Unveiling the potential of novel Metschnikowia yeast biosurfactants: triggering oxidative stress for promising antifungal and anticancer activity.

BACKGROUND: Sophorolipids are glycolipid biosurfactants with potential antibacterial, antifungal, and anticancer applications, rendering them promising for research. Therefore, this study hypothesizes that sophorolipids may have a notable impact on disrupting membrane integrity and triggering the production of reactive oxygen species, ultimately resulting in the eradication of pathogenic microbes. RESULTS: The current study resulted in the isolation of two Metschnikowia novel yeast strains. Sophorolipids production from these strains reached maximum yields of 23.24 g/l and 21.75 g/l, respectively, at the bioreactors level. Biosurfactants sophorolipids were characterized using FTIR and LC-MS techniques and found to be a mixture of acidic and lactonic forms with molecular weights of m/z 678 and 700. Our research elucidated sophorolipids' mechanism in disrupting bacterial and fungal membranes through ROS generation, confirmed by transmission electron microscopy and FACS analysis. The results showed that these compounds disrupted the membrane integrity and induced ROS production, leading to cell death in Klebsiella pneumoniae and Fusarium solani. In addition, the anticancer properties of sophorolipids were investigated on the A549 lung cancer cell line and found that sophorolipid-11D (SL-11D) and sophorolipid-11X (SL-11X) disrupted the actin cytoskeleton, as evidenced by immunofluorescence microscopy. The A549 cells were stained with Acridine orange/Ethidium bromide, which showed that they underwent necrosis. This was confirmed by flow cytometric analysis using Annexin/PI staining. The SL-11D and SL-11X molecules exhibited low levels of haemolytic activity and in-vitro cytotoxicity in HEK293, Caco-2, and L929 cell lines. CONCLUSION: In this work, novel yeast species CIG-11DT and CIG-11XT, isolated from the bee's gut, produce significant yields of sophorolipids without needing secondary oil sources, indicating a more economical production method. Our research shows that sophorolipids disrupt bacterial and fungal membranes via ROS production. They suggest they may act as chemo-preventive agents by inducing apoptosis in lung cancer cells, offering the potential for enhancing anticancer therapies.

Chromobacterium indicum sp. nov., a Pigment-Producing Bacterium Isolated from Soil.

A purple colony, designated as TRC1.1.SA was isolated from a tea garden soil sample. It was a Gram-negative, rod-shaped, non-spore-forming and motile bacterium. The strain TRC1.1.SAT grew aerobically at temperatures 15-37 â„ƒ and pH levels 5.0-9.0. It showed both oxidase and catalase activity. The 16S rRNA gene sequence blast analysis revealed identity with the members of the genus Chromobacterium. The maximum identity was with the type strains of species Chromobacterium piscinae CCM 3329T (99.8%), C. vaccinii MWU205T (99.7%), and C. violaceum ATCC 12472T (98.7%). However, the average nucleotide identity (ANI) of the genome sequence showed less than 96% similarity with all species of the genus Chromobacterium. Further, digital DNA-DNA hybridization (dDDH) revealed the highest identity of 63.4% with its phylogenetic relative C. piscinae CCM 3329T. The G + C content of the strain was 63.9%. The major polar lipids identified were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), and phosphoglyceraldehyde (PG). Fatty acid analysis showed C16:0, C16:1ω7c, C17:0 cyclo, and C18:1ω7c as the major fatty acids. RAST and antiSMASH analyses of the genome revealed the presence of a biosynthetic gene cluster (BGC) involved in the production of violacein pigment, as observed for type species C. violaceum ATCC 12472T. Considering the phenotypic differences and genomic identity, strain TRC1.1.SAT is assigned as a novel species of the genus Chromobacterium, for which the name Chromobacterium indicum is proposed. The type strain of prospective species is designated as TRC1.1.SAT (= MTCC 13391T; JCM 36723T; = KCTC 8324T).

Ion-pumping microbial rhodopsin protein classification by machine learning approach.

BACKGROUND: Rhodopsin is a seven-transmembrane protein covalently linked with retinal chromophore that absorbs photons for energy conversion and intracellular signaling in eukaryotes, bacteria, and archaea. Haloarchaeal rhodopsins are Type-I microbial rhodopsin that elicits various light-driven functions like proton pumping, chloride pumping and Phototaxis behaviour. The industrial application of Ion-pumping Haloarchaeal rhodopsins is limited by the lack of full-length rhodopsin sequence-based classifications, which play an important role in Ion-pumping activity. The well-studied Haloarchaeal rhodopsin is a proton-pumping bacteriorhodopsin that shows promising applications in optogenetics, biosensitized solar cells, security ink, data storage, artificial retinal implant and biohydrogen generation. As a result, a low-cost computational approach is required to identify Ion-pumping Haloarchaeal rhodopsin sequences and its subtype. RESULTS: This study uses a support vector machine (SVM) technique to identify these ion-pumping Haloarchaeal rhodopsin proteins. The haloarchaeal ion pumping rhodopsins viz., bacteriorhodopsin, halorhodopsin, xanthorhodopsin, sensoryrhodopsin and marine prokaryotic Ion-pumping rhodopsins like actinorhodopsin, proteorhodopsin have been utilized to develop the methods that accurately identified the ion pumping haloarchaeal and other type I microbial rhodopsins. We achieved overall maximum accuracy of 97.78%, 97.84% and 97.60%, respectively, for amino acid composition, dipeptide composition and hybrid approach on tenfold cross validation using SVM. Predictive models for each class of rhodopsin performed equally well on an independent data set. In addition to this, similar results were achieved using another machine learning technique namely random forest. Simultaneously predictive models performed equally well during five-fold cross validation. Apart from this study, we also tested the own, blank, BLAST dataset and annotated whole-genome rhodopsin sequences of PWS haloarchaeal isolates in the developed methods. The developed web server ( https://bioinfo.imtech.res.in/servers/rhodopred ) can identify the Ion Pumping Haloarchaeal rhodopsin proteins and their subtypes. We expect this web tool would be useful for rhodopsin researchers. CONCLUSION: The overall performance of the developed method results show that it accurately identifies the Ionpumping Haloarchaeal rhodopsin and their subtypes using known and unknown microbial rhodopsin sequences. We expect that this study would be useful for optogenetics, molecular biologists and rhodopsin researchers.

Marinobacterium lacunae sp. nov. isolated from estuarine sediment.

A novel motile bacterium was isolated from a sediment sample collected in Kochi backwaters, Kerala, India. This bacterium is Gram negative, rod shaped, 1.0-1.5 µm wide, and 2.0-3.0 µm long. It was designated as strain AK27T. Colonies were grown on marine agar displayed circular, off-white, shiny, moist, translucent, flat, margin entire, 1-2 mm in diameter. The major fatty acids identified in this strain were C18:1 ω7c, C16:0, and summed in feature 3. The composition of polar lipids in the strain AK27T included phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, one unidentified amino lipid, two unidentified aminophospholipids, two unidentified phospholipids, and six unidentified lipids. The genomic DNA of strain AK27T exhibited a G+C content of 56.4 mol%. Based on the analysis of 16S rRNA gene sequence, strain AK27T showed sequence similarity to M. ramblicola D7T and M. zhoushanense WM3T as 98.99% and 98.58%, respectively. Compared to other type strains of the Marinobacterium genus, strain AK27T exhibited sequence similarities ranging from 91.7% to 96.4%. When compared to Marinobacterium zhoushanense WM3T and Marinobacterium ramblicola D7T, strain AK27T exhibited average nucleotide identity values of 80.25% and 79.97%, and dDDH values of 22.9% and 22.6%, respectively. The genome size of the strain AK27T was 4.55 Mb, with 4,229 coding sequences. Based on the observed phenotypic and chemotaxonomic features, and the results of phylogenetic and phylogenomic analysis, this study proposes the classification of strain AK27T as a novel species within the genus Marinobacterium. The proposed name for this novel species is Marinobacterium lacunae sp. nov.

Brevicillin, a novel lanthipeptide from the genus Brevibacillus with antimicrobial, antifungal, and antiviral activity.

AIM: This study was aimed to determine antimicrobial and antiviral activity of a novel lanthipeptide from a Brevibacillus sp. for disinfectant application. METHODS AND RESULTS: The antimicrobial peptide (AMP) was produced by a bacterial strain AF8 identified as a member of the genus Brevibacillus representing a novel species. Whole genome sequence analysis using BAGEL identified a putative complete biosynthetic gene cluster involved in lanthipeptide synthesis. The deduced amino acid sequence of lanthipeptide named as brevicillin, showed >30% similarity with epidermin. Mass determined by MALDI-MS and Q-TOF suggested posttranslational modifications like dehydration of all Ser and Thr amino acids to yield Dha and Dhb, respectively. Amino acid composition determined upon acid hydrolysis is in agreement with core peptide sequence deduced from the putative biosynthetic gene bvrAF8. Biochemical evidence along with stability features ascertained posttranslational modifications during formation of the core peptide. The peptide showed strong activity with 99% killing of pathogens at 12 µg ml-1 within 1 minute. Interestingly, it also showed potent anti-SARS-CoV-2 activity by inhibiting ∼99% virus growth at 10 µg ml-1 in cell culture-based assay. Brevicillin did not show dermal allergic reactions in BALB/c mice. CONCLUSION: This study provides detailed description of a novel lanthipeptide and demonstrates its effective antibacterial, antifungal and anti-SARS-CoV-2 activity.

Isolation, purification, and characterization of a novel exopolysaccharide isolated from marine bacteria Brevibacillus borstelensis M42.

Marine microbes produce polysaccharides with unique physicochemical and functional properties that help them survive in harsh marine environments. However, only a handful of marine exopolysaccharides (EPSs) have been reported to date. The present study explored the seashore of Visakhapatnam, India, to report a novel exopolysaccharide designated as Br42 produced by Brevibacillus borstelensis M42. The isolate was identified through morphological, biochemical, phylogenetic, and genome sequencing analysis. The studies on fermentation kinetics revealed that EPS Br42 was a primary metabolite with a maximum production of 1.88 ± 0.02 g/L after 60 h when production broth was fortified with 2% glucose. Additionally, EPS Br42 was found to be a heteropolysaccharide consisting of glucose and galacturonic acid with a molecular weight of about 286 kDa. Interestingly, this molecule possesses industrially relevant functional properties such as water-holding (510 ± 0.35%), oil-holding (374 ± 0.12% for coconut oil and 384 ± 0.35% for olive oil), and swelling capacities (146.6 ± 5.75%). EPS Br42 could form an emulsion that was stable at a wide pH range for about 72 h and, in fact, performed better as compared to Span 20, a commercially used synthetic emulsifier. Moreover, this EPS was also found to be heat stable and exhibited non-Newtonian pseudoplastic behavior. These physicochemical and functional properties of polysaccharides suggest that the EPS Br42 has potential for multifarious industrial applications as an emulsifier, stabilizer, viscosifier, and binding agent.

Marinobacterium alkalitolerans sp. nov., with nitrate reductase and urease activity isolated from green algal mat collected from a solar saltern.

A novel Gram-staining-negative, rod-shaped, 0.6-0.8 µm wide and 2.0-3.0 µm in length, motile bacterium designated strain AK62T, was isolated from the green algal mat collected from saltpan, Kakinada, Andhra Pradesh, India. Colonies on ZMA were circular, off-white, shiny, moist, translucent, 1-2 mm in diameter, flat, with an entire margin. The major fatty acids include C16:0, C18:1 ω7c, and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c and/or iso-C14:0 3-OH). Polar lipids include diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminophospholipid, three unidentified phospholipids, and one unidentified lipid. Polyamine includes Spermidine. The DNA G + C content of the strain AK62T was 58.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain AK62T was closely related to the type strains Marinobacterium sediminicola, Marinobacterium coralli and Marinobacterium stanieri with a pair-wise sequence similarity of 96.9, 96.6 and 96.6%, respectively, forming a distinct branch within the genus Marinobacterium and clustered with M. stanieri, M. sediminicola, M. coralli and M. maritimum cluster. Strain AK62T shares average nucleotide identity (ANIb, based on BLAST) of 78.44, 76.69, and 76.95% with M. sediminicola CGMCC 1.7287T, M. stanieri DSM 7027T, and Marinobacterium halophilum Mano11T respectively. Based on the observed phenotypic, chemotaxonomic characteristics, and phylogenetic analysis, strain AK62T is described in this study as a novel species in the genus Marinobacterium, for which the name Marinobacterium alkalitolerans sp. nov. is proposed. The type strain of M. alkalitolerans is AK62T (= MTCC 12102T = JCM 31159T = KCTC 52667T).

Taxonomically Characterized and Validated Bacterial Species Based on 16S rRNA Gene Sequences from India During the Last Decade.

Microbial taxonomy dealing with identification and characterization of prokaryotes like bacteria and archaea has always been a major area of research all over the world. Exploring diversity of microbes and description of novel species with different genes and secondary compounds is of utmost importance for better future and sustenance of life. India having an enormous range of ecosystems and diverse species inhabiting these niches is considered to be one of the richest biodiversity regions of the world. During the last decade, with newer methodologies and better technology, the prokaryotic taxonomy from India has extended our inventory of microbial communities in specific niches. However, there still exist some limitations in classifying the microbes from India as compared to that is done world-over. This review enlists the taxonomic description of novel taxa of prokaryotes from India in the past decade. A total of 378 new bacterial species have been classified from different habitats in India in the last ten years and no descriptions of archaeal species is documented till date.

Arenibacter amylolyticus sp. nov., an amylase-producing bacterium of the family Flavobacteriaceae isolated from marine water in India.

A novel Gram-stain-negative, curved rod-shaped, 0.5-0.7 µm wide and 3.0-10.0 µm long, non-motile bacterium, designated strain AK53T, was isolated from a 5 m depth water sample collected from the Bay of Bengal, Visakhapatnam, India. Colonies on marine agar were circular, small, dark orange, shiny, smooth, translucent, flat, with an entire margin. The major fatty acids included iso-C15 : 0, iso-C15 : 0 3OH, anteiso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3OH and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c and/or iso-C15 : 0-2OH). Polar lipids included phosphatidylethanolamine and five unidentified lipids. The DNA G+C content of the strain AK53T was found to be 40.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain AK53T was closely related to Arenibacter latericius KMM 426T and Arenibacter certesii KMM3941T (pair-wise sequence similarity of 99.17 and 98.89 %, respectively), forming a distinct branch within the genus Arenibacter and clustering with A. latericius. Strain AK53T shared average nucleotide identity (ANIb, based on blast) of 78.07 and 77.44 % with A. latericius JCM 13508T and A. certesii JCM 13507T, respectively. Based on the observed phenotypic, chemotaxonomic characteristics and phylogenetic analysis, strain AK53T is described in this study as representing a novel species in the genus Arenibacter, for which the name Arenibacter amylolyticus sp. nov. is proposed. The type strain of Arenibacter amylolyticus is AK53T (=MTCC 12004T= JCM 19206T=KCTC 62553T).

Shewanella submarina sp. nov., a gammaproteobacterium isolated from marine water.

A curved-rod-shaped bacterium was isolated from a marine (100 m depth) water sample collected from Bay of Bengal, Visakhapatnam, India. Strain NIO-S14T, was Gram-stain-negative, motile and pale-yellow. NIO-S14T was able to grow aerobically and anaerobically and could utilize a number of organic substrates. Major fatty acids were C12 : 0, iso-C13 : 0, C14 : 0, iso-C15 : 0, C16 : 0 and C16 : 1ω7c and/or C16 : 1ω6c (summed feature 3). NIO-S14T contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminophospholipids and six unidentified lipids as polar lipids. The DNA G+C content of NIO-S14T was 47.9 mol%. The 16S rRNA gene sequence comparisons indicated that the isolate represented a member of the family Shewanellaceae within the class Gammaproteobacteria. According to the results of 16S rRNA gene sequence analysis, NIO-S14T was closely related to Shewanella coralliiwith a pair-wise sequence similarity of 99.26 %. On the basis of the sequence comparison, NIO-S14T clustered with Shewanella coralliiand together they clustered with Shewanella mangroviand seven other species of the genus Shewanella but were distantly related. DNA-DNA hybridization between NIO-S14T and Shewanella corallii DSM 21332Trevealed a relatedness of 35 %. Distinct morphological, physiological and genotypic differences from these previously described taxa supported the classification of NIO-S14T as a representative of a novel species of the genus Shewanella, for which the name Shewanellasubmarina sp. nov. is proposed. The type strain of Shewanellasubmarina is NIO-S14T (=MTCC 12524T=KCTC 52277T=LMG 30752T).

Virgicin, a novel lanthipeptide from Virgibacillus sp. strain AK90 exhibits inhibitory activity against Gram-positive bacteria.

There is a significant increase in the discovery of new antimicrobial compounds in recent past to combat drug resistant pathogens. Members of the genus Bacillus and related genera have been screened extensively due to their ability to produce wide range of antimicrobial compounds. In this study, we have isolated and characterized a new antimicrobial peptide from a marine bacterium identified as Virgibacillus species. The low molecular mass and stability of the antimicrobial substance pointed towards the bacteriocinogenic nature of the compound. The RAST analysis of genome sequence showed presence of a putative bacteriocin biosynthetic cluster containing genes necessary for synthesis of a lanthipeptide. Translated amino acid sequence of mature C-terminal propeptide showed identity with salivaricin A (52.2%) and lacticin A (33.3%). Accordingly, the mass (2417 Da) obtained by MALDI analysis was in agreement with posttranslational modifications of the leader peptide to yield three methyl lanthionine rings and a disulfide bond between two free cysteine residues. The lanthipeptide was named as virgicin, which selectively inhibited the growth of Gram-positive bacteria and biofilm formation by Enterococcus faecalis. Inhibition of biofilm formation by E. faecalis was also observed in in vitro model experiments using hydroxyapatite discs. Thus, virgicin appears to be a promising new bacteriocin to control oral biofilm formation by selective pathogens.

Bacillus shivajii sp. nov., isolated from a water sample of Sambhar salt lake, India.

A novel Gram-stain-positive, rod-shaped, motile, spore-forming, strictly aerobic, alkali- and halo- tolerant bacterium, designated strain AK72T, was isolated from a water sample collected from Sambhar salt lake, Rajasthan, India. The colony appears circular, shiny, smooth, translucent or slightly pale in colour and convex with an entire margin after 48 h incubation at 37 °C with pH 9. Growth of the bacterium occurred at 10-42 °C (optimum, 25-37 °C), at salinities of 0.5-10 % (w/v) NaCl (optimum 3-5 % NaCl) and pH of 6-10 (optimum pH 9). Strain AK72T was positive for oxidase, catalase, nitrate reductase, phenylalanine deaminase, ornithine decarboxylase, aesculinase, lipase and urease activities. The predominant fatty acids were iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0 and the cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The major polar lipids of the strain were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminophospholipid, three unidentified phospholipids and three unidentified lipids. The genomic DNA G+C content of the strain AK72T was 36.8 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain AK72T was closely related to Bacillus cellulosilyticus (96.5 %) and Bacillus vedderi (96.3 %), but the novel strain AK72T formed a separate clade with Bacillus aurantiacus whereas B. cellulosilyticus and B. vedderi were clustered in a separate clade. The above data in combination with the phenotypic characteristics and phylogenetic data inferred that strain AK72T represents a novel species of the genus Bacillus, for which the name Bacillusshivajii sp. nov. is proposed. The type strain is AK72T (=MTCC 12636T=KCTC 33981T=JCM 32183T).

Sphingobacterium bovisgrunnientis sp. nov., isolated from yak milk.

A novel Gram-negative, rod shaped, non-motile bacterium, designated strain YK2T, was isolated from yak milk from Leh, India. The strain was positive for oxidase- and catalase-activities and negative for starch hydrolysis, nitrate reduction, citrate utilization, urease, lysine decarboxylase and ornithine decarboxylase activities. The predominant fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, iso-C17 : 1ω9c and C16 : 1ω7c and/or C16 : 1ω6c and/or iso-C15 : 0 2-OH (summed feature 3). The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid and six unidentified lipids. The DNA G+C content of the strain was 38.9 mol%. The 16S rRNA gene sequence analysis indicated that strain YK2T was a member of the genus Sphingobacterium and closely related to Sphingobacterium alimentarium and Sphingobacterium composti with pair-wise sequence similarity of 98.3 and 97.9 %, respectively. The sequence similarity to other members of the genus Sphingobacterium was between 92.6 to 96.3 %. Phylogenetic analysis showed that strain YK2T clustered with Sphingobacterium alimentarium and together clustered with Sphingobacterium composti. DNA-DNA hybridization of strain YK2T with Sphingobacterium alimentarium WCC 4521T and Sphingobacterium composti T5-12T showed a relatedness of only 38 and 54 %, respectively. Based on the phenotypic characteristics and on phylogenetic inference, it appears that strain YK2T represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium bovisgrunnientis sp. nov. is proposed. The type strain of Sphingobacterium bovisgrunnientis sp. nov. is YK2T (=MTCC 12631T=KCTC 52685T=JCM 31951T).

Bacillus alkalilacus sp. nov., isolated from a sediment sample from a lake in India.

An aerobic, endospore-forming, haloalkali-tolerant, Gram-stain-positive, motile, rod-shaped bacterium, designated strain AK73T, was isolated from a sediment sample collected from Sambhar lake, Jaipur, Rajasthan, India. Colonies were circular, 1-2 mm in diameter, glossy, smooth, yellowish and convex with an entire margin after 48 h growth on marine agar at pH 9 and 37 °C. Growth occurred at 15-42 °C, 0-10 % (w/v) NaCl and at a pH range of 7-12. Strain AK73T was positive for catalase and arginine dihydrolase 2 activities, hydrolysis of Tweens 20, 40 and 80, and negative for esculinase, caseinase, gelatinase, ß-galactosidase, lipase (Tween 60) and urease activities. The fatty acids were dominated by branched iso-, anteiso-, saturated fatty acids with a high abundance of iso-C15 : 0, anteiso-C15 : 0, C16 : 0 and anteiso-C17 : 0; MK-7 was the major menaquinone. The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminophospholipid, four unidentified phospholipids and three unidentified lipids. The DNA G+C content of strain AK73T was 54 mol%. Analysis based on comparative 16S rRNA gene sequence analysis indicated that Bacillus alcalophilus was the nearest phylogenetic neighbour, with a pair-wise sequence similarity of 96.0 %. Phylogenetic analysis showed that strain AK73T formed a separate lineage but was loosely associated with a peripheral cluster of organisms that contained Bacillus gibsonii, Bacillus murimartini and Bacillus plakortidis with similarity values of 93.6, 93.5 and 93.4 %, respectively. Based on its phenotypic characteristics and on phylogenetic inference, strain AK73T represents a novel species of the genus Bacillus, for which the name Bacillus alkalilacus sp. nov. is proposed. The type strain is AK73T (=JCM 32184T=MTCC 12637T=KCTC 33880T).

Salibacterium nitratireducens sp. nov., a haloalkalitolerant bacterium isolated from a water sample from Sambhar salt lake, India.

A strictly aerobic, haloalkali-tolerant, Gram-stain-positive, non-motile, rod-shaped bacterium, designated strain SMB4T, was isolated from a water sample collected from Sambhar salt lake, Rajasthan, India. Growth occurred at 25-50 °C, 4-12 % (w/v) NaCl and pH of 5-9. Strain SMB4T was positive for ß-galactosidase, oxidase, catalase and urease activities. The fatty acids were dominated by branched forms of fatty acids with iso- and anteiso-saturated fatty acids, with a high abundance of anteiso-C15 : 0, anteiso-C17 : 0 and C18 : 0. The cell-wall peptidoglycan of strain SMB4T contained meso-diaminopimelic acid, while the polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid and three unidentified lipids. The DNA G+C content of strain SMB4T was 49.1 mol%. A blast sequence similarity search based on 16S rRNA gene sequence indicated that Salibacterium halochares, Salibacterium halotolerans and Salibacterium qingdaonense were the nearest phylogenetic neighbours, with a pair-wise sequence similarities of 98.4, 98.2 and 97.0 % respectively. Phylogenetic analysis showed that strain SMB4T was clustered with S. halochares and together clustered with S. halotolerans and S. qingdaonense. DNA-DNA hybridization of strain SMB4T with S. halochares DSM 21373T, S. halotolerans S7T and S. quigdaonense DSM 21621T showed a relatedness values of only 39.8, 26.3 and 42.8 %, respectively. Based on its phenotypic characteristics and on phylogenetic inference, strain SMB4T represents a novel species of the genus Salibacterium, for which the name Salibacterium nitratireducens sp. nov. is proposed. The type strain is SMB4T (=MTCC 12633T=KCTC 33876T=JCM 32187T).

Bacillus lacus sp. nov., isolated from a water sample of a salt lake in India.

A strictly aerobic, alkaliphilic, Gram-stain-positive, motile, rod-shaped bacterium, designated strain AK74T, was isolated from a water sample collected from Sambhar salt lake, Rajasthan, India. Colonies were circular, 1.2 mm in diameter, shiny, smooth, whitish and convex with an entire margin after 48 h growth at 37 °C with pH 9.0. Growth occurred at 25-42 °C, 0-4 % (w/v) NaCl and at a pH of 7-12. Strain AK74T was positive for aesculinase, caseinase, lipase activities and negative for oxidase, catalase, amylase, cellulase, DNase, gelatinase and urease activities. The fatty acids were dominated by branched iso-, anteiso- and saturated fatty acids with a high abundance of iso-C15 : 0, anteiso-C15 : 0, C16 : 0 and C16 : 1 and the cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The DNA G+C content of strain AK74T was 51.6 mol%. A blast sequence similarity search based on 16S rRNA gene sequences indicated that Bacillus niabensis, Bacillus idriensisand Bacillus halosaccharovorans were the nearest phylogenetic neighbours, with a pair-wise sequence similarities of 96.6, 96.6 and 96.5%, respectively. Phylogenetic analysis showed that strain AK74T clustered with Bacillus mangrove and together clustered with Bacillus idriensisand Bacillus indicus. Based on its phenotypic characteristics and on phylogenetic inference, strain AK74T represents a novel species of the genus Bacillus, for which the name Bacilluslacus sp. nov. is proposed. The type strain is AK74T (=MTCC 12638T=KCTC 33946T=JCM 32185T).

Bacillus mangrovi sp. nov., isolated from a sediment sample from a mangrove forest.

A facultatively anaerobic, endospore forming, alkali-tolerant, Gram-stain-positive, motile, rod-shaped bacterium, designated strain AK61T, was isolated from a sediment sample collected from Coringa mangrove forest, India. Colonies were circular, 1.5 mm in diameter, shiny, smooth, yellowish and convex with entire margins after 48 h growth at 30 °C. Growth occurred at 15-42 °C, with 0-3 % (w/v) NaCl and at pH 6-9. AK61T was positive for amylase activity and negative for oxidase, catalase, aesculinase, caseinase, cellulase, DNase, gelatinase, lipase and urease activities. The fatty acids were dominated by branched types with iso- and anteiso- saturated fatty acids with a high abundance of iso-C14 : 0, iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0; the cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid; and MK-7 was the major menaquinone. DNA-DNA hybridization between AK61T and Bacillus indicus MTCC 4374T and between AK61T and Bacillus indicus KCTC 3880 showed relatedness of 37.99 and 33.32 % respectively. The DNA G+C content of AK61T was 44 mol%. The results of a blast sequence similarity search based on 16S rRNA gene sequences indicated that Bacillus cibi and Bacillus indicus were the nearest phylogenetic neighbours, with a pair-wise sequence similarity of 97.69 and 97.55 % respectively. The results of phylogenetic analysis indicated that AK61T was clustered with Bacillus idriensis and Bacillus indicus. On the basis of its phenotypic characteristics and phylogenetic inference, AK61T represents a novel species of the genus Bacillus, for which the name Bacillus mangrovi sp. nov. is proposed. The type strain is AK61T (=JCM 31087T=MTCC 12015T=KCTC 33872T).

Oceanospirillum sanctuarii sp. nov., isolated from a sediment sample.

A novel Gram-staining-negative, spiral-shaped, pale-yellow, non-sporulating, motile, aerobic bacterium, designated strain AK56T, was isolated from a sediment sample collected at the Coringa Wildlife Sanctuary, India. Colonies on marine agar were circular, pale yellow, shiny, translucent, 1-2 mm in diameter, convex and had an entire margin. The major fatty acids included C16 : 1, C16 : 1ω7c/C16 : 1ω6c and C18 : 1ω7c. Polar lipids included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids, one unidentified phospholipid and five unidentified lipids. DNA-DNA hybridization between strain AK56T and Oceanospirillum linum LMG 5214T and 'Oceanospirillum nioense ' NIO-S6 showed relatedness values of 39.91 and 23.62 %, respectively. The DNA G+C content of strain AK56T was found to be 50.3 mol%. A sequence similarity search for the 16S rRNA gene sequence revealed that O. linum and O. nioense were the nearest phylogenetic neighbours, with a pair-wise sequence similarity of 98.9 and 98.2 %, respectively. Phylogenetic analysis also showed the formation of a cluster including strain AK56T with close relative O. linum and O. nioense. Based on the observed phenotypic, chemotaxonomic characteristics and phylogenetic analysis, strain AK56T is described in this study as a novel species in the genus Oceanospirillum, for which the name Oceanospirillum sanctuarii sp. nov. is proposed. The type strain of Oceanospirillumsanctuarii is AK56T (=MTCC 12005T=JCM 19193T=KCTC 52973T).

Imhoffiella gen. nov., a marine phototrophic member of the family Chromatiaceae including the description of Imhoffiella purpurea sp. nov. and the reclassification of Thiorhodococcus bheemlicus Anil Kumar et al. 2007 as Imhoffiella bheemlica comb. nov.

A coccoid-shaped phototrophic purple sulfur bacterium, strain AK35T, was isolated from a coastal surface water sample collected from Visakhapatnam, India. Cells were Gram-stain-negative, motile and purple, containing bacteriochlorophyll a and the carotenoid rhodopinal as major photosynthetic pigments. Strain AK35T was able to grow photoheterotrophically and could utilize a number of organic substrates. It was unable to grow photoautotrophically. Strain AK35T was able to utilize sulfide and thiosulfate as electron donors. The main fatty acids present were identified as C16 : 0, C18 : 1ω7c, and C16 : 1ω7c and/or iso-C15 : 0 2OH (summed feature 3). Strain AK35T contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and six unidentified lipids as polar lipids. The G+C content of the DNA of strain AK35T was 63.1 mol%. 16S rRNA gene sequence comparisons indicated that the isolate represented a member of the family Chromatiaceae. 16S rRNA gene sequence analysis indicated that strain AK35T is phylogenetically distinctly positioned outside the groups of most members of the genus Thiorhodococcus, clustered with members of the genera Marichromatium and Phaeochromatium, but was most closely related to Thiorhodococcus bheemlicus with a pairwise sequence similarity of 98.75 %. Based on DNA-DNA hybridization between strain AK35T and Thiorhodococcus bheemlicus MTCC 8120T a relatedness of 39.46 % was established. Distinct morphological, physiological and genotypic differences from these previously described taxa supported the classification of the new isolate as a representative of a novel species in a new genus, for which the name Imhoffiella purpurea gen. nov., sp. nov. is proposed. The type strain of Imhoffiella purpurea is AK35T (=JCM 18851T=KCTC 15575T=MTCC 12304T). In addition, Thiorhodococcus bheemlicus is recognized as another species of this genus and transferred to Imhoffiella bheemlica comb. nov.

TfoI produced by Tepidimonas fonticaldi PL17, a moderate thermophilic bacterium, is an isoschizomer of MseI.

A moderately thermophilic Gram-negative bacterium isolated from the Polok hot spring, Sikkim, India, was identified as a strain (PL17) of Tepidimonas fonticaldi by 16S rDNA sequencing. T. fonticaldi PL17 produces a Type IIP restriction endonuclease; named TfoI. Restriction mapping, run-off sequencing of TfoI-digests of dsDNA fragments, and end compatibility of TfoI with NdeI confirmed that the enzyme recognizes and cleaves the sequence 5'-T^TAA-3', and is thus an isoschizomer of MseI. The TfoI restriction-modification genes in the T. fonticaldi PL17 genome were identified, and the annotated TfoI protein encodes a protein of 181 amino acid residues that shares 47.2% sequence identity with MseI. The native enzyme was purified using a four-column chromatography protocol, and its functional homogeneity was confirmed by standard quality control tests. The ESI-MS measured molecular weight of purified TfoI (20.696 kDa) is in agreement with that of the calculated monomeric molecular weight of the predicted TfoI protein sequence (20.694 kDa). TfoI exhibits optimal activity in the temperature range of 55-70 °C with Mg+2 or Co+2 as cofactor. Similar to its isoschizomers, TfoI can be used as the frequent cutter for genome analysis.