Oceanobacillus aidingensis sp. nov., a moderately halophilic bacterium.

Two Gram-positive, rod-shaped moderately halophilic bacterial strains, designated AD7-25(T) and AB-11, were isolated from Aiding and Manasi salt lakes in Xinjiang of China, respectively. The strains were found to be able to grow at NaCl concentrations of 0-21 % (w/v), with optimum growth occurring at 6-8 % (w/v) NaCl. The optimal temperature and pH for growth were determined to be 33-37 °C and pH 7.0-7.5. Cells of the strains are motile by means of polar flagella. Both strains can produce ellipsoidal spores. The major cellular fatty acids were identified as anteiso-C15:0, iso-C15:0, iso-C14:0, anteiso-C17:0 and iso-C16:0. The diamino acid in the peptidoglycan and the major quinone system were determined to be meso-diaminopimelic acid (meso-DAP) and MK-7, respectively. The DNA G+C contents of stains AD7-25(T) and AB-11 were 39.8 and 40.0 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that these two novel strains are closely related to the genus Oceanobacillus showing 90-99.5 % similarity with respect to type strains. These two novel strains were most closely related to Oceanobacillus oncorhynchi subsp. incaldanensis DSM 16557(T) (99.1 and 99.5 %), followed by O. oncorhynchi subsp. oncorhynchi JCM 12661(T) (99.1 and 99.4 %), Oceanobacillus neutriphilus CGMCC 1.7693(T) (97.0 and 97.5 %), Oceanobacillus sojae JCM 15792(T) (97.6 and 98.0 %) and Oceanobacillus locisalsi KCTC 13253(T) (96.5 and 96.9 %). The DNA-DNA hybridization data indicated that DNA relatedness between strains AD7-25(T) and AB-11 was 91.0 %, and the genomic homology of representative strain AD7-25(T) with O. oncorhynchi subsp. incaldanensis DSM 16557(T), O. oncorhynchi subsp. oncorhynchi JCM 12661(T), O. neutriphilus CGMCC 1.7693(T), O. sojae JCM 15792(T) and O. locisalsi KCTC 13253(T) were 41, 39, 20, 23 and 17 %, respectively. On the basis of phenotypic and phylogenetic distinctiveness, strains AD7-25(T) and AB-11 should be assigned to the genus Oceanobacillus as a new species, for which the name Oceanobacillus aidingensis sp. nov. was proposed. The type strain is AD7-25(T) (=CGMCC 1.9106 (T) = NBRC 105904(T)).

Jeotgalicoccus halophilus sp. nov., isolated from salt lakes.

Two slightly halophilic bacterial strains, C1-52(T) and YD-9, were isolated from Daban and Aiding salt lakes in Xinjiang, China, respectively. The isolates were gram-positive, non-endospore-forming, non-motile, facultatively anaerobic cocci. Colonies were pale yellow, and a light pink, diffusible pigment was produced after a few additional days of incubation. The isolates grew optimally with 2-3 % (w/v) NaCl, at pH 7.5 and at 30-35 °C. The peptidoglycan type was L-Lys-Gly(3-4)-L-Ala(Gly). The menaquinones were MK-7 (83.2 %) and MK-6 (16.8 %). The major fatty acids (>10 %) were anteiso-C(15 : 0) and iso-C(15 : 0). The DNA G+C content of strains C1-52(T) and YD-9 was 41.2 and 41.0 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains C1-52(T) and YD-9 were closely related to Jeotgalicoccus psychrophilus YKJ-115(T) (98.0 and 97.1 % 16S rRNA gene sequence similarity, respectively), followed by Jeotgalicoccus halotolerans YKJ-101(T) (97.1 and 96.8 %). Strains C1-52(T) and YD-9 shared, respectively, 20 and 11 % DNA-DNA relatedness with J. halotolerans JCM 11198(T) and 8 and 13 % with J. psychrophilus JCM 11199(T). DNA-DNA relatedness between the isolates was 91 %. On the basis of phenotypic and phylogenetic distinctiveness, strains C1-52(T) and YD-9 belonged to the same species, which should be placed in the genus Jeotgalicoccus as a novel species. The name Jeotgalicoccus halophilus sp. nov. is proposed, with the type strain C1-52(T) ( = CGMCC 1.8911(T)  = NBRC 105788(T)).

Oceanobacillus manasiensis sp. nov., a moderately halophilic bacterium isolated from the salt lakes of Xinjiang, China.

Three Gram reaction positive, rod-shaped, moderately motile halophilic bacterial strains, designated YD3-56(T), YD16, and YH29, were isolated from the sediments of Manasi and Aiding salt lakes in the Xinjiang region of China, respectively. The strains grew optimally at 30-37 degrees C, pH 8-11, in the presence of 5-10% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strains were closely related to members of the genus Oceanobacillus, exhibiting 99.1-99.2% similarity to O. kapialis KCTC 13177(T), 99.2-99.3% to O. picturae KCTC 3821(T), and 94.2-96% sequence similarity to other described Oceanobacillus species. SDS-PAGE of whole cell proteins preparations demonstrated that the strains exhibited high similarity to each other, but distinguished from O. kapialis KCTC 13177(T) and O. picturae KCTC 3821(T) (75%). DNA-DNA hybridization revealed that the similarity between the representative strain YD3-56(T) and O. kapialis KCTC 13177(T) was 35.3%, and the similarity between YD3-56(T) and O. picturae KCTC 3821(T) was 22.3%. Chemotaxonomic analysis of the strains showed menaquinone-7 was the predominant respiratory quinine. Major cellular fatty acids were anteiso-C(15:0) and anteiso-C(17:0). The polar lipid pattern for strain YD3-56(T) predominantly contained phosphatidylcholine, and trace to moderate amounts of phosphatidyl ethanolamine and hydroxy-phosphatidyl ethanolamine. The diamino acid in murein was meso-diaminopimelic acid. The DNA G+C content of the strains was 39.7-40.1 mol%. On the basis of these results, the three strains should be classified as a novel species of the genus Oceanobacillus, for which the name Oceanobacillus manasiensis sp. nov. has been proposed, with the type strain as YD3-56(T) (=CGMCC 1.9105(T) =NBRC 105903(T)).

Terribacillus aidingensis sp. nov., a moderately halophilic bacterium.

Three Gram-positive, moderately halophilic bacteria, designated YI7-61(T), IA7 and DB2, were isolated from sediments of Aiding salt lake in the Xinjiang region of China. Cells of the strains were rod-shaped, motile by means of peritrichous flagella and produced ellipsoidal spores. Colonies were pale yellow in colour. The strains grew optimally at 30-37 °C, pH 6-7 and 3-7 % (w/v) NaCl. The diamino acid in the murein was meso-diaminopimelic acid and the major quinone system was MK-7. The major cellular fatty acids were anteiso-C(15 : 0) and anteiso-C(17 : 0). The DNA G+C content was 44.6-45.0 mol%. 16S rRNA gene sequence analysis revealed that strains YI7-61(T), IA7 and DB2 were closely related to members of the genus Terribacillus and showed 96.8-97.6, 96.4-97.2 and 95.4-95.5 % 16S rRNA gene sequence similarity with Terribacillus halophilus 002-051(T), Terribacillus saccharophilus RB589 and Terribacillus goriensis CL-GR16(T), respectively. DNA-DNA relatedness among the isolates was 88-92 % and strain YI7-61(T) shared 24, 18 and 18 % DNA-DNA relatedness with T. halophilus JCM 21760(T), T. saccharophilus JCM 21759(T) and T. goriensis DSM 18252(T), respectively. On the basis of phenotypic and phylogenetic distinctiveness, the three isolates should be placed in the genus Terribacillus as representatives of a novel species, for which the name Terribacillus aidingensis sp. nov. is proposed. The type strain is YI7-61(T) (=CGMCC 1.8913(T) =NBRC 105790(T)).

Cloning and characterization of a Na+/H+ antiporter gene of the moderately halophilic bacterium Halobacillus aidingensis AD-6T.

A gene encoding a Na(+)/H(+) antiporter was obtained from the genome of Halobacillus aidingensis AD-6(T), which was sequenced and designated as nhaH. The deduced amino acid sequence of the gene was 91% identical to the NhaH of H. dabanensis, and shared 54% identity with the NhaG of Bacillus subtilis. The cloned gene enable the Escherichia coli KNabc cell, which lack all of the major Na(+)/H(+) antiporters, to grow in medium containing 0.2 M NaCl or 10 mM LiCl. The nhaH gene was predicted to encode a 43.5 kDa protein (403 amino acid residues) with 11 putative transmembrane regions. Everted membrane vesicles prepared from E. coli KNabc cells carrying NhaH exhibited Na(+)/H(+) as well as Li(+)/H(+) antiporter activity, which was pH-dependent with the highest activity at pH 8.0, and no K(+)/H(+) antiporter activity was detected. The deletion of hydrophilic C-terminal amino acid residues showed that the short C-terminal tail was vital for Na(+)/H(+) antiporter activity.

Cloning and characterization of the gene cluster for biosynthesis of ectoine from Nesterenkonia halobia DSM 20541.

The ectABC genes encoding the biosynthesis of ectoine were identified from Nesterenkonia halobia DSM 20541. The intergenic regions of the ectABC genes from N. halobia DSM 20541 were more loosely spaced than those that had been reported before. The amino acid sequence deduced from ectABC of the strain was highly homologous to the EctABC of Brevibacterium linens BL2 (EctA 50%, EctB 70%, and EctC 68% identities). The osmoprotection of ectABC was studied in the Escherichia coli KNabc and E. coli XL1-Blue. The results revealed that ectABC could shorten the lag phase and enhance the final OD600 of E. coli XL1-Blue in MM63 medium containing 0.68 M NaCl, and could initiate KNabc growth in 0.2 M NaCl. Ectoine was proven to be accumulated in E. coli KNabc/pGEM-Nect using HPLC-UV, and validated by LC-MSD-Trap-VL.

Characterization of the glycine betaine biosynthetic genes in the moderately halophilic bacterium Halobacillus dabanensis D-8(T).

The gene cluster involved in the choline-glycine betaine conversion pathway was cloned from chromosomal DNA of the Gram-positive moderate halophile Halobacillus dabanensis D-8(T). Nucleotide sequence analysis revealed four genes, designated gbsT, gbsI, gbsA, and gbsB, which are clustered in a 5.1-kb fragment. After heterologous expression of gbsAB in the Escherichia coli mutant strain PD141, the transformed cells were able to grow in a selective M63 medium containing 0.7 M NaCl and 1 mM choline, in contrast to the mutant strain. Glycine betaine biosynthesis was restored and its accumulation was confirmed by using (13)C nuclear magnetic resonance spectroscopy.

[Study progress on compatible solutes in moderately halophilic bacteria].

Moderately halophilic bacteria which grow best in media with 3% to 15% salt constitute a heterogenous group of microorganisms which belong to different genera. These bacteria can inhabit the salt or soda lakes, coastal lagoons or man-made salterns. Moderately halophilc bacteria living in higher saline environments can not only cope with high osmotic stress but also adapt osmotic shock in short time. To adapt to these environments, all the species make a osmoprotection by the accumulation a restricted range of low molecular mass molecules, small, organic compatible solutes, such as sugars, amino acids, betaines and ectoines. Therefore, the osmoadaptation of moderately halophilc bacteria is regulated by the so-called "compatible solute" strategy. Compatible solutes are operationally defined as organic osmolytes that can be amassed by the cell in exceedingly high concentrations without disturbing vital cellular functions and the correct folding of proteins. As a result, compatible solutes can make important contributions to the restoration of the turgor under conditions of low water activity by counteracting the efflux of water from the cell. In addition, they have a stabilizing, both in vivo and vitro, on the native structure of proteins and cell components. This mechanism has a minimal requirement for genetic change and a high degree of flexibility in allowing moderate halophiles to adapt to saline environment. In this review, the adaptation to saline environments, the variety and characteristic of compatible solutes, and the functional mechanism of moderately halophilic bacteria are reviewed and discussed.

[Biosynthesis of polyhydroxyalkanoates and its metabolic pathway in Comamonas sp. strain CNB-1].

Comamonas sp. strain CNB-1 degrades chloronitrbenzene and nitrobenzene for carbon and nitrogen sources. In this study, accumulation of polyhydroxyalkanoic acids (PHAs) within strain CNB-1 cells was investigated under various conditions. Results indicated that strain CNB-1 was able to synthesize PHA from various short-chain fatty acid and alcohols, and 57 w% of the dry cell weight (DCW) PHA was obtained when valerate and 1,4-butanediol were co-fed. Supplements of short-chain alcohols stimulated the accumulation of PHAs, and this stimulatory effect was attributed to the more amount of reductant generated from alcohol dehydrogenation. The genes encoding for PHA polymerase (phaC), for acetoacetyl-CoA thiolase (phaA), and acetoacetyl-CoA reductase (phaB) were cloned in Escherichia coli, and the recombinant E. coli synthesized PHA and showed enzymatic activities of PHA polymerase, acetoacetyl-CoA thiolase, and acetoacetyl-CoA reductase. The three genes occurred as a cluster of pha(C-A-B). To optimize their expression, the three genes were cloned to the pET vector and expressed respectively. Mass of expressed protein was detected and the enzyme activities increased greatly in contrast to wild CNB-1 strain, which is about 4.1, 71, and 2882 folds of activities of CNB-1.

Wenxinia marina gen. nov., sp. nov., a novel member of the Roseobacter clade isolated from oilfield sediments of the South China Sea.

An aerobic and heterotrophic, Gram-negative bacterial isolate, strain HY34(T), was isolated from sediment of an oilfield in the South China Sea, China. The taxonomy of strain HY34(T) was studied by phenotypic and phylogenetic methods. Strain HY34(T) formed faint-pink colonies on marine agar 2216. Cells of strain HY34(T) were non-motile, ovoid or short rods. Strain HY34(T) was positive for catalase and oxidase, and nitrate was reduced to nitrite. The nearly complete 16S rRNA gene sequence of strain HY34(T) was obtained and sequence analysis showed that it, together with the genus Rubellimicrobium, formed a distinct clade close to some members of the Roseobacter clade in the family Rhodobacteraceae, and it showed highest sequence similarities to Oceanicola granulosus HTCC2516(T) (93.8 %), Silicibacter lacuscaerulensis ITI-1157(T) (93.3 %), Dinoroseobacter shibae DFL 12(T) (93.3 %) and Rubellimicrobium thermophilum C-lvk-R2A-2(T) (92.2 %). Bacteriochlorophyll a was not detected. The ubiquinone system was Q-10. The major polar lipids were phosphatidylglycerol, phosphatidylcholine and an unidentified glycolipid. The major fatty acids (>10 %) were C(18 : 1) omega 7c and C(16 : 0). The DNA G+C content of this strain was 69.4 mol%. A polyphasic analysis supported the conclusion that this strain represents a novel genus and species, which we designated Wenxinia marina gen. nov., sp. nov. The type strain of Wenxinia marina is HY34(T) (=CGMCC 1.6105(T) =JCM 14017(T)).

Salegentibacter catena sp. nov., isolated from sediment of the South China Sea, and emended description of the genus Salegentibacter.

A novel marine bacterial strain, HY1T, was isolated from sediment of the South China Sea. The strain was aerobic and heterotrophic and formed saffron yellow-pigmented colonies on marine agar 2216. Cells were non-motile, Gram-negative rods, frequently occurring in chains. BLASTN searches revealed that the 16S rRNA gene sequence of strain HY1T showed high similarity with those of members of the genera Gillisia (91.7-93.8 %) and Salegentibacter (92.6-93.5 %). Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain clustered with members of both Salegentibacter and Gillisia and phylogenetic trees constructed using three different methods (neighbour-joining, maximum-parsimony and minimum-evolution) indicated that strain HY1T clustered more frequently with members of the genus Salegentibacter. The DNA G+C content of strain HY1T was 44.4 mol% and its major cellular fatty acids (>or=5 % of the total fatty acids) were iso-15 : 1 (5.0 %), iso-15 : 0 (6.8 %), anteiso-15 : 0 (6.4 %), 15 : 0 (10.4 %), iso-16 : 0 (13.5 %), summed feature 3 (comprising iso-15 : 0 2-OH and/or 16 : 1omega7c; 6.3 %), iso-17 : 0 3-OH (5.2 %) and 17 : 0 2-OH (5.0 %). Cells contained menaquinone 6. Based on the phylogenetic and phenotypic analyses, strain HY1T should be classified as representing a novel species within the genus Salegentibacter, for which the name Salegentibacter catena sp. nov. is proposed. The type strain is HY1T (=CGMCC 1.6101T=JCM 14015T). Based on this study and on previously described Salegentibacter species, an emended description of the genus Salegentibacter is given.

Analysis of protein expression profiles of Halobacillus dabanensis D-8T under optimal and high salinity conditions.

Two-dimensional (2-D) gel electrophoresis was employed to display the expression profiles of proteins of Halobacillus dabanensis D-8(T) under 1%, 10%, and 20% salinities. Approximately 700 protein spots could be detected in the 2-D gels by Imagemastertrade mark 2D Platinum software. The molecular masses of the majority of intracellular proteins were distributed in the range of 17.5 kDa-66 kDa and isoelectric points of 4.0-5.9. In total 133 protein spots were observed with a changed expression level under different salinity conditions. Sixty-two protein spots showed upregulation and 26 new protein spots were found under high salinity conditions, while 25 protein spots were downregulated and 20 spots disappeared. Twenty-seven proteins with a markedly changed expression in hypersaline environments were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF/MS) and MASCOT. A changed expression pattern was observed for proteins related to energy-producing pathways, stress regulators, and proteins involved in the survival of strain D-8(T) under high salt challenges. Many proteins play necessary roles in the adaptation to high salt or as a general stress protein, and some proteins are salt-stressed specific proteins that improve the capability of salt-tolerance of strain D-8(T) growth under extremely hypersaline condition.

[Sodium ion transportation system and its possible mechanisms in bacteria].

Sodium ion with high concentration is toxic to living cells, and microorganisms adapt to the environment containing high concentration of salt by the strategies of salt-in-cytoplasm and compatible solutes. The Na+ extrusion system plays important roles in maintaining cytoplasmic Na+ homeostasis and pH level in microbial cells. Two possible mechanisms of Na+ circulation across the cytoplasmic membrane have been proposed, namely primary Na+ pump and secondary Na+/H+ antiporter. Primary sodium pumps coupled the extrusion of Na+ to respiration, and the activity of which was insensitive to uncoupler CCCP ( carbonyl-cyanide m-chlorophenylhydrazone). There were two types of secondary Na+/H+ antiporters-encoding genes designated single gene and multiple subunits, respectively. The types of transportation systems for Na+, possible mechanisms of Na+ extrusion, and projects for further study in bacteria are reviewed.

[Two-dimensional gel electrophoresis analysis of moderately halophilic bacterium Halobacillus dabanensis D-8T under hypoosmotic shock condition].

Halobacillus dabanensis D-8T was isolated from the saline deposits of Daban lake in Xinjiang of China, and is able to grow in complex medium containing 0.5% to 25% salt. To figure out the survival mechanisms of Gram-positive moderately halophilic bacteria under hypoosmotic shock conditions, two-dimensional gel electrophoresis (2-DE) was carried out to investigate differential protein expression profiles of H. dabanensis D-8T in response to low osmotic challenge. The 2-D gels were stored as dry gels and their images were taken by ImageScanner and analyzed by Imagemaster 2D Platinum software. About 650 protein spots were detected in 2-D gel. Most of proteins were distributed in molecular mass of 17.5 - 66kDa and the range of isoelectric point 4.0 - 5.9. A total of 34 protein spots were found to alter their expression after strain D-8T was subjected to hypoosmotic shock from 20% to 0% salinity for 5 min and 50 min. Among them, the expression of 20 protein spots is up-regulated including 6 new protein spots, while that of 14 protein spots is down-regulated in answer to sudden osmotic down-shift. Protein spots of interest were excised from the gels and digested by trypsin. By means of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) and MASCOT search engine, 4 up-regulated protein spots were identified with peptide mass fingerprint, and are similar to heat shock protein DanK, rod shape-determining protein, penicillin-binding protein (PBP-1A) and 5-enolpyruvoylshikimate-3-phosphate synthase, respectively. Noticeably, PBP-1A firstly was up-regulated after shock of 5 min but disappeared after shock of 50 min. This indicated that the strain activate a minor mechanism of peptidoglycan synthesis to compensate the major synthesis mechanism for cells survival through a down-shift challenge. In addition, this paper was the first report that heat shock proteins were up-regulated in response to sudden osmotic down-shift.

Cyclobacterium lianum sp. nov., a marine bacterium isolated from sediment of an oilfield in the South China Sea, and emended description of the genus Cyclobacterium.

The marine bacterial strain HY9(T) was isolated from sediment from the South China Sea. Strain HY9(T) is aerobic, heterotrophic and rose-pigmented. The cells are non-motile and curved, i.e. ring-like or horseshoe-shaped. The 16S rRNA gene sequence of strain HY9(T) was determined and blast searches revealed that it possessed significant sequence similarities with respect to Cyclobacterium species (92.8-93.6 %). Phylogenetic analysis confirmed that strain HY9(T) was tightly clustered with members of the genus Cyclobacterium. The cellular morphology and chemotaxonomic and phenotypic properties of strain HY9(T) showed that it should be classified as a member of the genus Cyclobacterium. Significant evolutionary distances and a range of phenotypic features distinguished strain HY9(T) from previously described Cyclobacterium species. Hence, strain HY9(T) represents a novel species in the genus Cyclobacterium, for which the name Cyclobacterium lianum sp. nov. is proposed. The type strain is HY9(T) (=CGMCC 1.6102(T)=JCM 14011(T)). On the basis of this study and previously described properties of Cyclobacterium species, an emended description of the genus Cyclobacterium is proposed.

Protein expression analysis of Halobacillus dabanensis D-8T subjected to salt shock.

To investigate the mechanism of salt tolerance of gram-positive moderately halophilic bacteria, two-dimensional gel electrophoresis (2-D PAGE) was employed to achieve high resolution maps of proteins of Halobacillus dabanensis D-8T. Approximately 700 spots of proteins were identified from these 2-D PAGE maps. The majority of these proteins had molecular weights between 17.5 and 66 kDa, and most of them were distributed between the isoelectric points (pI) 4.0 and 5.9. Some protein spots were distributed in the more acidic region of the 2-D gel (pI <4.0). This pattern indicated that a number of proteins in the strain D-8T are acidic. To understand the adaptation mechanisms of moderately halophilic bacteria in response to sudden environmental changes, differential protein profiles of this strain were investigated by 2-D PAGE and Imagemaster 2D Platinum software after the cells were subjected to salt shock of 1 to 25% salinity for 5 and 50 min. Analysis showed 59 proteins with an altered level of expression as the result of the exposure to salt shock. Eighteen proteins had increased expression, 8 proteins were induced, and the expression of 33 proteins was down-regulated. Eight of the up-regulated proteins were identified using MALDI-TOF/MS and MASCOT, and were similar to proteins involved in signal transduction, proteins participating in energy metabolism pathways and proteins involved in stress.

A primary sodium pump gene of the moderate halophile Halobacillus dabanensis exhibits secondary antiporter properties.

The primary sodium pump has been proved to be involved in Na(+) extrusion of bacteria. In our present study, a novel gene encoding a putative primary sodium pump was cloned from chromosomal DNA of moderate halophile Halobacillus dabanensis D-8 by functional complementation, which expression resulted in the growth of antiporter-deficient Escherichia coli strain KNabc in the presence of 0.2 M NaCl. The gene was sequenced and designated nap. The deduced amino acid sequence of Nap has 56% identity to NADH dehydrogenase of Bacillus cereus and 55% to NADH oxidase of Bacillus halodurans C-125. E. coli KNabc carrying nap exhibited resistance to uncoupler CCCP (carbonyl-cyanide m-chlorophenylhydrazone). Everted membrane vesicles prepared from E. coli KNabc carrying nap exhibited secondary Na(+)/H(+) antiporter activity, and nap also supported the growth of respiratory-deficient E. coli ANN0222 lacking NADH dehydrogenase. Based on these results, we proposed that Nap possessed both characteristics of secondary Na(+)/H(+) antiporter and primary sodium pump.

A Na+/H+ antiporter gene of the moderately halophilic bacterium Halobacillus dabanensis D-8T: cloning and molecular characterization.

A gene encoding a Na(+)/H(+) antiporter was cloned from a chromosomal DNA of Halobacillus dabanensis strain D-8(T) by functional complementation. Its presence enabled the antiporter-deficient Escherichia coli strain KNabc to survive in the presence of 0.2 M NaCl or 5 mM LiCl. The gene was sequenced and designated as nhaH. The deduced amino-acid sequence of NhaH consists of 403 residues with a calculated molecular mass of 43,481 Da, which was 54% identical and 76% similar to the NhaG Na(+)/H(+) antiporter of Bacillus subtilis. The hydropathy profile was characteristic of a membrane protein with 12 putative transmembrane domains. Everted membrane vesicles prepared from E. coli cells carrying nhaH exhibited Na(+)/H(+) as well as Li(+)/H(+) antiporter activity, which was pH-dependent with highest activities at pH 8.5-9.0 and at pH 8.5, respectively. Moreover, nhaH confers upon E. coli KNabc cells the ability to grow under alkaline conditions.

[Transcriptional regulation of noeAB from Sinorhizobium meliloti 042BM].

The expression regulation of S. meliloti 042BM noeAB was studied. The results showed that trigonelline could not elevate the level of noeAB expression, which indicated that these genes are not regulated by nodD2. Since association of nodD3 and syrM could not change the level of the genes expression, they aren't also controlled by nodD3-syrM system. However, induction of luteolin resulted in 16 times increase of noeAB expression, which indicated that noeAB was regulated by nodD1. Most interestingly, more than 30 times increase in its expression was observed on TY medium without any flavonoid. Thus, it was suggested that noeAB may be controlled by other unknown factors.

[Construction of the genomic library of Halobacillus sp. D8 and isolation of the glycine betaine transporter betH gene].

Halobacillus sp. D8 is a sporing-forming, gram-positive moderately halophilic bacterium which could tolerate up to 25% (W/V) NaCl. A genomic library of Halobacillus sp. D8 was constructed using pUC18 as vector, and 9000 recombinant plasmids were obtained. By dot blot hybridization, colony PCR and DNA sequencing, the entire glycine betaine transporter betH gene was isolated from the constructed library. Inspection of the sequenced 4.3 kb DNA region revealed the presence of three ORFs. The putative ORF of betH is 1515bp long, encoding a 505-residue protein (BetH) with a calculated molecular mass of 56.1kD. Hydrophobicity plot analysis of BetH indicated a transmembrane protein containing 12 transmembrane regions. Homology searches for BetH of strain D8 in the GenBank using the BLAST program revealed significant sequence identities to other glycine betaine transporters: the putative glycine betaine transporter of O. iheyensis (64% identity), OpuD of B. subtilis (51% identity), BetH of H. trueperi (49% identity), BetL of L. monocytogenes (48% identity), BetM of M. halophilus (43% identity) and the putative glycine betaine transporter of B. halodurans (44% identity).