Pradip Kumar Singh

Characterization of two antimicrobial peptides produced by a halotolerant Bacillus subtilis strain SK.DU.4 isolated from a rhizosphere soil sample

A bacterial strain producing two antimicrobial peptides was isolated from a rhizosphere soil sample and identified as Bacillus subtilis based on both phenotypic and 16S rRNA gene sequence phylogenetic analysis. It grew optimally up to 14% NaCl and produced antimicrobial peptide within 24 h of growth. The peptides were purified using a combination of chemical extraction and chromatographic techniques. The MALDI-TOF analysis of HPLC purified fractions revealed that the strain SK.DU.4 secreted a bacteriocin-like peptide with molecular mass of 5323.9 Da and a surface-active lipopeptide (m/z 1056 Da). The peptide mass fingerprinting of low-molecular-weight bacteriocin exhibited significant similarity with stretches of secreted lipoprotein of Methylomicrobium album BG8 and displayed 70% sequence coverage. MALDI MS/MS analysis elucidated the lipopeptide as a cyclic lipopeptide with a β-hydroxy fatty acid linked to Ser of a peptide with seven α-amino acids (Asp-Tyr-Asn-Gln-Pro-Asn-Ser) and assigned it to iturin-like group of antimicrobial biosurfactants. However, it differed in amino acid composition with other members of the iturin family. Both peptides were active against Gram-positive bacteria, suggesting that they had an additive effect.

Identification, Purification and Characterization of Laterosporulin, a Novel Bacteriocin Produced by Brevibacillus sp. Strain GI-9

Background Bacteriocins are antimicrobial peptides that are produced by bacteria as a defense mechanism in complex environments. Identification and characterization of novel bacteriocins in novel strains of bacteria is one of the important fields in bacteriology. Methodology/Findings The strain GI-9 was identified as Brevibacillus sp. by 16 S rRNA gene sequence analysis. The bacteriocin produced by strain GI-9, namely, laterosporulin was purified from supernatant of the culture grown under optimal conditions using hydrophobic interaction chromatography and reverse-phase HPLC. The bacteriocin was active against a wide range of Gram-positive and Gram-negative bacteria. MALDI-TOF experiments determined the precise molecular mass of the peptide to be of 5.6 kDa and N-terminal sequencing of the thermo-stable peptide revealed low similarity with existing antimicrobial peptides. The putative open reading frame (ORF) encoding laterosporulin and its surrounding genomic region was fished out from the draft genome sequence of GI-9. Sequence analysis of the putative bacteriocin gene did not show significant similarity to any reported bacteriocin producing genes in database. Conclusions We have identified a bacteriocin producing strain GI-9, belonging to the genus Brevibacillus sp. Biochemical and genomic characterization of laterosporulin suggests it as a novel bacteriocin with broad spectrum antibacterial activity.

Phosphorylation of PhoP Protein Plays Direct Regulatory Role in Lipid Biosynthesis of Mycobacterium tuberculosis

Background: PhoP is global regulator of Mycobacterium tuberculosis physiology. However, the role of phosphorylation of PhoP remains unknown. Results: PhoP activates complex lipid biosynthesis only upon phosphorylation. Conclusion: PhoP regulates lipid biosynthesis by a phosphorylation-dependent mechanism to contribute to morphology of the bacilli. Significance: This study sheds light on the unexplored role of phosphorylation of PhoP in regulating biosynthesis of lipids unique to M. tuberculosis. Mycobacterium tuberculosis PhoP is essential for virulence and intracellular growth of the tubercle bacilli. Genetic evidence suggests that PhoP regulates complex lipid biosynthesis, and absence of some of these lipid molecules in a phoP mutant partly accounts for its attenuated growth in macrophages and/or mice. To investigate the mechanism of regulation, here we demonstrate the essentiality of phosphorylation of PhoP in the regulation of complex lipid biosynthesis. We show that phosphorylated PhoP activates transcription of pks2 and msl3, gene(s) encoding polyketide β-ketoacyl synthases through direct DNA binding at the upstream regulatory region(s) of the target genes. Our results identify the genetic determinants recognized by PhoP and show that activation of target genes requires interaction(s) of the phosphorylated regulator at the cognate binding sites. The fact that these sites within the regulatory region of respective genes do not bind in vitro with either unphosphorylated or phosphorylation-deficient PhoP protein is consistent with phosphorylation-dependent assembly of the transcription initiation complex leading to in vivo transcriptional activation. Together, these results reveal so far unknown molecular mechanisms of how PhoP contributes to M. tuberculosis cell wall composition by regulating complex lipid biosynthesis.

Genome Sequence of Brevibacillus laterosporus Strain GI-9

We report the 5.18-Mb genome sequence of Brevibacillus laterosporus strain GI-9, isolated from a subsurface soil sample during a screen for novel strains producing antimicrobial compounds. The draft genome of this strain will aid in biotechnological exploitation and comparative genomics of Brevibacillus laterosporus strains.

Genome sequence of Pediococcus pentosaceus strain IE-3.

We report the 1.8-Mb genome sequence of Pediococcus pentosaceus strain IE-3, isolated from a dairy effluent sample. The whole-genome sequence of this strain will aid in comparative genomics of Pediococcus pentosaceus strains of diverse ecological origins and their biotechnological applications.

The intramolecular disulfide-stapled structure of laterosporulin, a class IId bacteriocin, conceals a human defensin-like structural module.

The growing emergence of antibiotic‐resistant bacteria has led to the exploration of naturally occurring defense peptides as antimicrobials. In this study, we found that laterosporulin (LS), a class IId bacteriocin, effectively kills active and nonmultiplying cells of both Gram‐positive and Gram‐negative bacteria. Fluorescence and electron microscopy suggest that growth inhibition occurs because of increased membrane permeability. The crystal structure of LS at 2.0 Å resolution reveals an all‐β conformation of this peptide, with four β‐strands forming a twisted β‐sheet. All six intrinsic cysteines are intramolecularly disulfide‐bonded, with two disulfides constraining the N terminus of the peptide and the third disulfide crosslinking the extreme C terminus, resulting in the formation of a closed structure. The significance of disulfides in maintaining the in‐solution peptide structure was confirmed by CD and fluorescence analyses. Despite a low overall sequence similarity, LS has disulfide connectivity [CI–CV, CII–CIV, and CIII–CVI] like that of β‐defensins and a striking architectural similarity with α‐defensins. Therefore LS presents a missing link between bacteriocins and mammalian defensins, and is also a potential antimicrobial lead, in particular against nonmultiplying bacteria.

Micrococcus lactis sp. nov., isolated from dairy industry waste

A Gram-positive, yellow-pigmented, actinobacterial strain, DW152(T), was isolated from a dairy industry effluent treatment plant. 16S rRNA gene sequence analysis indicated that strain DW152(T) exhibited low similarity with many species with validly published names belonging to the genera Micrococcus and Arthrobacter. However, phenotypic properties including chemotaxonomic markers affiliated strain DW152(T) to the genus Micrococcus. Strain DW152(T) had ai-C(15:0) and i-C(15:0) as major cellular fatty acids, and MK-8(H(2)) as the major menaquinone. The cell-wall peptidoglycan of strain DW152(T) had l-lysine as the diagnostic amino acid and the type was A4α. The DNA G+C content of strain DW152(T) was 68.0 mol%. In 16S rRNA gene sequence analysis, strain DW152(T) exhibited significant similarity with Micrococcus terreus NBRC 104258(T), but the mean value of DNA-DNA relatedness between these strains was only 42.3%. Moreover, strain DW152(T) differed in biochemical and chemotaxonomic characteristics from M. terreus and other species of the genus Micrococcus. Based on the above differences, we conclude that strain DW152(T) should be treated as a novel species of the genus Micrococcus, for which the name Micrococcus lactis sp. nov. is proposed. The type strain of Micrococcus lactis sp. nov. is DW152(T) (=MTCC10523(T) =DSM 23694(T)).

Characterization of heat induced spherulites of lysozyme reveals new insight on amyloid initiation

Here, we report results obtained during our experiments to visualize how heat transforms globular protein, lysozyme into building block of β-amyloids. Light scattering experiments showed formation of lower order associated species around 50–70 °C followed by rapid cooperativity to β-amyloid fibrils. Interestingly, crystallization drops set at higher temperatures either led to aggregates or spherulites. The latter possess an amorphous β-fibril rich core with thin crystalline needles projecting outwards. Diffraction of the crystalline outgrowths revealed novel dimers and trimers of lysozyme where individual chains were similar to monomer with marginal gain in β-sheet content. Importantly, analysis of Amide I stretching frequencies showed that protein loses its secondary structure at temperatures higher than where we obtained crystals followed by rapid gain in β-sheet content. Interestingly, attempts to use the needles as seeds for more crystals led to “broom-like” fibril formations at the ends. Further, aggregation inhibitors like arginine and benzyl alcohol completely obliterated spherulites formation during crystallization. Refinement of crystals of lysozyme in presence of these molecules showed these small molecules bind to the interfaces of heat associated dimers and trimers. Overall our work concludes that heat induced weakly associated structures of lysozyme are the first step towards its amyloid formation.

Fulvivirga imtechensis sp. nov., a member of the phylum Bacteroidetes

A novel, Gram-staining-negative, yellow-coloured, rod-shaped, obligately aerobic, non-motile bacterium, designated strain AK7(T), was isolated from seawater collected on the coast at Visakhapatnam, Andhra Pradesh, India. The predominant fatty acids of the novel strain were iso-C(15 : 0), iso-C(15 : 0) 3-OH, C(16 : 1)ω5c, iso-C(17 : 0) 3-OH and summed features 3 (C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH) and 4 (iso-C(17 : 1) I and/or anteiso-C(17 : 1) B). The major respiratory quinone was MK-7 and the polar lipid profile comprised phosphatidylethanolamine, two unidentified aminolipids and four other unidentified lipids. In phylogenetic analysis based on 16S rRNA gene sequences, strain AK7(T) appeared most closely related to Fulvivirga kasyanovii KMM 6220(T) (95.9 % sequence similarity), a member of the family Flammeovirgaceae in the phylum Bacteroidetes. The genomic DNA G+C content of strain AK7(T) was 55.1 mol%. Based on the morphological, biochemical, physiological, chemotaxonomic and phylogenetic evidence, strain AK7(T) represents a novel species of the genus Fulvivirga for which the name Fulvivirga imtechensis sp. nov. is proposed. The type strain is AK7(T) (= MTCC 11053(T) = JCM 17390(T)).

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).