Halobacillus salinarum sp. nov., Halobacillus shinanisalinarum sp. nov. and Halobacillus amylolyticus sp. nov., isolated from saltern soil.

Three bacterial strains, designated SSBR10-3T, SSTM10-2T and SSHM10-5T, were isolated from saltern soil sampled in Jeollanam-do, Republic of Korea. Cells were aerobic, Gram-stain-positive, flagellated and rod-shaped. The strains grew optimally at 28°C and at pH 7.0. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strains SSBR10-3T, SSTM10-2T and SSHM10-5T were placed within the genus Halobacillus, showing the highest similarity to Halobacillus alkaliphilus FP5T (98.6 %), 'Halobacillus ihumii' Marseille-Q1234T (98.5 %) and Halobacillus locisalis MSS-155T (98.6 %), respectively. The genomic similarity values between strains SSBR10-3T, SSTM10-2T and SSHM10-5T and their related species were 17.6-22.6 % for digital DNA-DNA hybridization (dDDH) and 69.6-78.5 % for orthologous average nucleotide identity (OrthoANI), which were lower than the thresholds recommended for species delineation. The dDDH and OrthoANI values among the three strains were below 38.3 and 89.4 %, respectively. Besides the differences in genomic features, strains SSBR10-3T, SSTM10-2T and SSHM10-5T were distinct from each other and from members of the genus in terms of phenotypic traits related to substrate assimilation. The cell-wall peptidoglycan contained meso-diaminopimelic acid, the major fatty acids were anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0, and the predominant menaquinone was MK-7 for all three strains. Diphosphatidylglycerol, phosphatidylglycerol and an unidentified phospholipid were present in their polar lipid profiles. Based on a polyphasic approach incorporating genomic data, strains SSBR10-3T, SSTM10-2T and SSHM10-5T represent novel species, for which the names Halobacillus salinarum sp. nov. (SSBR10-3T=DSM 114353T=KACC 21935T=NBRC 115504T), Halobacillus shinanisalinarum sp. nov. (SSTM10-2T=DSM 114354T=KACC 21936T=NBRC 115505T) and Halobacillus amylolyticus sp. nov. (SSHM10-5T=DSM 114355T= KACC 21937T=NBRC 115506T) are proposed.

Characterization of bioemulsifying EPS from Halobacillus trueperi MXM-16, a halophilic adhered bacterial isolate from the mangrove ecosystem.

Exopolymeric substances (EPS) produced by bacterial cells play a crucial role in the interaction of the cells with the surrounding environment. Halobacillus trueperi manxer mangrove-16, an adhered bacterial isolate from the mangrove ecosystem was found to produce EPS that was observed by Alcian blue staining and congo red-coomassie blue agar. The EPS of the bacterial isolate exhibited emulsifying properties. Purification of the EPS by dialysis showed an emulsification index of 80% with hexadecane. Qualitative analysis and Fourier's Infrared spectroscopy (FTIR) revealed that the EPS was a glycoprotein in nature. The EPS showed no surface-active properties. Further exploration of the potential of the EPS interaction with metal solutions showed the ability of the bioemulsifier to cause precipitation in the metal solutions and particularly change the color of the Chromium (VI) solution. The scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDS) of the cells and EPS particularly indicated the interaction of the EPS with the (Fe0 ) zerovalent iron nanoparticles and its effect on the cells and EPS of the bacteria. It is therefore concluded that the EPS is a crucial component that anchors the bacteria to particulate matter in the mangrove ecosystem and also plays an important role in interaction with metals and hydrocarbons.

Identification of antibacterial metabolites produced by a marine bacterium Halobacillus marinus HMALI004.

AIMS: This study examined and characterized the extract for metabolites of Halobacillus marinus HMALI004 to understand their antibacterial activities against opportunistic marine pathogens, that is, Vibrio parahaemolyticus and Vibrio cholerae. METHODS AND RESULTS: The bacterial strain HMALI004 was characterized as H. marinus, and an antibacterial spectral test revealed its inhibition against two opportunistic marine pathogens (V. parahaemolyticus and V. cholera). Fermentation broth of strain HMALI004 was subjected to column chromatography and high-performance liquid chromatography to separate antibacterial substances. Two compounds were successfully isolated and identified as 1H-pyrrole-2-carboxylic acid and 4-chloro-1H-pyrrole-2-carboxylic acid by mass spectrometry (MS) and nuclear magnetic resonance. The minimal inhibition concentration (MIC) values of 1H-pyrrole-2-carboxylic acid and 4-chloro-1H-pyrrole-2-carboxylic acid for V. parahaemolyticus were 25 µg/ml, while their MIC values for V. cholerae were 50 and 100 µg/ml, respectively. The reactive oxygen species (ROS) production of two pathogen strains treated with 1H-pyrrole-2-carboxylic acid and 4-chloro-1H-pyrrole-2-carboxylic acid were detected to investigate the antimicrobial mechanism. The results suggested that 4-chloro-1H-pyrrole-2-carboxylic acid exerted enhanced ROS production in V. parahaemolyticus, whereas 1H-pyrrole-2-carboxylic acid had a weaker effect. Both compounds caused a significant rise in ROS production in V. cholerae, causing severe damage to the cell wall and cytoplasm, leading to cell death. CONCLUSIONS: The bacterium H. marinus HMALI004 was isolated from a shrimp pond and was found to produce antimicrobial compounds, which could inhibit the growth of opportunistic marine pathogens V. parahaemolyticus and V. cholerae by increasing ROS. SIGNIFICANCE AND IMPACT OF THE STUDY: Successfully isolated antibacterial-producing strain, H. marinus HMALI004, and its antimicrobial compounds could be used as biological control agents for marine pathogens.

Inhibitory activity of Halobacillus trueperi S61 and its active extracts on potato dry rot.

This study investigated the inhibitory activity of Halobacillus trueperi S61 and its active extract on potato dry rot pathogens and aimed at contributing to biocontrol agent development during potato storage. Three kinds of pathogens were isolated as target pathogenic fungi from dry rot tubers and determined as Fusarium acuminatum (Qing 9A-2), Fusarium equisetai (Qing 9A-5-8) and Fusarium tricinctum (Qing 9A-1-1) by morphological and molecular identification. The strain Halobacillus trueperi S61 and its extract exhibited a higher inhibitory rate on both three pathogens (56.32-65.75 and 1.67-51.11%), notably the best suppression efficiency is presented in Halobacillus trueperi S61 and 40 mg/mL ethyl acetate extract. In terms of in vivo effects, both Halobacillus trueperi S61 and its ethyl acetate extract effectively reduced the decayed fruit and weight loss rate (0-20% and 7.59-16.56%) and enhanced the defensive enzymatic activities to improve resistance. In addition, strain S61 could be colonized on potato tubers, especially the highest amount of 1.55 × 107 CFU/mL on fifth day for variety Xiazhai 65. Overall, Halobacillus trueperi S61 and its ethyl acetate extract could be considered as potential approach for biocontrol potato dry rot.

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).

Inhibition of quorum sensing-controlled virulence factors with natural substances and novel protease, obtained from Halobacillus karajensis.

INTRODUCTION: In recent years, a challenge in clinical treatment has developed due to bacterial resistance to antibiotics. One of the new mechanisms against infections is virulence factor inhibition. Many virulence factors are controlled by quorum sensing pathways such as biofilm formation and pyocyanin production. The goal of the present study was to investigate the effect of an obligate halophilic bacterial strain on Pseudomonas aeruginosa and Staphylococcus aureus, due to its halo-tolerant substances and enzymes. METHODS: The effect of Halobacillus karajensis on bacterial growth and production of virulence factors was studied in this work. The obligate halophile cells and supernatant fractions were extracted by the methanol/chloroform method and characterized by Fourier Transform Infrared (FTIR) spectroscopy, X-ray diffraction (XRD), Gas Chromatography-Mass Spectrometry (GC-MS), and zymography. The effects of these fractions were studied on biofilm formation in P. aeruginosa and S. aureus as well as on pyocyanin production in P. aeruginosa. The effective protein in the fraction was analyzed by the SDS-PAGE method, and all protein fragments were studied for pyocyanin inhibition. RESULTS: The crude supernatant extract, MMS fraction, from H. karajensis was effective for the biofilm reduction in S. aureus (74%) and P. aeruginosa (27%). Two proteases in this fraction, which were recognized by zymography on skim milk, were the probable causes for extracellular polymeric substances (EPS) hydrolysis in the biofilm matrix. Also, halide crystals and branched fatty acids, 12methyl-tetradecanoic acid, in the other fractions decreased the biofilm by 18% in S. aureus. The results showed that a new 25 kD protein, which was obtained from MMS fraction, inhibited pyocyanin production by 60% in P. aeruginosa. The zymogram and bioinformatics studies showed that this protein was a serine alkaline metalloprotease and had an interaction with AHL molecules. CONCLUSION: The inhibitory effects of the non-toxic natural substances and proteases on biofilm formation and pyocyanin production, specifically the 25 kD protease, are novel in this study and make them a good candidate for infected wound healing and inhibiting the virulence factors.

Zwitterionic small molecule based fluorophores for efficient and selective imaging of bacterial endospores.

In the emerging scenario of increasing antibiotic resistance and pathogen transmission channels, the grave danger posed by bacterial endospores in critical fields like food industry, health and medicine highlights the urgent need to develop efficient probes for their detection; their sturdy and impermeable multilayer coat makes desirable methods like fluorescence imaging extremely difficult. Selective imaging of the endospores in the presence of the bacteria is even more challenging. Furthermore, it is preferable to maintain the dormant state of endospores through the imaging process, if extended monitoring is required; many of the available techniques involve lethal germination or destruction of the endospores. We show that simple zwitterionic diaminodicyanoquinodimethane (DADQ) molecules with selected functionalities are efficient dyes for fluorescence imaging due to their dipolar structure that facilitates the penetration into the endospore system, and the enhanced fluorescence in their rigid/aggregated state. The facile structural tailorability allows DADQs with various appendage moieties to be synthesized; a derivative with ionic substituents (BT2), and another with optimally long alkyl chains and the resultant hydrophobic character (BHADQ) are shown to be excellent fluorescent probes for endospores. Nanomolar amounts of dyes provide effective staining; while BT2 stains bacteria and endospores, most significantly, BHADQ stains endospores selectively. To the best of our knowledge, this is the first example of selective fluorescence imaging of endospores in their dormant state. A range of spectroscopy, microscopy and calorimetry studies provide insight into the molecular level interactions that enable efficient staining and bright images. DADQ fluorophores are photostable and non-cytotoxic, hence useful in practical applications. The versatile structural tailorability of these dye molecules holds great promise for targeted imaging.

Quantitative proteomic analysis provides insights into the algicidal mechanism of Halobacillus sp. P1 against the marine diatom Skeletonema costatum.

Algicidal behavior is a common interaction between marine microalgae and bacteria, especially in the dissipation phase of algal blooms. The marine bacterium Halobacillus sp. P1 was previously isolated and exhibits high algicidal activity against the diatom Skeletonema costatum. However, little is known about the mechanism underlying this algicidal process. Here, a tandem mass tag (TMT)-based proteomic approach was coupled with physiological analysis to investigate the cellular responses of S. costatum when treated with P1 culture supernatant. Among the 4582 proteins identified, 82 and 437 proteins were differentially expressed after treatment for 12 and 24 h, respectively. The proteomic results were in accordance with the results of verification by parallel reaction monitoring (PRM) assays. Proteins involved in reactive oxygen species scavenging, protein degradation and transport were upregulated, while proteins participating in nitrogen metabolism, protein translation, photosynthetic pigment biosynthesis and cell cycle regulation were significantly downregulated (p-value ≤0.05), corresponding to the increasing malondialdehyde content and the decreasing nitrogen, protein and chlorophyll a contents. A nutrient competitive relationship might exist between the bacterium P1 and S. costatum, and the inhibition of nitrogen metabolism by the P1 culture supernatant might be the key lethal factor that results in the dysfunction of S. costatum metabolism. Our study sheds light on the algicidal mechanism of P1 at the molecular level and provides new insights into algae-bacteria interactions.

Bioremediation potential of a halophilic Halobacillus sp. strain, EG1HP4QL: exopolysaccharide production, crude oil degradation, and heavy metal tolerance.

A halophilic bacterial strain, EG1HP4QL, was isolated from a salt sample from Lake Qarun, Fayoum Province, Egypt. Morphological, physiological, biochemical, and phylogenetic analyses indicated that the strain belonged to the genus Halobacillus. Strain EG1HP4QL produced an extracellular polysaccharide (EPS), with production peaking (5.9 g L-1) during growth on medium S-G containing 2% (w/v) sucrose at 35 °C (pH 8.0). The EPS had significant emulsifying activity (E24 %) against kerosene (65.7 ± 0.8%), o-xylene (64.0 ± 1%), and sunflower oil (44.7 ± 0.5%). The composition of the EPS included two polymers-a negatively charged and a neutral one (~ 3:1)-in which mannose and glucose were the main neutral monosaccharide constituents. Strain EG1HP4QL was able to utilize crude oil (35.3%) as the sole carbon source within 12 days. The minimum inhibitory concentrations of heavy metals [Zn(II), Cd(II), Pb(II), Ni(II), and Cu(II)] for strain EG1HP4QL were 1.0, 2.0, 2.0, 2.5, and 5 mM, respectively.

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.

Antagonism of Quorum Sensing Phenotypes by Analogs of the Marine Bacterial Secondary Metabolite 3-Methyl-N-(2'-Phenylethyl)-Butyramide.

Quorum sensing (QS) antagonists have been proposed as novel therapeutic agents to combat bacterial infections. We previously reported that the secondary metabolite 3-methyl-N-(2'-phenylethyl)-butyramide, produced by a marine bacterium identified as Halobacillus salinus, inhibits QS controlled phenotypes in multiple Gram-negative reporter strains. Here we report that N-phenethyl hexanamide, a structurally-related compound produced by the marine bacterium Vibrio neptunius, similarly demonstrates QS inhibitory properties. To more fully explore structure-activity relationships within this new class of QS inhibitors, a panel of twenty analogs was synthesized and biologically evaluated. Several compounds were identified with increased attenuation of QS-regulated phenotypes, most notably N-(4-fluorophenyl)-3-phenylpropanamide against the marine pathogen Vibrio harveyi (IC50 = 1.1 µM). These findings support the opportunity to further develop substituted phenethylamides as QS inhibitors.

Synthesis of salt-stable fluorescent nanoparticles (quantum dots) by polyextremophile halophilic bacteria.

Here we report the biological synthesis of CdS fluorescent nanoparticles (Quantum Dots, QDs) by polyextremophile halophilic bacteria isolated from Atacama Salt Flat (Chile), Uyuni Salt Flat (Bolivia) and the Dead Sea (Israel). In particular, a Halobacillus sp. DS2, a strain presenting high resistance to NaCl (3-22%), acidic pH (1-4) and cadmium (CdCl2 MIC: 1,375 mM) was used for QDs biosynthesis studies. Halobacillus sp. synthesize CdS QDs in presence of high NaCl concentrations in a process related with their capacity to generate S2- in these conditions. Biosynthesized QDs were purified, characterized and their stability at different NaCl concentrations determined. Hexagonal nanoparticles with highly defined structures (hexagonal phase), monodisperse size distribution (2-5 nm) and composed by CdS, NaCl and cysteine were determined by TEM, EDX, HRXPS and FTIR. In addition, QDs biosynthesized by Halobacillus sp. DS2 displayed increased tolerance to NaCl when compared to QDs produced chemically or biosynthesized by non-halophilic bacteria. This is the first report of biological synthesis of salt-stable QDs and confirms the potential of using extremophile microorganisms to produce novel nanoparticles. Obtained results constitute a new alternative to improve QDs properties, and as consequence, to increase their industrial and biomedical applications.

Flocculating properties and potential of Halobacillus sp. strain H9 for the mitigation of Microcystis aeruginosa blooms.

Microcystis aeruginosa can cause harmful algal blooms in freshwaters worldwide. It has already seriously affected human lives and prevented the use of water resources. Therefore, there is an urgent need to develop ecofriendly and effective methods to control and eliminate M. aeruginosa in aquatic environments. In this study, Halobacillus sp. strain H9, a bacterium that showed high M. aeruginosa flocculation activity, was isolated and selected to assess its potential for the removal of M. aeruginosa. The analyses of flocculation activity and mode indicated that the strain H9 induced M. aeruginosa flocculation by secreting active flocculating substance rather than by directly contacting algal cells. A 5% concentration of the H9 supernatant could efficiently flocculate M. aeruginosa cells with a density of up to 5 × 107 cells/mL. Dramatic increases in the zeta potential indicated that charge neutralization could be the mechanism of the flocculation process. The strain H9 flocculated M. aeruginosa with no damage to the algal cell membrane, and did not result in microcystin being released into the surrounding environment. The flocculated algal culture was less toxic to zebrafish larvae, suggesting an environmentally friendly benefit of the H9 supernatant. In addition to M. aeruginosa, the H9 strain was also able to flocculate two other species causing harmful algal blooms, Phaeocystis globose and Heterosigma akashiwo. Furthermore, the flocculation activity of the H9 supernatant was stable at different temperatures and over a wide pH range. These characteristics give the H9 strain great potential for mitigating the influences of harmful algal blooms.

PYRROLO isolated from marine sponge associated bacterium Halobacillus kuroshimensis SNSAB01 - Antifouling study based on molecular docking, diatom adhesion and mussel byssal thread inhibition.

In the present study, an attempt has been made to explore the antifouling potential of bioactive compound isolated from sponge associated bacterium Halobacillus kuroshimensis SNSAB01. The crude extract of SNSAB01 strongly inhibited the growth of fouling bacterial strains with least minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The bioactive compound was characterized through FT-IR, HPLC, GCMS and NMR predicted as 'pyrrolo". From the mass spectral library, structure was elucidated as pyrrolo [1, 2-a] pyrazine-1, 4-dione, hexahydro. The in silico studies provided encouraging docking scores with two interactions by GLN 200 and GLU 304. The extract inhibited 89% diatom adhesion at 350 µg/ml concentration against Amphora sp. An EC50 value of 150 µg/ml for 50% inhibition of byssal thread of Perna viridis and LC50 was found to be 500 µg/ml. The LC50/EC50 ratio of 3.0 indicated nontoxic to nature. The result suggested that pyrrolo[1,2-a]pyrazine-1,4-dione can be used for antifouling coating.

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).