Reidentification of the keratinase-producing facultatively alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans

Alkaliphilic Bacillus sp. AH-101 was characterized in terms of physiological and biochemical characteristics, and 16S rDNA sequence homology and DNA–DNA hybridization analyses were performed. Phylogenetic analysis of strain AH-101 based on comparison of 16S rDNA sequences revealed that this strain is closely related to Bacillus halodurans. DNA–DNA hybridization of AH-101 and related Bacillus reference strains showed that the highest level of DNA–DNA relatedness (88%) was found between strain AH-101 and the B. halodurans type strain (DSM497). Our findings demonstrate that strain AH-101 is a member of the species B. halodurans. Received: June 10, 1999 / Accepted: August 6, 1999     

Physical map of alkaliphilic Bacillus firmus OF4 and detection of a large endogenous plasmid

Extremely alkaliphilic Bacillus firmus OF4 is among the best characterized of this group of alkaliphiles. Together with alkaliphilic Bacillus C-125 and numerous non-alkaliphilic Bacillus species whose chromosomes and gene organizations are currently being studied in detail, work on B. firmus OF4 offers the opportunity to discern whether there are features of chromosome and gene organization that are associated with alkaliphily. A physical map of the B. firmus OF4 is consistent with a circular chromosome of approximately 4 Mb, with an extrachromosomal element of 110 kb also detected. The previously identified cadmium-resistance locus and transposition functions in B. firmus OF4 were localized to the extrachromosomal element, whose genes exhibit a slightly different pattern of codon usage from chromosomal genes. No clustering of genes thus far identified with roles in alkaliphily has been found. Direct repeat sequences (DRS) were previously reported upstream of a gene encoding a Na⁺/H⁺ antiporter that has a role in pH homeostasis. In the current analyses, these sequences were found to be present in multiple copies on the chromosome, most of which are present in one 920-kb fragment. Such sequences might play a role in DNA rearrangements that allow amplification of important genes in this region. Received: March 3, 1998 / Accepted May 12, 1998     

Identification of facultatively alkaliphilic Bacillus sp. strain YN-2000 and its fatty acid composition and cell-surface aspects depending on culture pH

Facultatively alkaliphilic Bacillus sp. strain YN-2000 was isolated from an indigo ball. Although the strain has been extensively investigated as a representative strain of alkaliphilic bacillus, its taxonomic position is not yet known. Morphological, biochemical, and physiological characteristics and chemotaxonomic properties indicated that the strain was closely related to Bacillus cohnii; this was confirmed by the high homology of the 16S rRNA sequence and the construction of a phylogenetic tree on the basis of the 16S rRNA sequence and DNA–DNA relatedness data. Strain YN-2000 contained a larger amount of unsaturated fatty acids compared with Bacillus subtilis and the obligate alkaliphile, Bacillus alcalophilus, regardless of its culture pH. When the cells were grown at pH 10, the unsaturated fatty acid content and anteiso-/iso-branched fatty acid ratio became lower than those at pH 7. This result suggests that membrane fluidity decreases when the cells are grown at pH 10 compared to those of pH 7. In the cells of strain YN-2000 grown at pH 10, the cell-surface aspect was rougher, the cell shape was longer, and the cell-surface layer was thicker compared with those of the cells grown at pH 7. The cell-surface structural change might be related to adaptation to an alkaline environment. Received: April 6, 2000 / Accepted: May 8, 2000     

New species in the genus <Emphasis Type="Italic">Francisella</Emphasis> (Gammaproteobacteria; Francisellaceae); <Emphasis Type="Italic">Francisella piscicida</Emphasis> sp. nov. isolated from cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella strain, GM2212, previously isolated from moribund farmed Atlantic cod (Gadus morhua) in Norway, is closely related to Francisella philomiragia among Francisella spp. according to its complete 16S rDNA, 16S-23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and hypothetical lipoprotein (LpnB) sequences. A comparison between GM2212 and the type strain of Francisella philomiragia were performed by DNA–DNA hybridization and fatty acid analysis. The DNA–DNA hybridization showed a 70% similarity. The fatty acid analysis showed only minor differences between the Francisella isolates. Due to the inconclusive result from the DNA–DNA hybridisation, major emphasis concerning the status of this isolate is made on previously published molecular, phenotypic and biochemical characters. All characteristics taken together support the establishment of GM2212 as a novel species, for which the name Francisella piscicida sp. nov. is proposed (=CNCM I-3511^T = DSM 18777^T = LMG registration number not yet available).     

Diverse genes of alkaliphilic Bacillus firmus OF4 that complement K+-uptake-deficient Escherichia coli include an ftsH homologue

Seven clones isolated from libraries of DNA from alkaliphilic Bacillus firmus OF4 restored the growth of a K⁺-uptake-deficient Escherichia coli mutant on only 10mM K⁺. None of the clones contained genes with apparent homology to known K⁺ transport systems in other organisms. Based on sequence homologies, the newly isolated alkaliphile loci included: ftsH; a dipeptide transport system; a gerC locus with hydrophobic open reading frames not found in the comparable locus of Bacillus subtilis; a sugar phosphotransferase enzyme; and a capBC homologue. The ftsH gene provided a new and striking example of a recognized property of extracellular and external regions of polytopic alkaliphile proteins: a significant paucity of basic amino acid residues relative to neutrophile counterparts. The alkaliphile ftsH gene was able to complement a mutant of E. coli with a temperature-sensitive ftsH gene product. Received: 5 August 1996 / Accepted: 14 October 1996     

Organization and nucleotide sequence of the atp genes encoding the ATP synthase from alkaliphilic Bacillus firmus OF4

Summary The atp operon from the extreme alkaliphile Bacillus firmus OF4 was cloned and sequenced, and shown to contain genes for the eight structural subunits of the ATP synthase, preceded by a ninth gene predicted to encode a 14 kDa hydrophobic protein. The arrangement of genes is identical to that of the atp operons from Escherichia coli, Bacillus megaterium, and thermophilic Bacillus PS3. The deduced amino acid sequences of the subunits of the enzyme are also similar to their homologs in other ATP synthases, except for several unusual substitutions, particularly in the a and c subunits. These substitutions are in domains that have been implicated in the mechanism of proton translocation through F₀-ATPase, and therefore could contribute to the gating properties of the alkaliphile ATP synthase or its capacity for proton capture.     

pH homeostasis and ATP synthesis: studies of two processes that necessitate inward proton translocation in extremely alkaliphilic Bacillus species

Alkaliphilic Bacillus species that are isolated from nonmarine, moderate salt, and moderate temperature environments offer the opportunity to explore strategies that have developed for solving the energetic challenges of aerobic growth at pH values between 10 and 11. Such bacteria share many structural, metabolic, genomic, and regulatory features with nonextremophilic species such as Bacillus subtilis. Comparative studies can therefore illuminate the specific features of gene organization and special features of gene products that are homologs of those found in non-extremophiles, and potentially identify novel gene products of importance in alkaliphily. We have focused our studies on the facultative alkaliphile Bacillus firmus OF4, which is routinely grown on malate-containing medium at either pH 7.5 or 10.5. Current work is directed toward clarification of the characteristics and energetics of membrane-associated proteins that must catalyze inward proton movements. One group of such proteins are the Na⁺/H⁺ antiporters that enable cells to adapt to a sudden upward shift in pH and to maintain a cytoplasmic pH that is 2–2.3 units below the external pH in the most alkaline range of pH for growth. Another is the proton-translocating ATP synthase that catalyzes robust production of ATP under conditions in which the external proton concentration and the bulk chemiosmotic driving force are low. Three gene loci that are candidates for Na⁺/H⁺ antiporter encoding genes with roles in Na⁺- dependent pH homeostasis have been identified. All of them have homologs in B. subtilis, in which pH homeostasis can be carried out with either K⁺ or Na⁺. The physiological importance of one of the B. firmus OF4 loci, nhaC, has been studied by targeted gene disruption, and the same approach is being extended to the others. The atp genes that encode the alkaliphile's F₁F_O-ATP synthase are found to have interesting motifs in areas of putative importance for proton translocation. As an initial step in studies that will probe the importance and possible roles of these motifs, the entire atp operon from B. firmus OF4 has been cloned and functionally expressed in an Escherichia coli mutant that has a full deletion of its atp genes. The transformant does not exhibit growth on succinate, but shows reproducible, modest increases in the aerobic growth yields on glucose as well as membrane ATPase activity that exhibits characteristics of the alkaliphile enzyme. Received: January 22, 1998 / Accepted: February 16, 1998     

Sequence analysis and functional studies of a chromosomal region of alkaliphilic Bacillus firmus OF4 encoding an ABC-type transporter with similarity of sequence and Na+ exclusion capacity to the Bacillus subtilis NatAB transporter

A 14.1-kb DNA fragment was cloned from a lambda library containing inserts of DNA from alkaliphilic Bacillus firmus OF4 on the basis of its hybridization to a probe from a previously sequenced alkaliphile homolog of the natA gene from Bacillus subtilis. Sequence analysis of the entire fragment revealed that, as in B. subtilis, the natA gene was part of a putative gene locus encoding an ABC-type transporter. In the alkaliphile, the transporter involved three genes, designated natCAB, that are part of a larger operon of unknown function. This is in contrast to the two-gene natAB operon and to another homolog from B. subtilis, the yhaQP genes. Like natAB, however, the alkaliphile natCAB catalyzes Na⁺ extrusion as assessed in a mutant of Escherichia coli that is deficient in Na⁺ extrusion. The full 14.1-kb fragment of alkaliphile DNA sequenced in this study contained several probable operons as well as likely monocistronic units. Among the 17 predicted ORFs apart from natCAB were acsA, a homolog of a halobacterial gene encoding acetylCoA synthetase; sspA, a homolog of a small acid-soluble spore protein; and malK, an ATP-binding component that was unaccompanied by candidates for other mal transport genes but was able to complement a malK-deficient mutant of E. coli. No strong candidates for genes encoding a secondary Na⁺/H⁺ antiporter were found in the fragment, either from the sequence analysis or from analyses of complementation of E. coli mutants by subclones of the 14.1-kb piece. There were a total of 12 ORFs whose closest and significant homologs were genes from B. subtilis; of these, one-third were in apparently different contexts, as assessed by the sequence of the neighboring genes, than the B. subtilis homologs. Received: August 30, 1998 / Accepted: November 13, 1998     

Na<Superscript>+</Superscript> and flagella-dependent swimming of alkaliphilic <Emphasis Type="Italic">Bacillus pseudofirmus</Emphasis> 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 < ∼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.     

<Emphasis Type="Italic">Bacillus berkeleyi</Emphasis> sp. nov., isolated from the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis>

A bacterial strain, designated KMM 6244^T, was isolated from the sea urchin Strongylocentrotus intermedius and subjected to a polyphasic taxonomic investigation. The bacterium was found to be heterotrophic, aerobic, non-motile and spore-forming. Comparative phylogenetic analysis based on 16S rRNA gene sequencing placed the marine isolate in the genus Bacillus. The nearest neighbor of strain KMM 6244^T was Bacillus decolorationis LMG 19507^T with a 16S rRNA gene sequence similarity of 98.0%. Sequence similarities with the other recognized Bacillus species were less than 96.0%. The results of the DNA–DNA hybridization experiments revealed a low relatedness (37%) of the novel isolate with the type strain of B. decolorationis LMG 19507^T. Strain KMM 6244^T grew at 4–45°C and with 0–12% NaCl. It produced catalase and oxidase and hydrolyzed aesculin, casein, gelatin and DNA. The predominant fatty acids were anteiso-C_15:0, iso-C_15:0, anteiso-C_17:0, C_15:0, iso-C_16:0 and iso-C_14:0. The DNA G + C content was 39.4 mol%. A combination of phylogenetic, genotypic and phenotypic data clearly indicated that strain KMM 6244^T represents a novel species in the genus Bacillus, for which the name Bacillus berkeleyi sp. nov. is proposed. The type strain is KMM 6244^T (KCTC 12718^T = LMG 26357^T).     

Action of antimicrobial substances produced by different oil reservoir Bacillus strains against biofilm formation

Microbial colonization of petroleum industry systems takes place through the formation of biofilms, and can result in biodeterioration of the metal surfaces. In a previous study, two oil reservoir Bacillus strains (Bacillus licheniformis T6-5 and Bacillus firmus H₂O-1) were shown to produce antimicrobial substances (AMS) active against different Bacillus strains and a consortium of sulfate-reducing bacteria (SRB) on solid medium. However, neither their ability to form biofilms nor the effect of the AMS on biofilm formation was adequately addressed. Therefore, here, we report that three Bacillus strains (Bacillus pumilus LF4—used as an indicator strain, B. licheniformis T6-5, and B. firmus H₂O-1), and an oil reservoir SRB consortium (T6lab) were grown as biofilms on glass surfaces. The AMS produced by strains T6-5 and H₂O-1 prevented the formation of B. pumilus LF4 biofilm and also eliminated pre-established LF4 biofilm. In addition, the presence of AMS produced by H₂O-1 reduced the viability and attachment of the SRB consortium biofilm by an order of magnitude. Our results suggest that the AMS produced by Bacillus strains T6-5 and H₂O-1 may have a potential for pipeline-cleaning technologies to inhibit biofilm formation and consequently reduce biocorrosion.     

<Emphasis Type="Italic">Bacillus rhizosphaerae</Emphasis> sp. nov., an novel diazotrophic bacterium isolated from sugarcane rhizosphere soil

A Gram-positive, non-pigmented, rod-shaped, diazotrophic bacterial strain, designated SC-N012^T, was isolated from rhizosphere soil of sugarcane and was subjected to a polyphasic taxonomic study. The strain exhibited phenotypic properties that included chemotaxonomic characteristics consistent with its classification in the genus Bacillus. Sequence analysis of the 16S rRNA gene of SC-N012^T revealed the closest match (98.9% pair wise similarity) with Bacillus clausii DSM 8716^T. However, DNA–DNA hybridization experiments indicated low levels of genomic relatedness (32%) with this strain. The major components of the fatty acid profile are iso-C_15:0, anteiso-C_15:0, iso-C_17:0 and anteiso-C_17:0. The diagnostic cell-wall diamino acid was meso-diaminopimelic acid. The G+C content of the genomic DNA is 43.0 mol%. The lipids present in strain SC-N012^T are diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol and two unknown phospholipids. Their predominant respiratory quinone was MK-7. Studies of DNA-DNA relatedness, morphological, physiological and chemotaxonomic analyses and phylogenetic data based on 16S rRNA gene sequencing allowed strain SC-N012^T to be described as members of novel species of the genus Bacillus, for which the name Bacillus rhizosphaerae sp. nov. is proposed. The type strain is SC-N012^T (=DSM 21911^T = NCCB 100267^T).     

Production of cyclodextrin from starch by cyclodextrin glycosyltransferase from Bacillus firmus and characterization of purified enzyme

During screening for cyclodextrin-forming microorganisms, an alkalophilic Bacillus sp, which produced high activity of cyclodextrin glycosyltransferase, was isolated and identified as Bacillus firmus. The crude enzyme transformed starch to mainly β-and γ-cyclodextrin. The purified enzyme had an optimum pH of 7.5–8.5 and its optimum temperature was 65°C, which is the highest optimum temperature as compared to other cyclodextrin glycosyltransferases except that produced by Bacillus amyloliquefaciens. Received 06 January 1997/ Accepted in revised form 20 March 1997     

<Emphasis Type="Italic">Bacillus deserti</Emphasis> sp. nov., a novel bacterium isolated from the desert of Xinjiang,China

A Gram-positive, rod-shaped, motile and spore-forming bacterium, designated ZLD-8^T, was isolated from a desert soil sample collected from Xinjiang Province in north-west China, and subjected to a polyphasic taxonomic analysis. This isolate grew optimally at 30°C and pH 7.0. It grew with 0–4% NaCl (optimum, 0–1%). Comparative 16S rRNA gene sequence analysis showed that strain ZLD-8^T was closely related to members of the genus Bacillus, exhibiting the highest 16S rRNA gene sequence similarity to Bacillus kribbensis DSM 17871^T (98.0%). The levels of 16S rRNA gene sequence similarity with respect to other Bacillus species with validly published names were less than 96.3%. The DNA G + C content of strain ZLD-8^T was 40.1 mol%. The strain contained MK-7 as the predominant menaquinone. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids (>5% of total fatty acids) were anteiso-C15:0 (39.56%), iso-C14:0 (25.69%), C16:1 ω7c alcohol (10.13%) and iso-C15:0 (5.27%). These chemotaxonomic results supported the affiliation of strain ZLD-8^T to the genus Bacillus. However, low DNA–DNA relatedness values and distinguishing phenotypic characteristics allowed genotypic and phenotypic differentiation of strain ZLD-8^T from recognized Bacillus species. On the basis of the polyphasic evidence presented, strain ZLD-8^T is considered to represent a novel species of the genus Bacillus, for which the name Bacillus deserti sp. nov. is proposed. The type strain is ZLD-8^T (=CCTCC AB 207173^T = KCTC 13246^T).     

Newly Isolated Bacillus gibsonii S-2 Capable of using Sugar Beet Pulp for Alkaline Pectinase Production

Summary A Bacillus strain capable of growing under highly alkaline conditions was isolated from a sample of alkaline soil. The isolate was a Gram-positive, spore-forming, aerobic, alkaliphilic bacterium and was designated as strain S-2. Growth of the strain was observed in the pH range of 7–12 and temperature range of 4–40 °C. Sequence analysis of the 16S rDNA of the strain revealed more than 99% homology with the strains of Bacillus gibsonii. The S-2 strain was confirmed as B. gibsonii by comparing its physiological and biochemical characteristics with the B. gibsonii DSM 8722 strain. The S-2 strain could use sugar beet pulp as the carbon source as well as the pectinase inducer to produce extracellular alkaline pectinase by solid-state fermentation. The maximum polygalacturonase yield of 3600 U/g dry sugar beet pulp was obtained at 35 °C after 48 h of incubation.     

Isolation and characterization of lipid-degrading Bacillus thermoleovorans IHI-91 from an icelandic hot spring

An efficient lipid-degrading thermophilic aerobic bacterium was isolated from an icelandic hot spring and classified as Bacillus thermoleovorans IHI-91. The aerobic bacterium grows optimally at 65°C and pH 6.0 and secretes a high level of lipase (300 U l⁻¹). The newly isolated strain utilizes several lipids such as palmitic acid, stearic acid, lanolin, olive oil, sunflower seed oil, soya oil, and fish oil as sole carbon and energy source without an additional supply of growth factors. The degradation of about 93% of triolein, which is present in olive oil, was observed after only 7 h of fermentation at a maximal growth rate of 1.0 h⁻¹. During growth at optimal conditions on yeast extract, the doubling time was only 15 min. Based on 16S rDNA studies, DNA–DNA hybridization and morphological and physiological properties, the isolate IHI-91 was identified as Bacillus thermoleovorans IHI-91 sp. nov. Because of its production of high concentrations of thermoactive lipases and esterases and the capability of degrading a wide range of lipids at high temperatures, the isolated strain is an ideal candidate for application in various biotechnological processes such as wastewater treatment. Received: August 25, 2000 / Accepted: September 26, 2000     

Genetic Characterization of a New Thermotolerant Bacillus licheniformis Strain

A potentially new thermotolerant B. licheniformis strain (code name I89), producer of an antibiotic active against Gram-positive bacteria, was genetically characterized and compared with the type strain B. licheniformis ATCC 10716, producer of bacitracin. Studies on DNA base composition (G + C content) and DNA reassociation revealed that the two strains show around 76% homology. Nevertheless, results obtained by rRNA hybridization, with a heterologous probe coding for most of the 16S region of the rRNA operon of Bacillus subtilis, revealed differences in the number of copies for that gene and in the hybridization pattern. Additionally, a different restriction digestion pattern was obtained when DNA was digested with the enzymes NotI, SmaI and analyzed by PFGE. The I89 strain holds a 7.6-kb plasmid not present in the reference strain. The existence of various unique restriction sites and also the stability of this plasmid make it ideal for the future development of a cloning and expression vector. Received: 29 June 1999 / Accepted: 1 September 1999     

Molecular phylogenetic diversity of Bacillus community and its temporal–spatial distribution during the swine manure of composting

In order to obtain the diversity and temporal–spatial distribution of Bacillus community during the swine manure composting, we utilized traditional culture methods and the modern molecular biology techniques of polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) and –denaturing gradient gel electrophoresis (PCR-DGGE). Bacillus species were firstly isolated from the composting. Based on temperature changes, the temporal–spatial characteristics of total culturable Bacillus were remarkable that the number of the culturable Bacillus detected at the high-temperature stage was the highest in each layer of the pile and that detected in the middle layer was the lowest at each stage of composting respectively. The diversity of cultivated Bacillus species isolated from different composting stages was low. A total of 540 isolates were classified by the RFLP method and partial 16S rDNA sequences. They affiliated to eight species including Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Bacillus anthracis, Bacillus megaterium, Bacillus licheniformis, Bacillus pumilus, and Bacillus circulans. The predominant species was B. subtilis, and the diversity of culturable Bacillus isolated in the middle-level samples at temperature rising and cooling stages was the highest. The DGGE profile and clone library analysis revealed that the temporal–spatial distribution of Bacillus community was not obvious, species belonging to the Bacillus were dominant (67%) with unculturable bacteria and B. cereus was the second major culturable Bacillus species. This study indicated that a combination of culture and culture-independent approaches could be very useful for monitoring the diversity and temporal–spatial distribution of Bacillus community during the composting process.     

Transfer of Tn925 and plasmids between Bacillus subtilis and alkaliphilic Bacillus firmus OF4 during Tn925-mediated conjugation.

Conjugative transposon Tn925 was transferred to alkaliphilic Bacillus firmus OF4 during mating experiments, as monitored by the acquisition of tetracycline resistance at pH 7.5 and confirmed by Southern analysis of chromosomal DNA from transconjugants. Tetracycline resistance could not be demonstrated at pH 10.5, but transconjugants retained resistance upon growth at pH 7.5 after having grown for several generations at pH 10. When the Bacillus subtilis donor strain contained plasmids, either pUB110 or pTV1, in addition to Tn925, transfer of the plasmid to the alkaliphile occurred during conjugation, either together with or independently of the transfer of the transposon. The plasmids were stable in B. firmus OF4, expressing their resistance markers for kanamycin or chloramphenicol at pH 7.5 after growth of the transformants at high pH. Transconjugant B. firmus OF4, which carried Tn925, could serve as the donor in mating experiments with B. subtilis lacking the transposon. These studies establish a basis for initiation of genetic studies in this alkaliphilic Bacillus species, including the introduction of cloned genes and the use of transposon-mediated insertional mutagenesis.     

Bacillus locisalis sp. nov., a new haloalkaliphilic species from hypersaline and alkaline lakes of China, Kenya and Tanzania

A polyphasic taxonomic study was performed on seven Bacillus-like bacteria isolated from three hypersaline and alkaline lakes located in China, Kenya and Tanzania. All strains were moderately halophilic and alkaliphilic, Gram positive, motile rods. The DNA G+C content from the seven isolates ranged from 42.2 to 43.4 mol% and their major fatty acid was anteiso-C_15:0. Strain CG1^T, selected as representative strain of the isolates, possesses meso-diaminopimelic acid in the cell wall peptidoglycan, MK-7 as the predominant menaquinone and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine as the major polar lipids. Comparative 16S rRNA gene sequence analysis indicated that the isolates belonged to the genus Bacillus. The seven isolates shared 97.7–99.9% 16S rRNA gene sequence similarity, and formed a branch that was distinct from the type strains of the recognized species of the genus Bacillus. They were most closely related to Bacillus agaradhaerens DSM 8721^T (92.6–93.8% 16S rRNA sequence similarity). DNA–DNA hybridization values between the seven isolates were 85–100%. According to the polyphasic characterization, the strains represent a novel species, for which the name Bacillus locisalis sp. nov. is proposed. The type strain is CG1^T (CCM 7370^T = CECT 7152^T = CGMCC 1.6286^T = DSM 18085^T).