Taxonomic description and genome sequence of Halobacillus marinus sp. nov., a novel strain isolated from Chilika Lake, India.

moderately halophilic spore forming, motile, Gram-positive, rod-shaped bacterial strain designated as KGW1T was isolated from water sample of Chilika Lake and characterized taxonomically using polyphasic approach. The strain grew in the presence of 0-25% (w/v) NaCl in marine salt agar media, hydrolyzes casein, and gelatin and shows presence of alkaline proteases. The major cell wall menaquinone was MK7 and major cellular fatty acids were anteiso-C15:0 (44.89%), anteiso-C17:0 (6.18%), isoC15:0 (19.38%), and iso-C16:0 (7.39%). Several chemotaxonomic features conform the isolate be a member of genus Halobacillus. The isolate KGW1T contained A1γ meso-Dpm-direct type of peptidoglycan which is different from its phylogenetically closest neighbours. The 16S rRNA gene sequence based phylogenetic analysis also revealed the strain KGW1T was affiliated to the genus Halobacillus and sequence similarity between the isolated strain and the type strains of Halobacillus species were found closest to, H. dabanensis D-8 DSM 18199T (99.08%) and H. faecis IGA7-4 DSM 21559T (99.01%), H. trueperi SL-5 DSM 10404T (98.94%). The in silico DDH showed that the values in a range of 14.2-17.5% with the most closest strain H. dabanensis D-8 DSM 18199T and other type strains of the genus Halobacillus for which whole genome sequence is reported. DNA-DNA relatedness between strain KGW1T and the closest type strain Halobacillus trueperi DSM 10404T was 11.75% (± 1.15). The draft genome sequence includes 3,683,819 bases and comprises of 3898 predicted coding sequences with a G + C content of 46.98%. Thus, the significant distinctiveness supported by phenotypic and genotypic data with its closest neighbors and other closely related species confirm the strain KGW1T to be classified as a novel species within the genus Halobacillus, for which the name Halobacillus marinus sp. nov. is proposed. The type strain is KGW1T (= DSM 29522 = JCM 30443).

A UPF0118 family protein with uncharacterized function from the moderate halophile Halobacillus andaensis represents a novel class of Na+(Li+)/H+ antiporter.

In this study, genomic DNA was screened from Halobacillus andaensis NEAU-ST10-40T by selection in Escherichia coli KNabc lacking three major Na+/H+ antiporters. One gene designated upf0118 exhibiting Na+(Li+)/H+ antiport activity was finally cloned. Protein alignment showed that UPF0118 shares the highest identity of 81.5% with an unannotated gene encoding a protein with uncharacterized protein function belonging to UPF0118 family from H. kuroshimensis, but shares no identity with all known specific Na+(Li+)/H+ antiporter genes or genes with Na+(Li+)/H+ antiport activity. Growth test, western blot and Na+(Li+)/H+ antiport assay revealed that UPF0118 as a transmembrane protein exhibits pH-dependent Na+(Li+)/H+ antiport activity. Phylogenetic analysis indicated that UPF0118 clustered with all its homologs belonging to UPF0118 family at a wide range of 22-82% identities with the bootstrap value of 92%, which was significantly distant with all known specific single-gene Na+(Li+)/H+ antiporters and single-gene proteins with the Na+(Li+)/H+ antiport activity. Taken together, we propose that UPF0118 should represent a novel class of Na+(Li+)/H+ antiporter. To the best of our knowledge, this is the first report on the functional analysis of a protein with uncharacterized protein function as a representative of UPF0118 family containing the domain of unknown function, DUF20.

Optimization of Cellulase Production by Halobacillus sp. QLS 31 Isolated from Lake Qarun, Egypt.

A halophilic cellulase-producing bacterium was isolated from a sediment sample collected from Lake Qarun (Fayoum Province, Egypt). Molecular identification based on 16S rDNA amplification and sequencing revealed 99% homology with Halobacillus sp. and hence was designated as Halobacillus sp. QLS 31. Medium composition and culture conditions were optimized for enhancing the production of cellulase enzyme using the Plackett-Burman statistical design. Ten variables were evaluated for their influence on cellulase production. Carboxymethyl cellulose (CMC), zinc sulfate (ZnSO4), and inoculum size were found to exert a significant effect on cellulase productivity by Halobacillus sp. QLS 31. The maximum specific activity of cellulase enzyme was 48.08 U/mg. Following the predicted conditions, a 7.5-fold increase in cellulase specific activity (175.47 U/mg) was achieved compared to the basal medium (23.19 U/mg) under the following optimized conditions: temperature (30 °C), fermentation time (2 days ), pH value (9), CMC concentration (1%), inoculum size (1%), yeast extract concentration (0.1%), ammonium sulfate ((NH3)2SO4) concentration (0.1%), sodium chloride (NaCl) concentration (20%), and metal inducers: ZnSO4 (0.1%) and Ca/Mg ratio (0.01%). Thus, the results of this study provide an important basis for more efficient, cheap industrial cellulase production from halophilic Halobacillus sp. QLS 31.

Characterization of thermo-solvent stable protease from Halobacillus sp. CJ4 isolated from Chott Eldjerid hypersaline lake in Tunisia.

About 110 newly isolated halophilic and halotolerant bacteria were screened for protease production. A moderately halophilic strain (CJ4), isolated from Chott Eldjerid Hypersaline lake in Tunisia, showed the highest activity on agar plate and was then selected. The biochemical and physiological characterization of the isolate along with the 16S rRNA sequence analysis placed it in the genus Halobacillus. Protease production was maximal at 120 g/L NaCl (2 M) and it started from the post-exponential phase reaching a maximum level at the early decline phase of bacterial growth. Protease activity was optimal at 0.4 M NaCl, pH 9 and 45 °C. It showed an excellent stability over wide ranges of temperatures (30-60 °C), NaCl concentrations (0-5 M), and pH values (5-10), which make it a good candidate for industrial applications at harsh conditions. Crude protease was strongly inhibited by PMSF revealing the dominance of serine proteases. Protease activity exhibited high stability in the presence of several organic solvents and detergent additives. These findings make Halobacillus sp. CJ4 protease with a great interest for many biotechnological applications at high salt or low water content such as peptide synthesis and detergent formulation.

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

A Gram-positive, moderately halophilic bacterium, designated strain TGS-15(T), was isolated from the sediment of a solar saltern pond located in Shinan, Korea. Strain TGS-15(T) was found to be a strictly aerobic, non-motile rod which can grow at pH 6.0-10.0 (optimum, pH 9.0), at 20-35 °C (optimum, 28 °C) and at salinities of 1-20 % (w/v) NaCl (optimum, 9 % NaCl). Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain TGS-15(T) belongs to the genus Halobacillus, with sequence similarity of 98.5-96.0 % to known type strains, showing high sequence similarity to Halobacillus locisalis MSS-155(T) (98.5 %), Halobacillus faecis IGA7-4(T) (98.2 %) and Halobacillus alkaliphilus FP5(T) (98.0 %), and less than 98.0 % sequence similarity to other currently recognised type strains of the genus. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified glycolipid and an unidentified lipid. The cell wall peptidoglycan was found to be based on L-Orn-D-Asp, the predominant isoprenoid quinone was identified as menaquinone-7 (MK-7) and the major fatty acids were identified as anteiso-C15:0, iso-C15:0, anteiso-C17:0 and C16:1 ω7c alcohol. The DNA G+C content of this novel isolate was determined to be 45.3 mol %. Levels of DNA:DNA relatedness between strain TGS-15(T) and the type strains of 13 other species of the genus ranged from 52 to 9 %. On the basis of the polyphasic analysis conducted in this study, strain TGS-15(T) is concluded to represent a novel species of the genus Halobacillus, for which the name Halobacillus salicampi sp. nov. is proposed. The type strain is TGS-15(T) (=KACC 18264(T) = NBRC 110640(T)).

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).

Halobacillus andaensis sp. nov., a moderately halophilic bacterium isolated from saline and alkaline soil.

A Gram-stain-positive, endospore-forming, moderately halophilic bacterial strain, NEAU-ST10-40T, was isolated from a saline and alkaline soil in Anda City, China. It was strictly aerobic, rod-shaped and motile by peritrichous flagella. It formed light yellow colonies and grew at NaCl concentrations of 3-15 % (w/v) (optimum, 8 %, w/v), at pH 7.0-9.0 (optimum, pH 8.0) and at 4-60 °C (optimum, 30 °C). It contained meso-diaminopimelic acid in the cell-wall peptidoglycan. Phylogenetic analysis based on 16S rRNA gene sequences indicated that it belonged to the genus Halobacillus. Levels of 16S rRNA gene sequence similarity between strain NEAU-ST10-40T and the type strains of related species of the genus Halobacillus ranged from 98.8 % (Halobacillus alkaliphilus FP5T) to 97.1 % (Halobacillus kuroshimensis IS-Hb7T). DNA-DNA hybridization relatedness values between strain NEAU-ST10-40T and H. alkaliphilus DSM 18525T, Halobacillus campisalis KCTC 13144T, Halobacillus yeomjeoni DSM 17110T, Halobacillus halophilus DSM 2266T, Halobacillus litoralis DSM 10405T, Halobacillus dabanensis DSM 18199T, Halobacillus salinus DSM 18897T, Halobacillus naozhouensis DSM 21183T, Halobacillus trueperi DSM 10404T and Halobacillus salsuginis DSM 21185T were from 43 ± 1 to 19 ± 1 % (mean ± sd). The DNA G+C content was 39.3 mol%. The major fatty acids (>10 %) were anteiso-C15:0, anteiso-C17:0 and iso-C16:0, the only respiratory quinone detected was MK-7, and polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unknown phospholipids and three unknown lipids. On the basis of the data presented, strain NEAU-ST10-40T is considered to represent a novel species, for which the name Halobacillus andaensis sp. nov. is proposed. The type strain is NEAU-ST10-40T ( = CGMCC 1.12153T = DSM 25866T).

Plant growth promoting properties of Halobacillus sp. and Halomonas sp. in presence of salinity and heavy metals.

Salinity and heavy metal stress are challenging problems in agriculture. Here we report the plant growth promoting ability of three moderate halophiles, Halobacillus sp. ADN1, Halomonas sp. MAN5, and Halobacillus sp. MAN6, in presence of both salinity and heavy metal stress. Halobacillus sp. ADN1, Halomonas sp. MAN5, and Halobacillus sp. MAN6 can tolerate 25, 21, and 29% NaCl, respectively and grow in presence of 1 mM cobalt, cadmium, and nickel and 0.04 mM mercury and 0.03 mM silver. Halobacillus sp. ADN1, Halomonas sp. MAN5, and Halobacillus sp. MAN6 produced 152.5, 95.3, and 167.3 µg/ml indole acetic acid (IAA) and could solubilize 61, 53, and 75 parts per million (ppm) phosphate, respectively in the presence of 15% NaCl. The production of IAA and solubilization of phosphate was well retained in the presence of salinity and heavy metals like 1 mM cadmium, 0.7 mM nickel, 0.04 mM mercury, and 0.03 mM silver. Besides, the strains showed amylase and protease activities and could produce hydrogen cyanide and ammonia in presence of salinity and heavy metals. A mixture of three strains enhanced the root growth of Sesuvium portulacastrum under saline and heavy metal stress, where the root length increased nearly 4.5 ± 0.6 times and root dry weight increased 5.4 ± 0.5 times as compared to control. These strains can thus be useful in microbial assisted phytoremediation of polluted saline soils.

Uneven distribution of Halobacillus trueperi species in arid natural saline systems of Southern Tunisian Sahara.

The genetic diversity of a collection of 336 spore-forming isolates recovered from five salt-saturated brines and soils (Chott and Sebkhas) mainly located in the hyper-arid regions of the southern Tunisian Sahara has been assessed. Requirements and abilities for growth at a wide range of salinities\ showed that 44.3 % of the isolates were extremely halotolerant, 23 % were moderate halotolerant, and 32.7 % were strict halophiles, indicating that they are adapted to thrive in these saline ecosystems. A wide genetic diversity was documented based on 16S-23S rRNA internal transcribed spacer fingerprinting profiles (ITS) and 16S rRNA gene sequences that clustered the strains into seven genera: Bacillus, Gracilibacillus, Halobacillus, Oceanobacillus, Paenibacillus, Pontibacillus, and Virgibacillus. Halobacillus trueperi was the most encountered species in all the sites and presented a large intraspecific diversity with a multiplicity of ITS types. The most frequent ITS type included 42 isolates that were chosen for assessing of the intraspecific diversity by BOX-PCR fingerprinting. A high intraspecific microdiversity was documented by 14 BOX-PCR genotypes whose distribution correlated with the strain geographic origin. Interestingly, H. trueperi isolates presented an uneven geographic distribution among sites with the highest frequency of isolation from the coastal sites, suggesting a marine rather than terrestrial origin of the strains. The high frequency and diversity of H. trueperi suggest that it is a major ecosystem-adapted microbial component of the Tunisian Sahara harsh saline systems of marine origin.

Bioenergetics of the moderately halophilic bacterium Halobacillus halophilus: composition and regulation of the respiratory chain.

In their natural environments, moderately halophilic bacteria are confronted not only with high salinities but also with low oxygen tensions due to the high salinities. The growth of H. halophilus is strictly aerobic. To analyze the dependence of respiration on the NaCl concentration and oxygen availability of the medium, resting cell experiments were performed. The respiration rates were dependent on the NaCl concentration of the growth medium, as well as on the NaCl concentration of the assay buffer, indicating regulation on the transcriptional and the activity level. Respiration was accompanied by the generation of an electrochemical proton potential (Δµ(H+)) across the cytoplasmic membrane whose magnitude was dependent on the external pH. Genes encoding proteins involved in respiration and Δµ(H+) generation, such as a noncoupled NADH dehydrogenase (NDH-2), complex II, and complex III, were identified in the genome. In addition, genes encoding five different terminal oxidases are present. Inhibitor profiling revealed the presence of NDH-2 and complex III, but the nature of the oxidases could not be resolved using this approach. Expression analysis demonstrated that all the different terminal oxidases were indeed expressed, but by far the most prominent was cta, encoding cytochrome caa3 oxidase. The expression of all of the different oxidase genes increased at high NaCl concentrations, and the transcript levels of cta and qox (encoding cytochrome aa3 oxidase) also increased at low oxygen concentrations. These data culminate in a model of the composition and variation of the respiratory chain of H. halophilus.

Isolation and characterization of halotolerant bacteria associated with the midgut of Culex quinquefasciatus Say (Diptera: Culicidae).

We show for the first time that the midgut of Culex quinquefasciatus (Say) mosquito larvae harbors halotolerant bacteria. The midgut from field collected Cx. quinquefasciatus larvae were dissected under aseptic conditions, homogenized and plated on LB agar medium with 2% (w/v) NaCl. Two different colonies were successfully isolated and bacterial isolates were identified by 16S rRNA sequences. The halotolerant bacterial isolates were: Halobacillus litoralis (CxH1) and Staphylococcus cohnii (CxH2). The gene sequence of these isolates has been deposited in GenBank (JN016804 and JN183986). These halotolerant bacteria grew in the absence of salt (0%) as well as in the presence of relatively high salt concentrations in culture medium (20%), and grew best in the presence of 8-10% (w/v) NaCl. H. litoralis and S. cohnii showed growth up to 18 and 20% (w/v) NaCl, respectively. Optimum growth temperatures for both the bacteria were between 30-37 degrees C. H. litoralis was resistant to the antibiotics oxacillin, penicillin, polymixin and S. cohnii was resistant to the antibiotic oxacillin.

Production and characterization of a novel extracellular metalloproteinase by a newly isolated moderate halophile, Halobacillus sp. LY6.

A moderately halophilic bacterium LY6 with high proteolytic activity was isolated. Biochemical and physiological characterization, along with 16S rDNA sequence analysis placed the isolate in the genus Halobacillus. The salinity of the culture medium strongly influenced the proteinase production of LY6. Maximum enzyme production was observed in the medium containing 5% Na(2)SO(4) or 10% NaCl. Proteinase production was synchronized with bacterial growth and reached a maximum level during the mid-stationary phase. Enzyme purification was carried out by a simple approach including a combination of ammonium sulfate precipitation and Sephacryl S-100 gel filtration chromatography. SDS-PAGE and gelatin zymography analysis revealed it was a monomer with high molecular weight of 69 kDa. Optimal proteinase activity was obtained at pH 10.0, 40°C, and 10% NaCl. It was high active over broad temperature (30-80°C), pH (6.0-12.0), and NaCl concentration (0-25%) ranges, indicating its thermostable, alkali-stable, and halotolerant nature. Moreover, the enzyme activity was markedly enhanced by Ca(2+) and Cu(2+), but strongly inhibited by EDTA, PAO, and DEPC, indicating that it probably was a metalloproteinase with cysteine and histidine residues located in its active site.

Development of a genetic system for the moderately halophilic bacterium Halobacillus halophilus: generation and characterization of mutants defect in the production of the compatible solute proline.

A procedure for markerless mutagenesis gene deletions was developed for the moderately halophilic model strain Halobacillus halophilus. Gene transfer was achieved by protoplast fusion and allelic replacement by a two-step procedure. In the first step the non-replicating plasmid integrated over the upstream or the downstream region of the target gene or operon into the chromosome to obtain single-crossover mutants. When cells were grown under non-selective conditions a second homologous recombination happened (segregation). This resulted in either the wild-type or the mutated allele. The method was used to delete the proHJA operon from H. halophilus. The mutant still produced proline and thus was not proline auxotroph but it completely lost the ability to produce proline as a compatible solute. However, growth was not impaired and the loss of the solute proline was compensated for by an increase in glutamate, glutamine and ectoine concentration. Expressions of the genes encoding the biosynthesis enzymes of theses solutes were upregulated and the activity of the key enzyme in glutamine biosynthesis, the glutamine synthetase, was increased. A model for the proline biosynthesis in the ΔproHJA mutant is discussed.

Photochemistry of a putative new class of sensory rhodopsin (SRIII) coded by xop2 of Haloarcular marismortui.

Baliga et al. (2004) [1] reported the existence of a functionally unpredictable opsin gene, named xop2, in Haloarcula marismortui, a holophilic archaeon. Ihara et al. [38] performed molecular phylogenetic analysis and determined that the product of xop2 belonged to a new class of opsins in the sensory rhodopsins. This microbial rhodopsin was therefore named H. marismortui sensory rhodopsin III (HmSRIII). Here, we functionally expressed HmSRIII in Escherichia coli cell membranes to examine the photochemistry. The wavelength of maximum absorption (λ(max)) for HmSRIII was 506nm. We observed a very slow photocycle that completed in ∼50s. Intermediates were defined as M (λ(max)∼380nm), N (λ(max)∼460nm) and O (λ(max)∼530nm) 0.01s after the flash excitation. The nomenclature for these intermediates was based on their locations along the absorption maxima of bacteriorhodopsin. Analysis of laser-flash-photolysis data in the presence and absence of azide gave the following results: (1) an equilibrium between N and O was attained, (2) the direct product of the M-decay was O but not N, and (3) the last photo-intermediate (HmSRIII') had a λ(max) similar to that of the original, and its decay rate was very slow. Resonance Raman spectroscopy revealed that this N-intermediate had 13-cis retinal conformation. Proton uptake occurred during the course of M-decay, whereas proton release occurred during the course of O-decay (or exactly N-O equilibrium). Very weak proton-pumping activity was observed whose direction is the same as that of bacteriorhodopsin, a typical light-driven proton pump.