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.     

The molecular chaperone hsp70 interacts with the cytosolic II-III loop of the Cav2.3 E-type voltage-gated Ca2+ channel.

Multiple types of voltage-activated Ca2+ channels (T, L, N, P, Q, R type) coexist in excitable cells and participate in synaptic differentiation, secretion, transmitter release, and neuronal plasticity. Ca2+ ions entering cells trigger these events through their interaction with the ion channel itself or through Ca2+ binding to target proteins initiating signalling cascades at cytosolic loops of the ion conducting subunit (Cava1). These loops interact with target proteins in a Ca2+-dependent or independent manner. In Cav2.3-containing channels the cytosolic linker between domains II and III confers a novel Ca2+ sensitivity to E-type Ca2+ channels including phorbol ester sensitive signalling via protein kinase C (PKC) in Cav2.3 transfected HEK-293 cells. To understand Ca2+ and phorbol ester mediated activation of Cav2.3 Ca2+ channels, protein interaction partners of the II-III loop were identified. FLAG-tagged II-III - loop of human Cav2.3 was over-expressed in HEK 293 cells, and the molecular chaperone hsp70, which is known to interact with PKC, was identified as a novel functional interaction partner. Immunopurified II-III loop-protein of neuronal and endocrine Cav2.3 splice variants stimulate autophosphorylation of PKCa, leading to the suggestion that hsp70--binding to the II-III loop--may act as an adaptor for Ca2+ dependent targeting of PKC to E-type Ca2+ channels.     

Production and characterization of a low‐molecular‐weight bacteriocin from Bacillus licheniformis MKU3

Aims: Enhancing production and characterization of a low‐molecular‐weight bacteriocin from Bacillus licheniformis MKU3. Methods and Results: The culture supernatant of B. licheniformis MKU3 exhibited bacteriocin‐like activity against Gram‐positive and ‐negative bacteria and different fungi and yeast. SDS–PAGE analysis of the extracellular proteins of B. licheniformis MKU3 revealed a bacteriocin‐like protein with a molecular mass of 1·5 kDa. This bacteriocin activity was found to be stable under a pH range of 3·0–10·0 and at temperatures up to 100°C for 60 min, but inactivated by proteinase K, trypsin or pronase E. An experimental fractional factorial design for optimization of production medium resulted in a maximum activity of bacteriocin (11 000 AU ml^?1) by B. licheniformis MKU3. Conclusions: A low‐molecular‐weight bacteriocin‐like protein from B. licheniformis MKU3 exhibited a wide spectrum of antimicrobial activity against several Gram‐positive bacteria, several fungi and yeast. A 3·6‐fold increase in the production of bacteriocin was achieved using the culture medium optimized through a fractional factorial design. Significance and Impact of the Study: A bacteriocin with wide spectrum of activity against Gram‐positive bacterial pathogens, filamentous fungi and yeast suggested its potential clinical use. Statistical method facilitated optimization of cultural medium for the improved production of bacteriocin.     

Antimicrobial Substance Produced by Pseudomonas aeruginosa Isolated from Slaughterhouse Sediment: Physicochemical Characterization,Purification, and Identification

International Journal of Peptide Research and Therapeutics - This study aimed to produce the novel bacteriocin from Pseudomonas aeruginosa 43. The bacteriocin was purified through 80% ammonium...     

The C-terminus of human Cav2.3 voltage-gated calcium channel interacts with alternatively spliced calmodulin-2 expressed in two human cell lines

Ca_v2.3 containing voltage-activated Ca^2 + channels are expressed in excitable cells and trigger neurotransmitter and peptide-hormone release. Their expression remote from the fast release sites leads to the accumulation of presynaptic Ca^2 + which can both, facilitate and inhibit the influx of Ca^2 + ions through Ca_v2.3. The facilitated Ca^2 + influx was recently related to hippocampal postsynaptic facilitation and long term potentiation. To analyze Ca^2 + mediated modulation of cellular processes more in detail, protein partners of the carboxy terminal tail of Ca_v2.3 were identified by yeast-2-hybrid screening, leading in two human cell lines to the detection of a novel, extended and rarely occurring splice variant of calmodulin-2 (CaM-2), called CaM-2-extended (CaM-2-ext). CaM-2-ext interacts biochemically with the C-terminus of Ca_v2.3 similar to the classical CaM-2 as shown by co-immunoprecipitation. Functionally, only CaM-2-ext reduces whole cell inward currents significantly. The insertion of the novel 46 nts long exon and the consecutive expression of CaM-2-ext must be dependent on a new upstream translation initiation site which is only rarely used in the tested human cell lines. The structure of the N-terminal extension is predicted to be more hydrophobic than the remaining CaM-2-ext protein, suggesting that it may help to dock it to the lipophilic membrane surrounding.     

Purification and characterization of a novel broad-spectrum bacteriocin from Bacillus licheniformis MKU3

A bacterial strain Bacillus licheniformis MKU3, isolated from slaughterhouse sediments showed a strong antimicrobial activity. The antimicrobial substance produced by this strain was found to be a protein that inhibited a broad range of bacterial strains, such as Bacillus sp., Staphylococcus sp., Streptococcus sp., and Listeria monocytogenes. The antimicrobial peptide was purified to homogeneity by cut off membrane filtration followed by gel filtration chromatography. The purified protein with low molecular mass (< 8 kDa) was resolved as single band on Tricine SDS-PAGE. This protein was stable at 100°C for 10 min, but lost its activity at 121°C in 15 min. It was resistant to the proteolytic action of trypsin, proteinase K, and pronase E and stable within a wide range of pH (3.0∼11.0). This protein exhibited lytic activity on selected indicator strain Kurthia gibsonii GCS6.     

The C-terminus of human Ca(v)2.3 voltage-gated calcium channel interacts with alternatively spliced calmodulin-2 expressed in two human cell lines

Ca(v)2.3 containing voltage-activated Ca(2+) channels are expressed in excitable cells and trigger neurotransmitter and peptide-hormone release. Their expression remote from the fast release sites leads to the accumulation of presynaptic Ca(2+) which can both, facilitate and inhibit the influx of Ca(2+) ions through Ca(v)2.3. The facilitated Ca(2+) influx was recently related to hippocampal postsynaptic facilitation and long term potentiation. To analyze Ca(2+) mediated modulation of cellular processes more in detail, protein partners of the carboxy terminal tail of Ca(v)2.3 were identified by yeast-2-hybrid screening, leading in two human cell lines to the detection of a novel, extended and rarely occurring splice variant of calmodulin-2 (CaM-2), called CaM-2-extended (CaM-2-ext). CaM-2-ext interacts biochemically with the C-terminus of Ca(v)2.3 similar to the classical CaM-2 as shown by co-immunoprecipitation. Functionally, only CaM-2-ext reduces whole cell inward currents significantly. The insertion of the novel 46 nts long exon and the consecutive expression of CaM-2-ext must be dependent on a new upstream translation initiation site which is only rarely used in the tested human cell lines. The structure of the N-terminal extension is predicted to be more hydrophobic than the remaining CaM-2-ext protein, suggesting that it may help to dock it to the lipophilic membrane surrounding.     

Genotypic and phenotypic diversity of PGPR fluorescent pseudomonads isolated from the rhizosphere of sugarcane (Saccharum officinarum L.)

The genetic diversity of plant growth-promoting rhizobacterial (PGPR) fluorescent pseudomonads associated with the sugarcane (Saccharum officinarum L.) rhizosphere was analyzed. Selected isolates were screened for plant growthpromoting properties including production of indole acetic acid, phosphate solubilization, denitrification ability, and production of antifungal metabolites. Furthermore, 16S rDNA sequence analysis was performed to identify and differentiate these isolates. Based on 16S rDNA sequence similarity, the isolates were designated as Pseudomonas plecoglossicida, P. fluorescens, P. libaniensis, and P. aeruginosa. Differentiation of isolates belonging to the same group was achieved through different genomic DNA fingerprinting techniques, including randomly amplified polymorphic DNA (RAPD), amplified ribosomal DNA restriction analysis (ARDRA), repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and bacterial repetitive BOX elements (BOX) analyses. The genetic diversity observed among the isolates and rep-PCR-generated fingerprinting patterns revealed that PGPR fluorescent pseudomonads are associated with the rhizosphere of sugarcane and that P. plecoglossicida is a dominant species. The knowledge obtained herein regarding the genetic and functional diversity of fluorescent pseudomonads associated with the sugarcane rhizosphere is useful for understanding their ecological role and potential utilization in sustainable agriculture.