Thangamani Rajesh

Influence of medium components and fermentation conditions on the production of bacteriocin(s) by Bacillus licheniformis AnBa9

Recently, antibacterial peptides are gaining more attention as an alternative therapeutics and food and other products from spoilage and deterioration. Antibacterial peptide producing strains were isolated from sediments of slaughterhouse sewage wastes. One among them, identified as Bacillus licheniformis inhibited the growth of several gram positive bacteria. Response surface methodology with central composite rotary design was used for optimization of fermentation medium and conditions for antibacterial peptide production. Lactose, NH(4)NO(3), yeast extract and NaCl and environmental factors such as pH, temperature and incubation period were selected as variables. Among ingredients, high concentration of yeast extract and NaCl had a positive effect on antibacterial peptide production and specific activity, respectively. Alkaline pH and high temperature favoured the production of antibacterial peptide by B. licheniformis AnBa9. Under optimized condition, B. licheniformis AnBa9 produced 25-fold higher production of antibacterial peptide than the un-optimized condition. Biochemical characteristics of the antibacterial peptides of B. licheniformis AnBa9 revealed that they are of bacteriocin type.

Identification and characterization of alkaline serine protease from goat skin surface metagenome

Metagenomic DNA isolated from goat skin surface was used to construct plasmid DNA library in Escherichia coli DH10B. Recombinant clones were screened for functional protease activity on skim milk agar plates. Upon screening 70,000 clones, a clone carrying recombinant plasmid pSP1 exhibited protease activity. In vitro transposon mutagenesis and sequencing of the insert DNA in this clone revealed an ORF of 1890 bp encoding a protein with 630 amino acids which showed significant sequence homology to the peptidase S8 and S53 subtilisin kexin sedolisin of Shewanella sp. This ORF was cloned in pET30b and expressed in E. coli BL21 (DE3). Although the cloned Alkaline Serine protease (AS-protease) was overexpressed, it was inactive as a result of forming inclusion bodies. After solubilisation, the protease was purified using Ni-NTA chromatography and then refolded properly to retain protease activity. The purified AS-protease with a molecular mass of ~63 kDa required a divalent cation (Co2+ or Mn2+) for its improved activity. The pH and temperature optima for this protease were 10.5 and 42°C respectively.

Characterization of a new ScbR-like γ-butyrolactone binding regulator (SlbR) in Streptomyces coelicolor

Abstractγ-Butyrolactones in Streptomyces are well recognized as bacterial hormones, and they affect secondary metabolism of Streptomyces. γ-Butyrolactone receptors are considered important regulatory proteins, and various γ-butyrolactone synthases and receptors have been reported in Streptomyces. Here, we characterized a new regulator, SCO0608, that interacted with SCB1 (γ-butyrolactone of Streptomyces coelicolor) and bound to the scbR/A and adpA promoters. The SCO0608 protein sequences are not similar to those of any known γ-butyrolactone binding proteins in Streptomyces such as ScbR from S. coelicolor or ArpA from Streptomyces griseus. Interestingly, SCO0608 functions as a repressor of antibiotic biosynthesis and spore formation in R5 complex media. We showed the existence of another type of γ-butyrolactone receptor in Streptomyces, and this SCO0608 was named ScbR-like γ-butyrolactone binding regulator (SlbR) in S. coelicolor.

Superantigen profiles of emm and emm-like typeable and nontypeable pharyngeal streptococcal isolates of South India

BackgroundThe major virulence factors determining the pathogenicity of streptococcal strains include M protein encoded by emm and emm-like (emmL) genes and superantigens. In this study, the distribution of emm, emmL and superantigen genes was analyzed among the streptococcal strains isolated from the patients of acute pharyngitis.MethodsThe streptococcal strains were isolated from the throat swabs of 1040 patients of acute pharyngitis. The emm and emmL genes were PCR amplified from each strain and sequenced to determine the emm types. The dot-blot hybridization was performed to confirm the pathogens as true emm nontypeable strains. The presence of eleven currently known superantigens was determined in all the strains by multiplex PCR.ResultsTotally, 124 beta-hemolytic streptococcal strains were isolated and they were classified as group A streptococcus (GAS) [15.3% (19/124)], group C streptococcus (GCS) [59.7% (74/124)] and group G streptococcus (GGS) [25.0% (31/124)]. Among 124 strains, only 35 strains were emm typeable and the remaining 89 strains were emm nontypeable. All GAS isolates were typeable, whereas most of the GCS and GGS strains were nontypeable. These nontypeable strains belong to S. anginosus [75.3% (67/89)] and S. dysgalactiae subsp. equisimilis [24.7% (22/89)]. The emm and emmL types identified in this study include emm12.0 (28.6%), stG643.0 (28.6%), stC46.0 (17.0%), emm30.11 (8.5%), emm3.0 (2.9%), emm48.0 (5.7%), st3343.0 (2.9%), emm107.0 (2.9%) and stS104.2 (2.9%). Various superantigen profiles were observed in typeable as well as nontypeable strains.ConclusionsMultiplex PCR analysis revealed the presence of superantigens in all the typeable strains irrespective of their emm types. However, the presence of superantigen genes in emm and emmL nontypeable strains has not been previously reported. In this study, presence of at least one or a combination of superantigen coding genes was identified in all the emm and emmL nontypeable strains. Thus, the superantigens may inevitably play an important role in the pathogenesis of these nontypeable strains in the absence of the primary virulence factor, M protein.

Functional analysis of a putative holin-like peptide-coding gene in the genome of Bacillus licheniformis AnBa9.

BhlA, a putative holin-like protein of Bacillus licheniformis AnBa9 expressed in Escherichia coli BL21(DE3) showed antibacterial activity against several gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and Micrococcusluteus. Deletion analysis of bhlA suggests that a hydrophobic transmembrane domain, BhlATM is essential for antibacterial activity. Though the minimum inhibitory concentration (MIC) of BhlA was sevenfold lower than BhlATM, transmembrane domain deleted construct (BhlA∆TM) had no antibacterial activity. The expression of BhlA in E. coli was found to be toxic to cells. Therefore, the bhlA was cloned in yeast surface display vector pYD1 and expressed in Saccharomyces cerevisiae. The surface displayed yeast showed inhibition of several gram-positive bacteria. This recombinant yeast expressing BhlA may be used as biodrug for efficient control of multiple drug-resistant bacterial infections.

Functional characterization of a new holin-like antibacterial protein coding gene tmp1 from goat skin surface metagenome

We have identified a holin-like gene from a goat skin surface metagenome. The ORF designated tmp1 coding for 34 amino acids shared sequence similarity with putative holin-like toxin genes. To analyze the antibacterial activity of tmp1 encoded protein, this ORF was cloned and expressed in Escherichia coli BL21(DE3). The expressed gene product Tmp1 exhibited antibacterial activity against Gram-positive bacteria but not to Gram-negative bacteria. A single transmembrane domain (TMD) was identified within Tmp1 and deletion analysis of the N-terminal region and TMD indicated TMD to be responsible for antibacterial activity. The TMD-dependent antibacterial activity was validated using a synthetic peptide with the amino acid sequence of TMD. Besides antibacterial activity, Tmp1 also complemented the function of holin in a lysis-defective bacteriophage lambda. To broaden the spectrum of antibacterial activity, a mutant library of tmp1 was generated by random mutagenesis. Four mutants with amino acid substitutions at the N-terminus of Tmp1 exhibited increased antibacterial activity against Gram-positive and Gram-negative bacteria and were not hemolytic. An improved activity of these mutant proteins is attributed to their increased hydrophobicity.

Deletion of an architectural unit, leucyl aminopeptidase (SCO2179), in Streptomyces coelicolor increases actinorhodin production and sporulation

Several reports state that three architectural units, including integration host factor, leucyl aminopeptidase (PepA), and purine regulator, are involved in transcriptional process with RNA polymerase in Escherichia coli. Similarly, Streptomyces species possess the same structural units. We previously identified a protein, Streptomyces integration host factor (sIHF), involved in antibiotic production and sporulation. Subsequently, the function of PepA (SCO2179) was examined in detail. PepA is highly conserved among various Streptomyces spp., but it has not yet been characterized in Streptomyces coelicolor. While it is annotated as a putative leucyl aminopeptidase because it contains a peptidase M17 superfamily domain, this protein did not exhibit leucyl aminopeptidase activity. SCO2179 deletion mutant showed increased actinorhodin production and sporulation, as well as more distinct physiological differences, particularly when cultured on N-acetylglucosamine (GlcNAc) minimal media. The results of two-dimensional gel analysis and reverse transcription PCR showed that the SCO2179 deletion increased protein and mRNA levels of ftsZ, ssgA, and actinorhodin (ACT)-related genes such as actII-ORF4, resulting in increased actinorhodin production and spore formation in minimal media containing GlcNAc.

A new flow path design for multidimensional protein identification technology using nano-liquid chromatography electrospray ionization mass spectrometry

Multidimensional protein identification technology (MudPIT) is one of the most versatile methods for separating and identifying highly complex peptides or proteins. However, there are still inherent problems resulting from salt in eluent systems and instrumentation with MudPIT. We designed a novel and simple flow path using two-valve system and successfully performed a fully automated peptide analysis using MudPIT coupled with nano-liquid chromatography electrospray ionization mass spectrometry (nLC-ESI-MS). It enables to generate a remarkably stable nanospray during the MudPIT analysis and realize the fully automated MudPIT system. This column arrangement could be easily installed to avoid laborious loading steps and unstable ionization from discontinuous flow. Consequently, the new flow path design for MudPIT system guarantees the detection of more peptides and higher protein coverage in proteome analysis.

Genomic Technologies in Environmental Bioremediation

Environmental pollution is a serious global problem. In recent years, microorganisms have been successfully employed in bioremediation of polluted environments. Studies on microorganisms have gained momentum due to the availability of complete genome sequences for microbes in well studied ecosystems. Due to limitations in understanding the culture conditions of environmental microbes, culture independent approaches are gaining much attention in the field of bioremediation. High-throughput technologies coupled with metagenomic analyses of soil microbes have led to the identification of catabolic pathways involved in degradation of organic and inorganic contaminants in soil. The novel properties of identified gene(s) clusters suggested metagenomics studies to have a real impact in bioremediation. Identified organisms have not only proved important in development of bioremediation processes but also in the design of new biosensors to detect the presence of pollutants in the environment. With the advent of environmental arrays, it is highly possible to understand the global analysis of gene expression of soil microbes in response to environmental changes. The application of genomics in understanding soil microorganisms involved in bioremediation has been presented in this review.

Functional analysis of the gene SCO1782 encoding Streptomyces hemolysin (S-hemolysin) in Streptomyces coelicolor M145.

In the process of evaluating the growth of Streptomyces coelicolor on rich media such as blood agar, we found that S. coelicolor a non-pathogenic, well-known antibiotic producer had the ability to grow and produce a prominent hemolytic zone. By comparing the growth with an agarase gene mutant of S. coelicolor, a similar prominent hemolytic zone was found to develop due to the organisms hemolytic activity. After the confirmation of hemolytic activity from S. coelicolor, the genome was searched for hemolysin-coding genes; consequently, SCO1782, SCO2534, and SCO3882 were identified, whose products were annotated as a putative, membrane, and hypothetical proteins, respectively. Functional characterization of all the recombinant proteins expressed in Escherichia coli BL21(DE3) revealed that only SCO1782 exhibited hemolytic activity. This S. coelicolor protein, designated as S-hemolysin, showed sequence similarity toward hemolysins from Brachyspira hyodysenteriae (35%) and Mycobacterium tuberculosis (62%). Recombinant hemolysin exhibited activity against sheep blood erythrocytes and cytolytic activity against human fibroblast cells. Deletion of SCO1782 resulted in complete loss of hemolysin activity in S. coelicolor.