Halobacillus Dabanensis Research Articles

Halobacillus rhizosphaerae sp. nov., a moderately halophilic bacterium with protease activities isolated from the rhizosphere soil of the mangrove Acanthus ebracteatus.

A Gram-positive, moderately halophilic, rod-shaped and facultatively anaerobic strain T66T, was isolated from the rhizosphere soil of mangrove Acanthus ebracteatus in Guangxi, China. Strain T66T was observed to grow at 15-40℃ (optimum 30℃), pH 5-8 (optimum 6-7) and with 2-20% (w/v) NaCl (optimum 6%). The phylogenetic tree of the 16S rRNA gene sequences depicted a cluster of strain T66T to be closely related to Halobacillus litoralis SL-4T (98.72%), Halobacillus dabanensis D-8T (98.70%) and Halobacillus campisalis ASL-17T (98.66%). The phylogenetic analysis based on the 16S rRNA gene sequence showed that strain T66T belonged to the genus Halobacillus and formed a separate branch. The digital DNA-DNA hybridisation and average nucleotide identify values between strain T66T and its related species were 17.80-19.80% and 70.78-73.22%, respectively, which were lower than the threshold recommended for species delineation. The genomic DNA G + C content was 40.9%. The predominant isoprenoid quinone was MK-7, the cell-wall peptidoglycan contained meso-diaminopimelic acid, and the major fatty acids that accounted for more than 10.0% were anteiso-C15:0 (68.4%) and anteiso-C17:0 (13.4%). Phosphatidylglycerol, diphosphatidylglycerol, three unidentified phospholipids and an unidentified glycolipid were found in the polar lipid extraction. Strain T66T could hydrolyse casein and contains several genes (e. g. vanY, vpr, pbpG and elpC) involved in protease. Therefore, strain T66T is salt-tolerant and produces protease, and it may have development and utilisation value in flavouring agents and food curing. Based on phenotypic, chemotaxonomic data and genotypic characteristics, strain T66T represents a novel species of the genus Halobacillus, for which the name Halobacillus rhizosphaerae sp. nov. is proposed. The type strain is T66T (= JCM 36534T = MCCC 1K08701T).

Isolation, molecular characterization and extracellular enzymatic activity of culturable halophilic bacteria from hypersaline natural habitats

Bin-Salman SA, Amasha RH, Jastaniah SD, Aly MM, Altaif K. 2018. Isolation, molecular characterization and extracellular enzymatic activity of culturable halophilic bacteria from hypersaline natural habitats. Biodiversitas 19: 1828-1834. Saline habitats, like the Dead Sea, are unusual extreme environments, due to their extreme salinity. Most saline habitats originate from the evaporation of seawater, and have a nearly neutral to slightly alkaline pH (such as the Red Sea (pH8.3) and Arabian Gulf, pH8.3). Ten halophilic bacterial strains (two Gram-negative) belonging to the family of Halomonadaceae and (eight Gram-positive), belonging to the family of Bacillaceae, were isolated from the Red Sea, Arabian Gulf, and Dead Sea by subjecting the isolates to a high salinity medium, followed by identification using 16S rRNA gene sequencing. Four of isolates were designated on the basis of their tolerance to high salinity; SBR1 exhibited 97% homology to Halomonas aquamarina, SBR2 showed 97% homology to Sediminibacillus sp., (Red Sea), SBA9 exhibited 94% homology to Halobacillus sp., (Arabian Gulf) and SBD17 gave 98% homology to Halobacillus dabanensis (Dead Sea). The isolates were also characterized by their physiological parameters, SBR1 showed optimum growth at 30°C, pH8.5 and1.5M NaCl, SBR2 at 30°C, pH6.0 and 1M NaCl. Optimum conditions for SBA9 were 35°C, pH6.5 and 1M NaCl and for SBD17, 37°C, pH7.0 and 1M NaCl.

Bacteria endemic to saline coastal belt and their ability to mitigate the effects of salt stress on rice growth and yields

Increase in soil salinity adversely affects the metabolism and lowers the yield of rice (Oryza sativa L). Application of plant growth-promoting rhizobacteria (PGPR) to ameliorate the effects of salt stress on sensitive rice can be both effective and sustainable. In this study, 20 bacterial strains were isolated from the soil of saline-prone regions of Satkhira, north of the Sundarbans in coastal Bangladesh. Three bacteria among these grew well in the presence of 3 M salt (NaCl) and were Gram positive and non-motile. Their 16S rRNA sequence revealed that they belong to the Halobacillus genus. Two of them were identified as Halobacillus dabanensis strain SB-26 and the other one as Halobacillus sp. GSP 34. A couple of mechanisms by which these microbes could play beneficial role if associated with plants, such as nitrogen fixation and indole acetic acid (IAA) production, were identified. The two bacterial strains showed positive results for nitrogen fixation and indole acetic acid (IAA) activity under salt stress. Their effect on the physiology and yield of a farmer popular but sensitive BRRI dhan 28 rice variety was investigated under both control and salt stress. At the seedling stage, inoculated plants had significantly greater root length, shoot height, total weight, chlorophyll content, but lower electrolyte leakage both in control and salt stress (0, 40, and 80 mM). Performance of the plants was even better when both bacteria were used in combination. At the reproductive stage, the plants also showed better phenology in presence of the inoculated bacteria. Under stress (50 mM NaCl), these plants showed significantly greater plant height, lower spikelet damage, and yield reduction compared to untreated plants. The identified Halobacillus strains can therefore be used to improve the yield of rice by exploiting their plant growth promotion activities in coastal areas affected by moderate salinity, such as those with an ionic conductivity of up to 5 dS m−1.

Removal of BOD5 and COD From Saline Wastewater Using Fixed Bed Column of Aspergillus Oryzae and Halobacillus Dabanensis

5-day BOD and COD can be removed by biological aerobic treatment of saline wastewater. In this research, halophilic microorganisms, namely Aspergillus oryzae and Halobacillus dabanensis were isolated from a return sludge basin of a wastewater treatment plant in the City of Bandar Abbas in southern Iran , that contained a Total Dissolved Solid (TDS) of about 7500 mg l-1. These microorganisms (bacteria and fungi) could tolerate 20% concentration of salt (NaCl) in Sabouraud-4% dextrose agar and Sabouraud-2% dextrose broth medium and brain heart (BHI) agar and BHI broth medium. The films of Aspergillus oryzae and Halobacillus dabanensis were formed around the Ca-alginate. These bioflims were introduced to a fixed bed column, on top of which saline wastewater was released with flow rates of 2-6 ml min-1. According to the results of Stover-Kincannon model, the constant values of maximum BOD and COD were estimated at 0.066 mg BOD5 l-1min-1 and 0.1449 mg COD l-1 min-1, respectively. The saturation constant values, at the flow rate of 2ml min-1, by Aspegillus oryzae were 0.00003 mg BOD5 l-1min-1 and 0.00038 mg COD l-1min-1. The removal process in fixed bed column was stopped after 1200 minutes.

Halobacillus sediminis sp. nov., a moderately halophilic bacterium isolated from a solar saltern sediment.

A Gram-staining-positive, moderately halophilic bacterium, designated strain NGS-2T, was isolated from sediment of a solar saltern pond located in Shinan, Korea. Strain NGS-2T was a strictly aerobic, non-motile rod that grew at pH 5.0-10.0 (optimum, pH 8.0), at 10-30 °C (optimum, 28 °C) and in the presence of 1-20 % (w/v) NaCl (optimum, 10 % NaCl). Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain NGS-2T belonged to the genus Halobacillus, with sequence similarity of 98.4-95.8 % to existing type strains, showing the highest sequence similarity to Halobacillus dabanensis D-8T (98.4 %), H. litoralis SL-4T (98.4 %), H. trueperi SL-5T (98.2 %), H. faecis IGA7-4T (98.2 %), H. profundi IS-Hb4T (98.1 %) and H. mangrovi MS10T (98.0 %). The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidyl-N-methylethanolamine and an unknown glycolipid. The cell-wall peptidoglycan was based on l-Orn-d-Asp, the predominant isoprenoid quinone was menaquinone 7 (MK-7) and the major fatty acids were anteiso-C15: 0 and anteiso-C17: 0. The DNA G+C content of the novel isolate was 45.0 mol%. Levels of DNA-DNA relatedness between strain NGS-2T and the type strains of 12 other species of the genus ranged from 32 to 3 %. On the basis of the polyphasic analysis conducted in this study, strain NGS-2T represents a novel species of the genus Halobacillus, for which the name Halobacillus sediminis sp. nov. is proposed. The type strain is NGS-2T ( = KACC 18263T = NBRC 110639T).

Characterization of biosurfactants produced by Halobacillus dabanensis and Pontibacillus chungwhensi isolated from oil-contaminated mangrove ecosystem in Egypt

The production and properties of biosurfactants synthesized by Halobacillus dabanensis and Pontibacillus chungwhensi, isolated from oil-contaminated mangrove sediments of Avicennia marina, Red Sea (Egypt) have been investigated. The two strains were classified as moderate halophile and halotolerant, respectively. The produced biosurfactants were considered as metabolites associated with the growth of H. dabanensis and P. chungwhensi and were able to reduce the interfacial tension to 20 and 19 mN/m, respectively. The partially purified biosurfactants showed high emulsiflcation value (E24) against n-hexadecane (68 and 72%, respectively). Biological degradation of n-alkane mixture and linear alkyl benzene after incubation of bacteria in Buslinell-Haas medium in presence and absence of the partially purified biosurfactants has been monitored. H. dabanensis and P. chungwhensi were capable of degrading 92.3 and 94.7% of n- alkane mixture and 76.5 and 76.7% of linear alkyl benzene, respectively. The biodegradation of n-alkane, achieved in the presence of biosurfactants was better than its degradation in control. The structural analysis of the biosurfactants proved them to be lipopeptides. Biosurfactant-producing halophilic/halotolerant microorganisms may play a significant role in accelerating remediation of oil-polluted saline environments.

Identification of important charged residues for alkali cation exchange or pH regulation of NhaH, a Na+/H+ antiporter of Halobacillus dabanensis

NhaH is a novel Na+/H+ antiporter identified from the moderate halophile Halobacillus dabanensis. In this study, six conserved charged residues located in the putative transmembrane segments (TMS) including TMSV, TMSVI, TMSVIII and TMSXI of NhaH as well as two His residues in Loop III were replaced by site-directed mutagenesis for the identification of their potential roles in the antiport activity and pH regulation. Substitutions D137A, D166A and R325A caused a complete loss of Na+(Li+)/H+ antiport activity, revealing that D137, D166 and R325 are indispensable for the antiport activity. Substitution D137E led to a significant increase of the apparent Km values for Na+ and Li+ without affecting the changes of pH profile, confirming that D137 plays vital roles in alkali cation binding/translocation. Substitution D166E resulted in not only a significant increase of the apparent Km values for Na+ and Li+ but also an alkaline shift of pH profile, suggesting that D166 is involved in alkali cation binding/translocation as well as H+ binding or pH regulation. Substitutions E161N, D224A and D224E caused a significant increase of Km for Na+ and Li+, indicating that E161 and D224 partly contribute to alkali cation binding/translocation. Substitution E229K caused an over 50% elevation of the apparent Km for Li+, without affecting that for Na+, suggesting that E229 may be mainly responsible for Li+ binding/translocation. Substitutions H87A and H88A resulted in an acidic shift of pH profile without an effect on Km for Na+ and Li+, indicating that H87 and H88 are involved in H+ binding or pH regulation.

Screening a novel Na+/H+antiporter gene from a metagenomic library of halophiles colonizing in the Dagong Ancient Brine Well in China

Metagenomic DNA libraries constructed from the Dagong Ancient Brine Well were screened for genes with Na(+)/H(+) antiporter activity on the antiporter-deficient Escherichia coli KNabc strain. One clone with a stable Na(+)-resistant phenotype was obtained and its Na(+)/H(+) antiporter gene was sequenced and designated as m-nha. The deduced amino acid sequence of M-Nha protein consists of 523 residues with a calculated molecular weight of 58 147 Da and a pI of 5.50, which is homologous with NhaH from Halobacillus dabanensis D-8(T) (92%) and Halobacillus aidingensis AD-6(T) (86%), and with Nhe2 from Bacillus sp. NRRL B-14911 (64%). It had a hydropathy profile with 10 putative transmembrane domains and a long carboxyl terminal hydrophilic tail of 140 amino acid residues, similar to Nhap from Synechocystis sp. and Aphanothece halophytica, as well as NhaG from Bacillus subtilis. The m-nha gene in the antiporter-negative mutant E. coli KNabc conferred resistance to Na(+) and the ability to grow under alkaline conditions. The difference in amino acid sequence and the putative secondary structure suggested that the m-nha isolated from the Dagong Ancient Brine Well in this study was a novel Na(+)/H(+) antiporter gene.

Erratum to: Characterization of the Glycine Betaine Biosynthetic Genes in the Moderately Halophilic Bacterium Halobacillus dabanensis D-8T

Erratum to: Characterization of the Glycine Betaine Biosynthetic Genes in the Moderately Halophilic Bacterium Halobacillus dabanensis D-8T

The short C-terminal hydrophilic domain of NhaH Na+/H+ antiporter from Halobacillus dabanensis with roles in resistance to salt and in pH sensing

The Na+/H+ antiporter plays key roles in maintaining low cytoplasmic Na+ level and pH homeostasis, while little is known about the Carboxyl-terminal hydrophilic tails of prokaryotic antiporters. In our previous study, the first Na+/H+ antiporter gene nhaH from moderate halophiles was cloned from Halobacillus dabanensis D-8 by functional complementation. A topological model suggested that only nine amino acid residues (395PLIKKLGMI403) existed in the hydrophilic C-terminal domain of NhaH. The C-terminal truncated mutant of NhaH was constructed by PCR strategy and designated as nhaHΔC. Salt tolerance experiment demonstrated that the deletion of hydrophilic C-terminal nine amino acid residues significantly inhibited the complementation ability of E. coli KNabc, in which three main Na+/H+ antiporters nhaA, nhaB and chaA were deleted. Everted membrane vesicles prepared from E. coli KNabc/nhaHΔC decreased both Na+/H+ and Li+/H+ exchange activities of NhaH, and also resulted in an acidic shift of its pH profile for Na+, indicating a critical role of the short C-terminal domain of NhaH antiporter in alkali cation binding/translocation and pH sensing.

Characterization of the Glycine Betaine Biosynthetic Genes in the Moderately Halophilic Bacterium Halobacillus dabanensis D-8T

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.

Purification of two putative type II NADH dehydrogenases with different substrate specificities from alkaliphilic Bacillus pseudofirmus OF4

A putative Type II NADH dehydrogenase from Halobacillus dabanensis was recently reported to have Na +/H + antiport activity (and called Nap), raising the possibility of direct coupling of respiration to antiport-dependent pH homeostasis. This study characterized a homologous type II NADH dehydrogenase of genetically tractable alkaliphilic Bacillus pseudofirmus OF4, in which evidence supports antiport-based pH homeostasis that is mediated entirely by secondary antiport. Two candidate type II NADH dehydrogenase genes with canonical GXGXXG motifs were identified in a draft genome sequence of B. pseudofirmus OF4. The gene product designated NDH-2A exhibited homology to enzymes from Bacillus subtilis and Escherichia coli whereas NDH-2B exhibited homology to the H. dabanensis Nap protein and its alkaliphilic Bacillus halodurans C-125 homologue. The ndh-2A, but not the ndh-2B, gene complemented the growth defect of an NADH dehydrogenase-deficient E. coli mutant. Neither gene conferred Na +-resistance on an antiporter-deficient E. coli strain, nor did they confer Na +/H + antiport activity in vesicle assays. The purified hexa-histidine-tagged gene products were approximately 50 kDa, contained noncovalently bound FAD and oxidized NADH. They were predominantly cytoplasmic in E. coli, consonant with the absence of antiport activity. The catalytic properties of NDH-2A were more consistent with a major respiratory role than those of NDH-2B.

Halobacillus mangrovi sp. nov., a moderately halophilic bacterium isolated from the black mangrove Avicennia germinans

A moderately halophilic, spore-forming, Gram-positive, short-rod-shaped bacterium, designated strain MS10(T), was isolated from the surface of leaves of the black mangrove Avicennia germinans and was subjected to a polyphasic taxonomic study. Strain MS10(T) was able to grow at NaCl concentrations in the range 5-20% (w/v) with optimum growth at 10% (w/v) NaCl. Growth occurred at temperatures of 10-50 degrees C (optimal growth at 33-35 degrees C) and pH 6.0-9.0 (optimal growth at pH 7.0). Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain MS10(T) fell within the branch encompassing members of the genus Halobacillus and was most closely related to Halobacillus dabanensis JCM 12772(T) (99.2% 16S rRNA gene sequence similarity). The DNA G+C content of strain MS10(T) was 45.7 mol%, the major respiratory isoprenoid quinone was MK-7 and the cell-wall peptidoglycan was of the L-Orn-D-Asp type, characteristics consistent with its affiliation to the genus Halobacillus. Strain MS10(T) showed a level of DNA-DNA hybridization with H. dabanensis JCM 12772(T) of 29% and levels below 70% were also obtained with respect to other recognized members of the genus Halobacillus. The major fatty acids of strain MS10(T) were iso-C(16:0), anteiso-C(15:0), iso-C(14:0) and iso-C(15:0). Overall, the phenotypic, genotypic and phylogenetic results presented in this study demonstrate that strain MS10(T) represents a novel species of the genus Halobacillus, for which the name Halobacillus mangrovi sp. nov. is proposed. The type strain is MS10(T) (=CECT 7206(T)=CCM 7397(T)).

Halobacillus profundi sp. nov. and Halobacillus kuroshimensis sp. nov., moderately halophilic bacteria isolated from a deep-sea methane cold seep

Two Gram-positive, rod-shaped, moderately halophilic bacteria were isolated from a deep-sea carbonate rock at a methane cold seep in Kuroshima Knoll, Japan. These bacteria, strains IS-Hb4(T) and IS-Hb7(T), were spore-forming and non-motile. They were able to grow at temperatures as low as 9 degrees C and hydrostatic pressures up to 30 MPa. Based on high sequence similarity of their 16S rRNA genes to those of type strains of the genus Halobacillus, from 96.4 % (strain IS-Hb7(T) to Halobacillus halophilus NCIMB 9251(T)) to 99.4 % (strain IS-Hb4(T) to Halobacillus dabanensis D-8(T)), the strains were shown to belong to this genus. DNA-DNA relatedness values of 49.5 % and 1.0-33.0 %, respectively, were determined between strains IS-Hb4(T) and IS-Hb7(T) and between these strains and other Halobacillus type strains. Both strains showed the major menaquinone MK7 and L-orn-D-Asp cell-wall peptidoglycan type. Straight-chain C(16 : 0), unsaturated C(16 : 1)omega7c alcohol and C(18 : 1)omega7c and cyclopropane C(19 : 0) cyc fatty acids were predominant in both strains. The DNA G+C contents of IS-Hb4(T) and IS-Hb7(T) were respectively 43.3 and 42.1 mol%. Physiological and biochemical analyses combined with DNA-DNA hybridization results allowed us to place strains IS-Hb4(T) (=JCM 14154(T)=DSM 18394(T)) and IS-Hb7(T) (=JCM 14155(T)=DSM 18393(T)) in the genus Halobacillus as the respective type strains of the novel species Halobacillus profundi sp. nov. and Halobacillus kuroshimensis sp. nov.

Cloning and Characterization of the Genes for Biosynthesis of the Compatible Solute Ectoine in the Moderately Halophilic Bacterium Halobacillus dabanensis D-8T

A 11.2-kb fragment containing the ectABC genes of the biosynthetic pathway of ectoine from the Gram-positive, moderately halophilic bacterium Halobacillus dabanensis D-8(T) was obtained by inverse polymerase chain reaction. Subsequently, the entire ectABC cluster was cloned and analyzed. It revealed that the intergenic regions of the ectABC genes from H. dabanensis D-8(T) are more tightly spaced than those of Chromohalobacter salexigens, Halomonas elongata, Marinococcus halophilus, and Salibacillus pasteurii. The amino-acid sequence deduced from ectABC was highly homologous that from Virgibacillus pantethenticus (EctA 52%, EctB 60%, EctC 67%, respectively). The ectABC genes were cloned in the expression plasmid pMXB10 resulting in pMXB10ectABC. The ectoine was detected from cell extract in Escherishia coli ER2566 containing pMXB10ectABC using (13)C nuclear magnetic resonance spectroscopy.

Cloning and Characterization of the Genes for Biosynthesis of the Compatible Solute Ectoine in the Moderately Halophilic Bacterium Halobacillus dabanensis D-8T

Cloning and Characterization of the Genes for Biosynthesis of the Compatible Solute Ectoine in the Moderately Halophilic Bacterium Halobacillus dabanensis D-8T

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 bacteriumHalobacillus dabanensisD-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.

Halobacillus dabanensis sp. nov. and Halobacillus aidingensis sp. nov., isolated from salt lakes in Xinjiang, China

Two moderately halophilic spore-forming bacteria were isolated from salt lakes in the Xinjiang region of China. The two strains, designated AD-6(T) and D-8(T), were aerobic, Gram-positive, rod-shaped and motile by means of peritrichous flagella. Strains AD-6(T) and D-8(T) grew in the presence of 0.5-20% and 0.5-25% (w/v) NaCl in complex medium, respectively. Their cell-wall peptidoglycan was of the L-Orn-D-Asp type. The major menaquinone found in both strains was menaquinone-7 (MK-7). The fatty acid profile contained a large amount of branched fatty acids; the main fatty acids were anteiso-C(15:0), anteiso-C(17:0), iso-C(15:0) and iso-C(16:0). The DNA G+C content of strains D-8(T) and AD-6(T) was 41.4 and 42.2 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains D-8(T) and AD-6(T) were located in the genus Halobacillus. Levels of 16S rRNA gene sequence similarity between the isolated strains and the type strains of Halobacillus species were in the range 96.2-99.5%. DNA-DNA relatedness values of 17.0-52.2% were found between the two strains and other Halobacillus species. The DNA-DNA relatedness value between D-8(T) and AD-6(T) was 50.6%. On the basis of phenotypic and chemotaxonomic properties, phylogenetic analysis and genomic distinctiveness, strains D-8(T) and AD-6(T) should be placed in the genus Halobacillus as two novel species, for which the names Halobacillus dabanensis sp. nov. (type strain=JCM 12772(T)=CGMCC 1.3704(T)) and Halobacillus aidingensis sp. nov. (type strain=JCM 12771(T)=CGMCC 1.3703(T)) are proposed, respectively.