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
4NO
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.
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 (Co
2+
or Mn
2+
) for its improved activity. The pH and temperature optima for this protease were 10.5 and 42°C respectively.
Corynebacterium glutamicum is one of the well-studied industrial strain that is used for the production of nucleotides and amino acids. Recently, it has also been studied as a possible producer of ...organic acids such as succinic acid, based on its ability to produce organic acids under an oxygen deprivation condition. In this study, we conducted the optimization of medium components for improved succinate production from C. glutamicum under an oxygen deprivation condition by Plackett-Burman design and applied a response surface methodology. A Plackett-Burman design for ten factors such as glucose, ammonium sulfate, magnesium sulfate, potassium phosphate (K2HPO4 and KH2PO4), iron sulfate, manganese sulfate, biotin, thiamine, and sodium bicarbonate was applied to evaluate the effects on succinate production. Glucose, ammonium sulfate, magnesium sulfate, and dipotassium phosphate were found to have significant influence on succinate production, and the optimal concentrations of these four factors were sequentially investigated by the response surface methodology using a Box-Behnken design. The optimal medium components obtained for achieving maximum concentration of succinic acid were as follows: glucose 10 g/l, magnesium sulfate 0.5 g/l, dipotassium phosphate (K2HPO4) 0.75 g/l, potassium dihydrogen phosphate (KH2PO4) 0.5 g/l, iron sulfate 6 mg/l, manganese sulfate 4.2 mg/l, biotin 0.2 mg/l, thiamine 0.2 mg/l, and sodium bicarbonate 100 mM. The parameters that differed from a normal BT medium were glucose changed from 40 g/l to 10 g/l, dipotassium phosphate (K2HPO4) 0.5 g/l changed to 0.75 g/l, and ammonium sulfate ((NH4)2SO4) 7 g/l changed to 0 g/l. Under these conditions, the final succinic acid concentration was 16.3 mM, which is about 1.46 fold higher than the original medium (11.1 mM) at 24 h. This work showed the improvement of succinate production by a simple change of media components deduced from sequential optimization.
The 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.
The 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.
Totally, 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.
Multiplex 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.
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.
Increased Sensitivity to Chloramphenicol by Inactivation of manB in Streptomyces coelicolor Rajesh, Thangamani, Konkuk University, Seoul, Republic of Korea; Song, E.J., Seoul National University, Seoul, Republic of Korea; Lee, B.R., Seoul National University, Seoul, Republic of Korea ...
Journal of microbiology and biotechnology,
10/2012, Volume:
22, Issue:
10
Journal Article
Peer reviewed
Phosphomannomutase (ManB) is involved in the biosynthesis of GDP-mannose, which is vital for numerous processes such as synthesis of carbohydrates, production of alginates and ascorbic acid, and ...post-translational modification of proteins. Here, we discovered that a deletion mutant of manB (BG101) in Streptomyces coelicolor (S. coelicolor) showed higher sensitivity to bacteriostatic chloramphenicol (CM) than the wild-type strain (M145), along with decreased production of CM metabolites. Deletion of manB also decreased the mRNA expression level of drug efflux pumps (i.e., cmlR1 and cmlR2) in S. coelicolor, resulting in increased sensitivity to CM. This is the first report on changes in antibiotic sensitivity to CM by deletion of one glycolysis-related enzyme in S. coelicolor, and the results suggest different approaches for studying the antibiotic-resistant mechanism and its regulation.
•A CODEHOP PCR based screening method was employed for type III polyketide synthase gene identification in fungal endophytes.•By this approach, partial type III PKS genes from eight fungal endophytes ...were amplified and sequenced.•FiPKS gene from Fusarium incarnatum BMER1, an endophyte of Bacopa monnieri was cloned and functionally characterized.•FiPKS produced pyrones and resorcinols with the highest catalytic efficiency of 7.6 x 104 s-1 M-1towards stearoyl CoA.
Endophytic fungi provide benefits to host plants by producing a diverse class of secondary metabolites (natural products). Arrays of polyketide natural products are synthesized by specific classes of polyketide synthases (PKS I, II and III) in host organisms. In the present study, we attempt to screen and identify type III PKSs in culturable fungal endophytes isolated from the ethno medicinal plants including Arbus precatorius, Bacopa monnieri,Citrus aurantifolia and Datura metel to detect the genetic potential of endophytic fungi in producing bioactive compounds. A total of seventeen endophytic fungal strains belonging to eight genera were identified using fungal morphology and rDNA-ITS phylogenetic analyses. A CODEHOP-PCR based strategy was followed to design degenerate primers for the screening of type III PKS genes from fungal endophytes. We had successfully amplified partial PKS genes from eight endophytes. The amplified PKS sequences showed 60–99% identity to already characterized/putative PKS genes. From the partial sequence of FiPKS from Fusarium incarnatum BMER1, a full-length gene was amplified, cloned and characterized. FiPKScDNA was cloned and expressed in E. coli Lemo21 (DE3) and the purified protein was shown to produce pyrones and resorcinols using acyl-CoA thioesters as substrates. FiPKS showed the highest catalytic efficiency of 7.6 × 104 s−1 M-1 with stearoyl CoA as a starter unit. This study reports the identification and characterization of type III PKS from endophytes of medicinal plants by CODEHOP PCR.
•The study reports the functional characterization of two fungal type III polyketide synthases, SmPKS and CtPKS with high catalytic efficiency.•Both the recombinant PKSs efficiently synthesize ...pyrones, resorcylic acids and resorcinols using various acyl- CoA (C4–C20) as starter units.•TrCtPKS produces pentaketide and hexaketide resorcinols from arachidoyl-CoA with a high catalytic efficiency of 6.2 × 104 s−1 M−1.•Enhanced binding interactions of CtPKS residues forming intermolecular contacts at the active site could be attributed to its high specificity.
Two putative type III polyketide synthase genes (PKS) were identified from Sordariomycetes fungi. These two type III PKS genes from Sordaria macrospora (SmPKS) and Chaetomium thermophilum (CtPKS), shared 59.8% sequence identity. Both, full-length and truncated versions of type III PKSs were successfully cloned and overexpressed in a bacterial host, Escherichia Coli BL21 (DE3) using a N-terminus hexa-histidine tag. The full-length and the truncated construct of PKSs showed similar activity profiles, suggesting that additional amino acid residues at the C-terminal of both SmPKS and CtPKS may not be involved in catalytic functions. We demonstrate that these two recombinant polyketide synthases could efficiently synthesize tri- and tetraketide pyrones, resorcinols and resorcylic acids using various acyl-CoAs (C4–C20) as starter units. The truncated S. macrospora polyketide synthases (TrSmPKS) showed a maximum of 7.0 × 104 s−1 M−1 catalytic efficiency towards stearoyl-CoA.Whereas, truncated C. thermophilum polyketide synthases (TrCtPKS) preferred the long-chain acyl-CoA starter arachidoyl-CoA, to produce pentaketide and hexaketide resorcinols with a high catalytic efficiency of 6.2 × 104 s−1 M−1. Homology model and substrate docking analyses suggest a shorter distance between sulfur of catalytic Cys152 and thioester carbonyl group of arachidoyl-CoA as well as stronger imidazolium–thiolate ion pair distance in TrCtPKS between catalytic Cys152-His309 compared to TrSmPKS- arachidoyl CoA complex. Enhanced binding interactions of CtPKS residues forming intermolecular contacts at the active site could be attributed to its high specificity towards arachidoyl-CoA. This study reports the functional characterization of two fungal type III polyketide synthases, SmPKS and CtPKS with high catalytic efficiency from S. macrospora and C. thermophilum respectively. Furthermore, the results suggested that the both SmPKS and CtPKS could be attractive targets for protein engineering to discern the unique substrate specificity and catalytic efficiency.
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.
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