Halobacillus fulvus sp. nov., a moderately halophilic bacterium isolated from shrimp paste (Ka-pi) in Thailand.

An aerobic, Gram-stain-positive, endospore-forming, rod-shaped and moderately halophilic strain SKP4-6T, was isolated from shrimp paste (Ka-pi) collected from Samut Sakhon Province, Thailand. Phylogenetic analysis revealed that strain SKP4-6T belonged to the genus Halobacillus and was most closely related to Halobacillus salinus JCM 11546T (98.6 %), Halobacillus locisalis KCTC 3788T (98.6 %) and Halobacillus yeomjeoni KCTC 3957T (98.6 %) based on 16S rRNA gene sequence similarity. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain SKP4-6T and its related species were 18.2-19.3 % and 69.84-84.51 %, respectively, which were lower than the threshold recommended for species delineation. The strain grew optimally at 30-40 °C, at pH 7.0 and with 10-15 % (w/v) NaCl. It contained l-Orn-d-Asp in the cell wall peptidoglycan. The DNA G+C content was 44.8 mol%. The major fatty acids were iso-C15 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. The predominant isoprenoid quinone was MK-7. Phosphatidylglycerol and diphosphatidylglycerol were present as major polar lipids. Based on this polyphasic approach, digital DNA-DNA relatedness and ANI values, strain SKP4-6T represents a novel species of the genus Halobacillus, for which the name Halobacillus fulvus sp. nov. is proposed. The type strain is SKP4-6T (=JCM 32624T=TISTR 2595T).

Carotenoids produced by halophilic bacterial strains on mural paintings and laboratory conditions.

Due to the presence of efflorescences and improper microclimate conditions for conservation, pink-pigmented areas were reported in two historic monuments in Northern and Central part of Romania. The aims of the present study were to find the nature of pink pigments observed on the pictorial layer, original and infilling mortar, to investigate the presence of carotenoids both on mural paintings and in the isolated halophilic bacterial strains and to preliminary characterize and identify the producing strains. Their role in the aesthetical biodeterioration of historic monuments was also pointed out. Obtained Raman spectra of the pink pigments extracted both from the isolated bacterial cultures (molecularly identified as mostly related to Halobacillus hunanensis and Halobacillus naozhouensis) and from the mural painting samples contain diagnostic bands of carotenoids. These results were confirmed by FTIR spectroscopy. The strong Raman signal of bacterial carotenoids detected on mural painting indicated their potential use as biomarker molecules in the evaluation of contamination and state of conservation of mural paintings and lithic monuments. Our results contribute to opening a new direction in cultural heritage restoration to assess the conservation status on the basis of interdisciplinary research, starting with spectroscopic methods (Raman, FTIR) and confirmed by microbiological analysis.

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).

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.

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).

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.

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.

Extracellular production of beta-amylase by a halophilic isolate, Halobacillus sp. LY9.

A moderately halophilic strain LY9 with high amylolytic activity was isolated from soil sample obtained from Yuncheng, China. Biochemical and physiological characterization along with 16S rRNA sequence analysis placed the isolate in the genus Halobacillus. Amylase production started from the post-exponential phase of bacterial growth and reached a maximum level during the early-stationary phase. The isolate LY9 was found to secrete the amylase, the production of which depended on the salinity of the growth medium. Maximum amylase production was observed in the presence of 10% KCl or 10% NaCl. Maltose was the main product of soluble starch hydrolysis, indicating a ß-amylase activity. The enzyme showed optimal activity at 60°C, pH 8.0, and 10-12.5% of NaCl. It was highly active over broad temperature (50-70°C), NaCl concentration (5.0-20.0%), and pH (4.0-12.0) ranges, indicating its thermoactive and alkali-stable nature. However, activity dropped off dramatically at low NaCl concentrations, showing the amylase was halophilic. Ca(2+) was found to stimulate the ß-amylase activity, whereas ethylenediaminetetraacetic acid (EDTA), phenylarsine oxide (PAO), and diethyl pyrocarbonate (DEPC) strongly inhibited the enzyme, indicating it probably was a metalloenzyme with cysteine and histidine residues located in its active site. Moreover, the enzyme exhibited remarkable stability towards sodium dodecyl sulfate (SDS) and Triton X-100. This is the first report of ß-amylase production from moderate halophiles. The present study indicates that the extracellular ß-amylase of Halobacillus sp. LY9 may have considerable potential for industrial application owing to its properties.