expression level of the Mbl protein at pH 7.0 was about one-fourth of that at pH 10. Immunofluorescence microscopy (IFM) showed that the Mbl protein was localized as a helical structure in the rod-shaped cell grown at pH 10, whereas a helical structure was not observed in the cells grown at pH 7.0. Fluorescent vancomycin staining showed insertion of new peptidoglycan strands of sidewalls occurred in the cells grown at pH 7.0. These data suggested that a decrease in the expression level of the Mbl protein can influence the morphology of B. halodurans C-125 grown at pH 7.0 without influencing insertion of new peptidoglycan strands.

Industrial applications of alkaliphiles and their enzymes--past, present and future.

Alkaliphiles are microorganisms that can grow in alkaline environments, i.e. pH >9.0. Their enzymes, especially extracellular enzymes, are able to function in their catalytic activities under high alkaline pH values because of their stability under these conditions. Proteases, protein degrading enzymes, are one of the most produced enzymes in industry. Among proteases, alkaline proteases, which are added to some detergents, are the most produced. Other alkaline enzymes, e.g. alkaline cellulases, alkaline amylases, and alkaline lipases, are also adjuncts to detergents for improving cleaning efficiency. Alkaline enzymes often show activities in a broad pH range, thermostability, and tolerance to oxidants compared to neutral enzymes. Alkaliphilic Bacillus species are the most characterized organisms among alkaliphiles. They produce so many extracellular alkaline-adapted enzymes that they are often good sources for industrial enzymes. As a patent strain, the whole genome sequence of alkaliphilic Bacillus halodurans C-125 has been sequenced for the first time. In addition, an increasing number of whole genomic sequences and structural analyses of proteins in alkaliphiles, development of genetic engineering techniques and physiological analyses will reveal the alkaline adaptation mechanisms of alkaliphilic Bacillus species and the structural basis of their enzymatic functions. This information opens up the possibility of new applications. In this paper we describe, first, the physiologies of environmental adaptations, and then the applications of enzymes and microorganisms themselves in alkaliphilic Bacillus species.

Draft Genome Sequence of Calcium-Dependent Novosphingobium sp. Strain TCA1, Isolated from a Hot Spring Containing a High Concentration of Calcium Ions.

Calcium-dependent Novosphingobium sp. strain TCA1 was newly isolated from a water sample from a hot spring containing a high concentration of calcium ions. Here, we report the draft genome sequence of this bacterium, which may be the basis for research on calcium ion homeostasis.

The Surface Layer Homology Domain-Containing Proteins of Alkaliphilic Bacillus pseudofirmus OF4 Play an Important Role in Alkaline Adaptation via Peptidoglycan Synthesis.

It is well known that the Na+ cycle and the cell wall are essential for alkaline adaptation of Na+-dependent alkaliphilic Bacillus species. In Bacillus pseudofirmus OF4, surface layer protein A (SlpA), the most abundant protein in the surface layer (S-layer) of the cell wall, is involved in alkaline adaptation, especially under low Na+ concentrations. The presence of a large number of genes that encode S-layer homology (SLH) domain-containing proteins has been suggested from the genome sequence of B. pseudofirmus OF4. However, other than SlpA, the functions of SLH domain-containing proteins are not well known. Therefore, a deletion mutant of the csaB gene, required for the retention of SLH domain-containing proteins on the cell wall, was constructed to investigate its physiological properties. The csaB mutant strain of B. pseudofirmus OF4 had a chained morphology and alkaline sensitivity even under a 230 mM Na+ concentration at which there is no growth difference between the parental strain and the slpA mutant strain. Ultra-thin section transmission electron microscopy showed that a csaB mutant strain lacked an S-layer part, and its peptidoglycan (PG) layer was disturbed. The slpA mutant strain also lacked an S-layer part, although its PG layer was not disturbed. These results suggested that the surface layer homology domain-containing proteins of B. pseudofirmus OF4 play an important role in alkaline adaptation via peptidoglycan synthesis.

Properties of motility in Bacillus subtilis powered by the H+-coupled MotAB flagellar stator, Na+-coupled MotPS or hybrid stators MotAS or MotPB.

Bacillus subtilis has a single set of flagellar rotor proteins that interact with two distinct stator-force generators, the H+-coupled MotAB complex and the Na+-coupled MotPS complex, that energize rotation. Here, motility on soft agar plates and in liquid was assayed in wild-type B.subtilis and strains expressing only one stator, either MotAB, MotPS or hybrid MotAS or MotPB. The strains expressing MotAB or MotAS had an average of 11 flagella/cell while those expressing MotPS or MotPB had an average of seven flagella/cell, and a Mot-less double mutant had three to four flagella/cell. MotAB had a more dominant role in motility than MotPS under most conditions, but MotPS supported comparable motility to MotAB on malate-containing soft agar plating media at elevated pH and Na+. MotAB supported much faster swimming speeds in liquid than MotPS, MotAS or MotPB under all conditions, but a contribution of MotPS to wild-type swimming was discernible from differences in swimming speeds of wild-type and MotAB at elevated viscosity, pH and Na+. Swimming supported by MotPS and MotAS was stimulated by Na+ and elevated pH whereas the converse was true of MotAB and MotPB. This suggests that MotAS is Na+-coupled and MotPB is H+-coupled and that MotB and MotS are major determinants of ion-coupling. However, the swimming speed supported by MotPB, as well as MotPS and MotAS, was inhibited severely at Na+ concentrations above 300 mM whereas MotAB-dependent swimming was not. The presence of either the MotP or MotS component in the stator also conferred sensitivity to inhibition by an amiloride analogue. These observations suggest that MotP contributes to Na+-coupling and inhibition by Na+ channel inhibitors. Similarly, a role for MotA in H+-dependent stator properties is indicated by the larger effects of pH on the Na+-response of MotAS versus MotPS. Finally, optimal function at elevated viscosity was found only in MotPS and MotPB and is therefore conferred by MotP.

Draft Genome Sequence of Methylobacterium sp. ME121, Isolated from Soil as a Mixed Single Colony with Kaistia sp. 32K.

Methylobacterium sp. ME121 was isolated from soil as a mixed single colony with Kaistia sp. 32K, and its growth was enhanced by coculture. Here, we report the draft genome sequence of Methylobacterium sp. ME121, which may contribute to the study of the molecular mechanisms underlying this phenomenon.

Draft Genome Sequence of Potassium-Dependent Alkaliphilic Bacillus sp. Strain TS-2, Isolated from a Jumping Spider.

The potassium-dependent alkaliphilic Bacillus sp. strain TS-2 was isolated from the mashed extract of a jumping spider, and its draft genome sequence was obtained. Comparative genomic analysis with a previously sequenced sodium-dependent alkaliphilic Bacillus species may reveal potassium-dependent alkaline adaptation mechanisms.

Genome Sequence of the Deep-Sea Denitrifier Pseudomonas sp. Strain MT-1, Isolated from the Mariana Trench.

Pseudomonas sp. strain MT-1 was the first deep-sea denitrifier isolated and characterized from mud recovered from a depth of 11,000 m in the Mariana Trench. We report here the genome sequence of this bacterium, which contributes to our understanding of denitrification and bioenergetics in the deep sea.

Draft Genome Sequence of Calcium-Dependent Paenibacillus sp. Strain TCA20, Isolated from a Hot Spring Containing a High Concentration of Calcium Ions.

Calcium-dependent Paenibacillus sp. strain TCA20 was isolated from a water sample of a hot spring containing a high concentration of calcium ions. Here, we report the draft genome sequence of this bacterium, which may be the basis for the research of calcium ion homeostasis.

Draft Genome Sequence of Sodium-Independent Alkaliphilic Microbacterium sp. Strain TS-1.

Alkaliphilic Microbacterium sp. strain TS-1, newly isolated from the jumping spider, showed Na(+)-independent growth and motility. Here, we report the draft genome sequence of this bacterium, which may provide beneficial information for Na(+)-independent alkaline adaptation mechanisms.

Complete genome sequence of Ilumatobacter coccineum YM16-304(T.).

Ilumatobacter coccineum YM16-304(T) (=NBRC 103263(T)) is a novel marine actinobacterium isolated from a sand sample collected at a beach in Shimane Prefecture, Japan. Strain YM16-304(T) is the type strain of the species. Phylogenetically, strain YM16-304(T) is close to Ilumatobacter nonamiense YM16-303(T) (=NBRC 109120(T)), Ilumatobacter fluminis YM22-133(T) and some uncultured bacteria including putative marine sponge symbionts. Whole genome sequence of these species has not been reported. Here we report the complete genome sequence of strain YM16-304(T). The 4,830,181 bp chromosome was predicted to encode a total of 4,291 protein-coding genes.

Complete genome sequence of Oscillibacter valericigenes Sjm18-20(T) (=NBRC 101213(T)).

Oscillibacter valericigenes is a mesophilic, strictly anaerobic bacterium belonging to the clostridial cluster IV. Strain Sjm18-20(T) (=NBRC 101213(T) =DSM 18026(T)) is the type strain of the species and represents the genus Oscillibacter Iino et al. 2007. It was isolated from the alimentary canal of a Japanese corbicula clam (Corbicula japonica) collected on a seacoast in Shimane Prefecture in Japan. Phylogenetically, strain Sjm18-20(T) is closest to uncultured bacteria in digestive tracts, including the enriched cells thought to represent Oscillospira guilliermondii Chatton and Perard 1913. The isolated phylogenetic position and some distinct characteristics prompted us to determine the complete genome sequence. The 4,410,036 bp chromosome and the 60,586 bp plasmid were predicted to encode a total of 4,723 protein-coding genes.

Motility and chemotaxis in alkaliphilic Bacillus species.

Alkaliphilic Bacillus species grow at pH values up to approximately 11. Motile alkaliphilic Bacillus use electrochemical gradients of Na(+) (sodium-motive force) to power ion-coupled, flagella-mediated motility as opposed to the electrochemical gradients of H(+) (proton-motive force) used by most neutralophilic bacteria. Membrane-embedded stators of bacterial flagella contain ion channels through which either H(+) or Na(+) flow to energize flagellar rotation. Stators of the major H(+)-coupled type, MotAB, are distinguishable from Na(+)-coupled stators, PomAB of marine bacteria and MotPS of alkaliphilic Bacillus. Dual ion-coupling capacity is found in neutralophilic Bacillus strains with both MotAB and MotPS. There is also a MotAB variant that uses both coupling ions, switching as a function of pH. Chemotaxis of alkaliphilic Bacillus depends upon flagellar motility but also requires a distinct voltage-gated NaChBac-type channel. The two alkaliphile Na(+) channels provide new vistas on the diverse adaptations of sensory responses in bacteria.

Na(+) and flagella-dependent swimming of alkaliphilic Bacillus pseudofirmus OF4: a basis for poor motility at low pH and enhancement in viscous media in an "up-motile" variant.

Flagella-based motility of extremely alkaliphilic Bacillus species is completely dependent upon Na(+). Little motility is observed at pH values < approximately 8.0. Here we examine the number of flagella/cell as a function of growth pH in the facultative alkaliphile Bacillus pseudofirmus OF4 and a derivative selected for increased motility on soft agar plates. Flagella were produced by both strains during growth in a pH range from 7.5 to 10.3. The number of flagella/cell and flagellin levels of cells were not strongly dependent on growth pH over this range in either strain although both of these parameters were higher in the up-motile strain. Assays of the swimming speed indicated no motility at pH < 8 with 10 mM Na(+), but significant motility at pH 7 at much higher Na(+) concentrations. At pH 8-10, the swimming speed increased with the increase of Na(+) concentration up to 230 mM, with fastest swimming at pH 10. Motility of the up-motile strain was greatly increased relative to wild-type on soft agar at alkaline pH but not in liquid except when polyvinylpyrrolidone was added to increase viscosity. The up-motile phenotype, with increased flagella/cell may support bundle formation that particularly enhances motility under a subset of conditions with specific challenges.

The voltage-gated Na+ channel NaVBP co-localizes with methyl-accepting chemotaxis protein at cell poles of alkaliphilic Bacillus pseudofirmus OF4.

Na(V)BP, found in alkaliphilic Bacillus pseudofirmus OF4, is a member of the bacterial voltage-gated Na(+) channel superfamily. The alkaliphile requires Na(V)BP for normal chemotaxis responses and for optimal pH homeostasis during a shift to alkaline conditions at suboptimally low Na(+) concentrations. We hypothesized that interaction of Na(V)BP with one or more other proteins in vivo, specifically methyl-accepting chemotaxis proteins (MCPs), is involved in activation of the channel under the pH conditions that exist in the extremophile and could underpin its role in chemotaxis; MCPs transduce chemotactic signals and generally localize to cell poles of rod-shaped cells. Here, immunofluorescence microscopy and fluorescent protein fusion studies showed that an alkaliphile protein (designated McpX) that cross-reacts with antibodies raised against Bacillus subtilis McpB co-localizes with Na(V)BP at the cell poles of B. pseudofirmus OF4. In a mutant in which Na(V)BP-encoding ncbA is deleted, the content of McpX was close to the wild-type level but McpX was significantly delocalized. A mutant of B. pseudofirmus OF4 was constructed in which cheAW expression was disrupted to assess whether this mutation impaired polar localization of McpX, as expected from studies in Escherichia coli and Salmonella, and, if so, whether Na(V)BP would be similarly affected. Polar localization of both McpX and Na(V)BP was decreased in the cheAW mutant. The results suggest interactions between McpX and Na(V)BP that affect their co-localization. The inverse chemotaxis phenotype of ncbA mutants may result in part from MCP delocalization.