Pyocin S2 and S4 in Pseudomonas aeruginosa use the same uptake channels as the pyoverdine does in bacteria, indicating a possible connection between them. In this study, we characterized the single bacterial gene expression distribution of three S-type pyocins (Pys2, PA3866, and PyoS5) and examined the impact of pyocin S2 on bacterial uptake of pyoverdine. The findings demonstrated that the expression of the S-type pyocin genes was highly differentiated in bacterial population under DNAdamage stress. Moreover, exogenous addition of pyocin S2 reduces the bacterial uptake of pyoverdine so that the presence of pyocin S2 prevents the uptake of environmental pyoverdine by non-pyoverdine synthesizing 'cheaters', thereby reducing their resistance to oxidative stress. Furthermore, we discovered that overexpression of the SOS response regulator PrtN in bacteria significantly decreased the expression of genes involved in the synthesis of pyoverdine, significantly decreasing the overall synthesis and exocytosis of pyoverdine. These findings imply a connection between the function of the iron absorption system and the SOS stress response mechanism in bacteria.
Pyocins/metabolism* ; Pseudomonas aeruginosa/metabolism*

The ban on addition of antibiotics in animal feed in China has made the search for new antibiotics substitutes, e.g. bacteriocin, a hot topic in research. The present study successfully isolated an antibacterial substance producing strain of Bacillus sp. from alpaca feces by agar diffusion method, using Escherichia coli, Salmonella enterica, Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus and Listeria monocytogenes as indicator bacteria. The isolated strain was named as B. licheniformis SXAU06 based on colony morphology, Gram staining and 16S rRNA gene sequence. The antibacterial substance was isolated and purified through a series of procedures including (NH4)2SO4 precipitation, chloroform extraction, molecular interception and SDS-PAGE analysis. Bioinformatics analysis of the LC-MS/MS data indicated that the antibacterial substance was a bacteriocin-like substance (BLIS) with an approximate molecular weight of 14 kDa, and it was designated as BLIS_SXAU06. BLIS_SXAU06 exhibited high resistance to treatment of proteinase K, high temperature, high acidity and alkalinity. BLIS_SXAU06 was heterologously expressed in E. coli and the recombinant BLIS_SXAU06 exhibited effective antibacterial activity against S. aureus, S. epidermidis, M. luteus, and L. monocytogenes, showing potential to be investigated further.
Animals ; Anti-Bacterial Agents/pharmacology* ; Bacillus licheniformis ; Bacteriocins/pharmacology* ; China ; Chromatography, Liquid ; Escherichia coli/genetics* ; Listeria monocytogenes ; RNA, Ribosomal, 16S ; Staphylococcus aureus ; Tandem Mass Spectrometry

Aims: The menace of antibiotic resistance has led to the search for alternatives, which in turn has diverted the attention to bacteriocins, antimicrobial peptides (AMPs) produced by bacteria for their bactericidal properties. The aim of our study was to isolate and partially purify bacteriocin from lactic acid bacteria (LAB) and comparing its antimicrobial activity with antibiotics. Methodology and results: Among 38 LAB screened using agar spot assay, LAB 28D1 showed the highest antimicrobial activity against the test bacterial strains. The proteinaceous nature of the antimicrobial compound extracted from LAB 28D1 was confirmed by its inactivation after treatment with proteolytic enzymes. The crude bacteriocin was found to be stable over a wide range of temperatures (60-100 °C) and pH (4-9). The bacteriocin was partially purified by ammonium sulfate precipitation (ASP) and the activity units were 204,800 AU/mL. The total protein and the specific activity of partially purified bacteriocin were found to be 24.585 mg and 124,954.24 AU/mg respectively. The molecular weight of partially purified bacteriocin was determined to be 8.5 kDa approximately. The efficacy of the partially purified bacteriocin against indicator bacterial strains was compared with antibiotics by the disc diffusion method and minimum inhibitory concentration (MIC). According to our study, the hospital waste isolate Enterococcus spp. was found to be multidrug-resistant (MDR) but sensitive to bacteriocin from LAB (MIC 0.06 ± 0 µg/mL). Conclusion, significance and impact of the study Bacteriocin from LAB has potential in combating MDR enterococcal infections.
Bacteriocins--isolation & ; purification ; Lactobacillales ; Drug Resistance, Microbial

Aims: To characterize the plantaricin IIA-1A5 crude extract that biosynthesized by Lactobacillus plantarum IIA-1A5 using corn steep liquor (CSL) based medium. Methodology and results: Lactobacillus plantarum IIA-1A5 was grown in several media containing different components including corn steep liquor (CSL), molasses and MRS (de Man Rogosa Sharpe) as control medium for 24 h at 37 °C. Antibacterial activities of the cell-free supernatant were expressed as diameter of inhibition zones observed by paper disc method. The results showed that CSL medium produced cell-free supernatant of L. plantarum IIA-1A5 with significantly higher antibacterial activity againts Staphylococcus aureus ATCC 25923 (9.81 mm), Lactobacillus monocytogenes ATCC 7644 (9.61 mm), Bacillus cereus (8.97 mm) and Escherichia coli ATCC 25922 (9.23 mm) were not significantly different compared to control MRS broth media (9.59 mm). CSL medium added only with 3% yeast extract and Tween 80 produced supernatant which showed similar antibacterial activity either to 10% molasses or control medium (Medium K and B). The CSL medium was considered more efficient and low cost, therefore this medium was selected for production and characterization of plantaricin IIA-1A5 crude extract. Further characterization performed by SDS PAGE analysis showed that crude plantaricin had molecular weight of approximately 9.9 kDa, higher than that produced in control medium (8.0 kDa). However, both plantaricins were categorized under the same class for small bacteriocin (class II). This study also revealed the plantaricin IIA-1A5 produced in CSL medium was stable to heat and pH and not significantly different compared to control MRS broth media. The antibacterial activity of plantaricin IIA-1A5 crude extract against S. aureus ATCC 25923 (10.09 mm) was not significantly different with 1000 ppm sodium benzoate (9.70 mm) and 300 ppm sodium nitrite (9.82 mm). Conclusion, significance and impact of study The CSL medium produced cell-free supernatant of L. plantarum IIA 1A5 had significant antibacterial activity characterization againts S. aureus ATCC 25923, L. monocytogenes ATCC 7644, B. cereus and E. coli ATCC 25922. Comparison of the inhibition activity of plantaricin IIA-1A5 crude extract against pathogen with synthetic preservatives indicated that plantaricin IIA-1A5 crude extract have the potency to replace synthetic preservatives. CSL based medium is potential to be used for low-cost plantaricin IIA-1A5 production.
Anti-Bacterial Agents--metabolism ; Bacteriocins--metabolism ; Lactobacillus plantarum ; Microbial Viability--drug effects ; Staphylococcus aureus

Aims: Burkholderia pseudomallei, the human pathogen that causes melioidosis, is intrinsically resistant towards a wide range of antibiotics and there have been reports of acquired resistance towards antibiotics used for melioidosis treatments. Antimicrobial peptides (AMP) such as bacteriocins are gaining the interests of researchers as alternative for treating infections caused by multidrug resistant bacteria. In this study, we aimed to identify Burkholderia spp. isolated from soil in Sarawak that possess the potential in inhibiting the growth of B. pseudomallei and to further characterize the antagonistic compound produced. Methodology and results: A total of 50 Burkholderia spp. isolates of environmental origin and two isolates of Ralstonia solanacearum were screened against five clinical isolates of B. pseudomallei using spot-on-lawn assay and flip streak method. Burkholderia stagnalis isolate K23/3 showed clear zones of inhibition (ZOI) in both preliminary tests. Cell-free supernatant (CFS) was obtained from B. stagnalis K23/3 broth culture and was tested via agar well diffusion assay (AWDA). The antagonistic compound secreted at the early log phase of the bacterial growth was shown to be stable in a wide range of temperatures and pH. Treatment with different enzymes revealed that it was sensitive towards proteinase K, suggesting that it is proteinaceous. The bacteriocin-like-substance (BLIS) was subjected to ammonium sulfate precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE gel was overlaid with indicator B. pseudomallei isolates where the active protein was shown to be less than 7.1 kDa. Conclusion, significance and impact of study Burkholderia stagnalis isolate K23/3 was able to secrete bacteriocin-like-substance (BLIS) that has the potential in biocontrol of B. pseudomallei in the environment or as potential treatment for melioidosis.
Bacteriocins ; Burkholderia ; Burkholderia pseudomallei

Aims: This study aims to predict the presence of bacteriocin- and probiotic-associated genes in the genome of Weissella cibaria NM1 isolated from Asian sea bass using a machine learning-based NeuBI prediction approach, followed by the investigation of the crude bacteriocin antimicrobial and probiotic properties via in vitro analysis. Methodology and results: This study utilized the machine learning-based NeuBI prediction approach with a homology search of highly conserved bacteriocin-associated genes present in the genome of W. cibaria NM1. This approach discovered a putative bacteriocin gene (WC_2064) and bacteriocin operon with complete immunity, transporter, regulator and modifier genes. Furthermore, the genome of W. cibaria NM1 was found to harboured specific probiotic*associated genes that would contribute to acid and bile tolerance, adhesion on thehost cell and exhibited cholesterolreducing ability. On top of that, the genome also shows the absence of virulence and antibiotic resistance genes, which signifies the safety of W. cibaria NM1 as a potential probiotic candidate. In vitro study has confirmed the antipseudomonal activity of crude bacteriocin NM1 with MIC of 62.5 mg/mL. Weissella cibaria NM1 can tolerate 0.3% (v/v) of bile salt condition and the transit through the simulated gastric (pH 3 and 4) and small intestinal (pH 8) tract. Conclusion, significance and impact of study Current findings suggested in silico approach can speed up the search for putative bacteriocin and probiotic-associated genes from the genome of W. cibaria NM1. Nevertheless, further verification through experimental works will be deemed essential.
Bacteriocins ; Probiotics ; Weissella ; Asian ; Computer Simulation ; In Vitro Techniques

Polyhydroxyalkanoates (PHAs), as a novel class of biopolymer, are attracting more attention due to their diverse material properties and environment-independent biodegradability. Here we report the preparation of PHA exhibiting efficient antibacterial activity by embedding Nisin, a food additive generally recognized as safe, into poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), a type of PHA with high biocompatibility. We first prepared Nisin-containing PHBHHx films using solvent casting method. Confocal laser scanning microscopy analysis showed that a well-mixed integrated structure of the films with an even distribution of the Nisin particles in the PHBHHx matrices. Then the antimicrobial activity of PHBHHx/Nisin films against Micrococcus luteus was quantified on agar plate by measuring the size of inhibition zone. Cultivation in liquid media further confirmed the releasing of Nisin from the films and the long-time antibacterial activity. Results showed that the threshold of Nisin concentration for long-time and effective inhibition against bacteria growth is 25 μg/g. These results altogether establish a technological foundation for the application of PHA in biomedicine and food industry.
3-Hydroxybutyric Acid ; chemistry ; Anti-Bacterial Agents ; chemistry ; Caproates ; chemistry ; Micrococcus luteus ; drug effects ; Nisin ; chemistry ; Polyhydroxyalkanoates ; chemistry

Biological Products ; Biotechnology ; history ; China ; Food Preservatives ; History, 20th Century ; History, 21st Century ; Nisin

Nisin is an antimicrobial peptide widely used in food industry. In this study, Nisin A production in Lactococcus lactis ATCC 11454 was improved by overexpression of Nisin A structural gene nisA through introducing a shuttle expression vector pMG36ek-nisA and an integrated vector pDG780-nisA into the host strain. The differences of growth profiles and Nisin A production level between the two obtained genetic engineering strains FMM1/FMM2 and the parent strain were investigated. Our results show that while the growth profile (the growth rate, biomass and pH) of FMM1 was similar to the parent strain, its Nisin A production increased 31%. In contrast, the biomass of FMM2 was notably lower than the parent strain, while its yield of Nisin A enhanced slightly. The transcription level of genes involved in Nisin A biosynthesis in both engineering strains was further detected by RT-PCR. We found that all the 11 Nisin A biosynthetic genes in FMM1 and FMM2 had a higher transcription level than those in the parent strain, and these genes exhibited more significant increasing degree of transcription level in FMM1 which hosted the autonomous replicating nisA gene. These data suggest that expression of nisA may act as a rate-limit factor in Nisin A biosynthesis. In conclusion, this work provides a new method to improve Nisin A production by increasing the transcription level of nisA, paving the way to further large-scale industrial production of Nisin A.
Bacterial Proteins ; genetics ; Genes, Bacterial ; Genetic Engineering ; Genetic Vectors ; Lactococcus lactis ; genetics ; metabolism ; Nisin ; biosynthesis ; genetics ; Transcription, Genetic

The possibility of heterologous expression of human Stearoyl-CoA Desaturase (scd1) was investigated. The scd1 encoding sequence was inserted into the pNZ8149 to generate the pNZ8149-scd1 expression plasmids. Then we introduced the pNZ8149-scd1 construct into the Lactococcus lactis NZ3900 to investigate its enzyme activity. The results show that heterologous expressed SCD1 enzyme resulted in a 92%-169% increase in the C16:1n-7 and a 53-127% increase in the C18:1n-7 (P<0.05). The SCD1 enzyme was capable of producing n-7 fatty acids in Lactococcus lactis efficiently. It also suggests that the fatty acid desaturases can be heterologous expressed in Lactococcus lactis to produce the helpful fatty acids.
Electroporation ; Humans ; Lactococcus lactis ; genetics ; metabolism ; Mutagenesis, Insertional ; Nisin ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; Stearoyl-CoA Desaturase ; biosynthesis ; genetics