RESUMO
Enterococci are lactic acid bacteria (LAB) that play a role in the aroma formation, maturation, and sensory development of fermented foods such as meat and dairy products. They also contribute to the improvement of the extended shelf life of fermented foods by producing bacteriocin. The aim of this study was to isolate bacteriocin-producing LAB from sheep and goat colostrum, to characterize the bacteriocin-producing strains, and determine the technological properties of the strains. A total of 13 bacteriocin-producing LAB was isolated and identified as 11 Enterococcus mundtii and two Enterococcus faecium. The strains were found to be genetically different from each other by phylogenetic analysis of 16S rRNA gene sequences and random amplified polymorphic-DNA (RAPD-PCR). It has been determined that bacteriocins show activity in a wide pH range and are resistant to heat, lose their activity with proteolytic enzymes and α-amylase, but are resistant to detergents. While the presence of the munKS gene was detected in all of the strains, it was determined that E. faecium HC121.4, HC161.1, E. mundtii HC147.1, HC166.5, and HC166.8 strains contained multiple enterocin genes. Trisin-SDS-PAGE analysis revealed two active protein bands of approximately 5.1 and 5.5 kDa in E. faecium HC121.4 and one active protein band with a weight of approximately 4.96 kDa in other strains. E. mundtii strains and E. faecium HC161.1 were identified as mundticin KS producers, and E. faecium HC121.4 was defined as an enterocin A and B producer. Except for E. mundtii HC166.8, acid production of strains was found to be slow at 6 h and moderate at 24 h. None of them showed extracellular proteolytic and lipolytic activities. It was found that the strains had esterase, esterase lipase, leucine arylamidase, acid phosphatase, and naphthol-AS-Bl-phosphohydrolase activities, while protease activities were low and peptidase activities were high. In conclusion, bacteriocin producer 13 Enterococcus strains isolated from sheep and goat colostrum were found to have the potential to be included in starter culture combinations.
Assuntos
Bacteriocinas , Enterococcus faecium , Animais , Ovinos , Feminino , Gravidez , Enterococcus faecium/genética , Colostro , Técnica de Amplificação ao Acaso de DNA Polimórfico/veterinária , RNA Ribossômico 16S/genética , Cabras/genética , Filogenia , Enterococcus/genética , Bacteriocinas/genética , Esterases/genética , Esterases/metabolismo , Antibacterianos/químicaRESUMO
BACKGROUND: Pediocin PA-1 is a bacteriocin of recognized value with applications in food bio-preservation and the medical sector for the prevention of infection. To date, industrial manufacturing of pediocin PA-1 is limited by high cost and low-performance. The recent establishment of the biotechnological workhorse Corynebacterium glutamicum as recombinant host for pediocin PA-1 synthesis displays a promising starting point towards more efficient production. RESULTS: Here, we optimized the fermentative production process. Following successful simplification of the production medium, we carefully investigated the impact of dissolved oxygen, pH value, and the presence of bivalent calcium ions on pediocin production. It turned out that the formation of the peptide was strongly supported by an acidic pH of 5.7 and microaerobic conditions at a dissolved oxygen level of 2.5%. Furthermore, elevated levels of CaCl2 boosted production. The IPTG-inducible producer C. glutamicum CR099 pXMJ19 Ptac pedACDCg provided 66 mg L-1 of pediocin PA-1 in a two-phase batch process using the optimized set-up. In addition, the novel constitutive strain Ptuf pedACDCg allowed successful production without the need for IPTG. CONCLUSIONS: The achieved pediocin titer surpasses previous efforts in various microbes up to almost seven-fold, providing a valuable step to further explore and develop this important bacteriocin. In addition to its high biosynthetic performance C. glutamicum proved to be highly robust under the demanding producing conditions, suggesting its further use as host for bacteriocin production.
Assuntos
Bacteriocinas , Corynebacterium glutamicum , Pediocinas , Peptídeos Antimicrobianos , Cálcio , Corynebacterium glutamicum/genética , Isopropiltiogalactosídeo , Bacteriocinas/genética , Íons , Concentração de Íons de HidrogênioRESUMO
Bovine mastitis infection in dairy cattle is a significant economic burden for the dairy industry globally. To reduce the use of antibiotics in treatment of clinical mastitis, new alternative treatment options are needed. Antimicrobial peptides from bacteria, also known as bacteriocins, are potential alternatives for combating mastitis pathogens. In search of novel bacteriocins against mastitis pathogens, we screened samples of Norwegian bovine raw milk and found a Streptococcus uberis strain with potent antimicrobial activity toward Enterococcus, Streptococcus, Listeria, and Lactococcus. Whole-genome sequencing of the strain revealed a multibacteriocin gene cluster encoding one class IIb bacteriocin, two class IId bacteriocins, in addition to a three-component regulatory system and a dedicated ABC transporter. Isolation and purification of the antimicrobial activity from culture supernatants resulted in the detection of a 6.3-kDa mass peak by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, a mass corresponding to the predicted size of one of the class IId bacteriocins. The identification of this bacteriocin, called ubericin K, was further confirmed by in vitro protein synthesis, which showed the same inhibitory spectrum as the purified antimicrobial compound. Ubericin K shows highest sequence similarity to the class IId bacteriocins bovicin 255, lactococcin A, and garvieacin Q. We found that ubericin K uses the sugar transporter mannose phosphotransferase (PTS) as a target receptor. Further, by using the pHlourin sensor system to detect intracellular pH changes due to leakage across the membrane, ubericin K was shown to be a pore former, killing target cells by membrane disruption. IMPORTANCE Bacterial infections in dairy cows are a major burden to farmers worldwide because infected cows require expensive treatments and produce less milk. Today, infected cows are treated with antibiotics, a practice that is becoming less effective due to antibiotic resistance. Compounds other than antibiotics also exist that kill bacteria causing infections in cows; these compounds, known as bacteriocins, are natural products produced by other bacteria in the environment. In this work, we discover a new bacteriocin that we call ubericin K, which kills several species of bacteria known to cause infections in dairy cows. We also use in vitro synthesis as a novel method for rapidly characterizing bacteriocins directly from genomic data, which could be useful for other researchers. We believe that ubericin K and the methods described in this work will aid in the transition away from antibiotics in the dairy industry.
Assuntos
Antibacterianos/uso terapêutico , Bacteriocinas/uso terapêutico , Doenças dos Bovinos/tratamento farmacológico , Mastite Bovina/tratamento farmacológico , Streptococcus/metabolismo , Animais , Membrana Externa Bacteriana/efeitos dos fármacos , Membrana Externa Bacteriana/patologia , Bacteriocinas/genética , Bovinos , Doenças dos Bovinos/microbiologia , Enterococcus/efeitos dos fármacos , Enterococcus/crescimento & desenvolvimento , Feminino , Lactococcus/efeitos dos fármacos , Lactococcus/crescimento & desenvolvimento , Listeria/efeitos dos fármacos , Listeria/crescimento & desenvolvimento , Mastite Bovina/microbiologia , Testes de Sensibilidade Microbiana , Fosfotransferases/metabolismo , Percepção de Quorum , Streptococcus/genéticaRESUMO
Bacteriocins are regarded as important factors mediating microbial interactions, but their exact role in community ecology largely remains to be elucidated. Here, we report the characterization of a mutant strain, derived from Pseudomonas syringae pv. tomato DC3000 (Pst), that was incapable of growing in plant extracts and causing disease. Results showed that deficiency in a previously unannotated gene saxE led to the sensitivity of the mutant to Ca2+ in leaf extracts. Transposon insertions in the bacteriocin gene syrM, adjacent to saxE, fully rescued the bacterial virulence and growth of the ΔsaxE mutant in plant extracts, indicating that syrM-saxE encode a pair of bacteriocin immunity proteins in Pst. To investigate whether the syrM-saxE system conferred any advantage to Pst in competition with other SyrM-sensitive pathovars, we compared the growth of a SyrM-sensitive strain co-inoculated with Pst strains with or without the syrM gene and observed a significant syrM-dependent growth reduction of the sensitive bacteria on plate and in lesion tissues upon desiccation-rehydration treatment. These findings reveal an important biological role of SyrM-like bacteriocins and help to understand the complex strategies used by P. syringae in adaptation to the phyllosphere niche in the context of plant disease.
Assuntos
Bacteriocinas , Dessecação , Pseudomonas syringae/crescimento & desenvolvimento , Bacteriocinas/genética , Doenças das Plantas , Folhas de Planta , Pseudomonas syringae/genéticaRESUMO
Spore forming Bacillus species are widely used as probiotics for human dietary supplements and in animal feeds. However, information on genetic basis of their probiotic action is obscure. Therefore, the present investigation was undertaken to elucidate probiotic traits of B. coagulans HS243 through its genome analysis. Genome mining revealed the presence of an arsenal of marker genes attributed to genuine probiotic traits. In silico analysis of HS243 genome revealed the presence of multi subunit ATPases, ADI pathway genes, chologlycine hydrolase, adhesion proteins for surviving and colonizing harsh gastric transit. HS243 genome harbored vitamin and essential amino acid biosynthetic genes, suggesting the use of HS243 as a nutrient supplement. Bacteriocin producing genes highlighted the disease preventing potential of HS243. Thus, this work established that HS243 possessed the genetic repertoire required for surviving harsh gastric transit and conferring health benefits to the host which were further validated by wet lab evidences.
Assuntos
Bacillus coagulans/genética , Genoma Bacteriano , Probióticos , Proteínas de Bactérias/genética , Bacteriocinas/biossíntese , Bacteriocinas/genética , Anotação de Sequência MolecularRESUMO
Lantibiotics represent a large untapped pipeline of attractive scaffolds for the development of novel antibiotics. Saturation mutagenesis was employed to substitute every amino acid of a lantibiotic called mutacin 1140 (MU1140), creating an unbiased expression library of 418 variants that was used to study the permissiveness to mutagenesis and the "drugability" of several compounds. Contrasting previous reports, the results from this study supported that not all residues involved in lanthionine bridge formation were critical for maintaining optimal activity. While substitutions in lanthionine bridges in Ring A, C, and D invariably lead to inactive variants, permissive substitutions in Abu8 and Ala11 (Ring B) were observed, albeit infrequently. Further, the data generated suggested that the unsaturated bond from Dha5 (Ser5) may not be critically involved in Lipid-II binding but still important for conferring optimal activity. This study identified additional permissive mutations of Ser5, including Ser5His, Ser5Met, Ser5Gln, and Ser5Leu. In contrast, no permissive substitutions were identified for Dhb14, which suggested that this residue may be critical for optimal activity. Novel blueprints are proposed for directing further development of MU1140 variants and other lantibiotics, which may enable the rational design, development, manufacture, and formulation of an entirely new class of anti-infectives.
Assuntos
Bacteriocinas/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Bacteriocinas/genética , Bacteriocinas/farmacologia , Biblioteca Gênica , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Peptídeos/genética , Peptídeos/farmacologia , Plasmídeos/genética , Plasmídeos/metabolismo , Streptococcus/química , Streptococcus/genética , Streptococcus/metabolismo , Relação Estrutura-AtividadeRESUMO
Highly pathogenic Escherichia coli strains that belong to the phylogenetic group B2 have developed a greater ability to acquire iron (heme receptor and numerous siderophores), to produce the genotoxin colibactin and to synthesize antimicrobial siderophore-microcins. There is an increased prevalence of these E. coli strains over the last 30 years in the intestinal microbiota in industrialized countries. Integrating the regulation of fitness/virulence factors, such as siderophores, colibactin and siderophore-microcins into networks that respond to specific environmental signals, such as the local iron concentration, could result in an accurate production of specific fitness/virulence factors, so that the E. coli can adapt to the competitive environment that is the gut and/or the blood. Iron deficiency is common in infancy, even in industrialized countries. Usual strategies for anemia correction are iron supplementation and iron fortification of foods. The long-term consequences and risks associated with high iron supply in the light of this iron-dependent network described in this review could explain at least in part the increased prevalence of E. coli B2 in the gut of people in industrialized countries. © 2017 IUBMB Life, 69(6):435-441, 2017.
Assuntos
Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Peptídeos/metabolismo , Policetídeos/metabolismo , Sideróforos/biossíntese , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriocinas/biossíntese , Bacteriocinas/genética , Suplementos Nutricionais , Enterobactina/biossíntese , Enterobactina/genética , Escherichia coli/classificação , Escherichia coli/genética , Microbioma Gastrointestinal/genética , Homeostase/genética , Humanos , Ferro/administração & dosagem , Peptídeos/genética , Filogenia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sideróforos/genética , Fatores de Virulência/metabolismoRESUMO
Lactococcus lactis KTH0-1S isolated from Thai traditional fermented shrimp (Kung-som) is able to produce heat-stable bacteriocin and inhibits food spoilage bacteria and food-borne pathogens. The inhibitory effect of bacteriocin remained intact after treatment with different pHs and after heating, but was sensitive to some proteolytic enzymes. Addition of bacteriocin KTH0-1S to Staphylococcus aureus cultures decreased viable cell counts by 2.8 log CFU/ml, demonstrating a bactericidal mode of action. Furthermore, the growth of S. aureus decreased significantly after 12-h co-cultivation with bacteriocinogenic strain. The molecular mass of bacteriocin KTH0-1S was found to be 3.346 kDa after ammonium sulfate precipitation, reversed phase (C8 Sep-Pak), cation-exchange chromatography, RP-HPLC on C8 column and mass spectrometry (MS/MS) analysis. Bacteriocin KTH0-1S was identified as nisin Z using PCR amplification and sequencing. The majority of tested virulence factors were absent, confirming the safety. Evidenced inhibitory effect of this strain, the absence of virulence factors creates the possibility for its application as protective culture to inhibit pathogenic bacteria in the several fermented seafood products.
Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Lactococcus lactis/fisiologia , Nisina/análogos & derivados , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Bacteriocinas/química , Bacteriocinas/genética , Bacteriocinas/isolamento & purificação , Fermentação , Lactococcus lactis/efeitos dos fármacos , Lactococcus lactis/isolamento & purificação , Lactococcus lactis/patogenicidade , Interações Microbianas , Nisina/genética , Nisina/isolamento & purificação , Nisina/farmacologia , Penaeidae/microbiologia , Frutos do Mar/microbiologia , Tailândia , Fatores de Virulência/genéticaRESUMO
Construction of Listeria monocytogenes mutants by allelic exchange has been laborious and time-consuming due to lack of proficient selection markers for the final recombination event, that is, a marker conveying substance sensitivity to the bacteria bearing it, enabling the exclusion of merodiploids and selection for plasmid loss. In order to address this issue, we engineered a counterselection marker based on a mutated phenylalanyl-tRNA synthetase gene (pheS*). This mutation renders the phenylalanine-binding site of the enzyme more promiscuous and allows the binding of the toxic p-chloro-phenylalanine analog (p-Cl-phe) as a substrate. When pheS* is introduced into L. monocytogenes and highly expressed under control of a constitutively active promoter, the bacteria become sensitive to p-Cl-phe supplemented in the medium. This enabled us to utilize pheS* as a negative selection marker and generate a novel, efficient suicide vector for allelic exchange in L. monocytogenes We used this vector to investigate the monocin genomic region in L. monocytogenes strain 10403S by constructing deletion mutants of the region. We have found this region to be active and to cause bacterial lysis upon mitomycin C treatment. The future applications of such an effective counterselection system, which does not require any background genomic alterations, are vast, as it can be modularly used in various selection systems (e.g., genetic screens). We expect this counterselection marker to be a valuable genetic tool in research on L. monocytogenesIMPORTANCEL. monocytogenes is an opportunistic intracellular pathogen and a widely studied model organism. An efficient counterselection marker is a long-standing need in Listeria research for improving the ability to design and perform various genetic manipulations and screening systems for different purposes. We report the construction and utilization of an efficient suicide vector for allelic exchange which can be conjugated, leaves no marker in the bacterial chromosome, and does not require the use of sometimes leaky inducible promoters. This highly efficient genome editing tool for L. monocytogenes will allow for rapid sequential mutagenesis, introduction of point mutations, and design of screening systems. We anticipate that it will be extensively used by the research community and yield novel insights into the diverse fields studied using this model organism.
Assuntos
Bacteriocinas/genética , Listeria monocytogenes/genética , Mitomicina/farmacologia , Fenilalanina-tRNA Ligase/genética , Fenilalanina/análogos & derivados , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Marcadores Genéticos/genética , Listeria monocytogenes/crescimento & desenvolvimento , Fenilalanina/metabolismo , Regiões Promotoras Genéticas/genética , Seleção Genética/genética , Deleção de Sequência/genéticaRESUMO
This study focuses on the potential of Pectobacterium carotovorum subsp. carotovorum (Pcc) strains producing bacteriocin as a tool to control potato soft rot disease. Thirty out of 48 purified bacterial strains were characterized as Pcc using specific PCR and phenotypic tests. The pathogenicity and pectate degrading assays were recorded positive for 13 strains. Bacteriocin typing clustered producers into three groups according to their antimicrobial spectra. Majority of the producers except strains of group II showed antibacterial activity toward relative genus and the role of UV or mitomycin C was inductive. In addition, none of the distant genus was sensitive to Pcc bacteriocins except Rhizobium vitis. Molecular detection of four bacteriocins including carotovoricin, carosin S1, S2 and carosin D was performed. Overall, 54.5% of group I, 47.3 and 70% of groups II and III strains carried carotovoricin and four strains harbored gene corresponding to carosin S1. According to our data divers antimicrobial patterns obtained by Pcc strains and existence of new bateriocines could be possible. Moreover, our findings recommended that direct application of P29 or expression of corresponding genes of Pog22 or P21 in a nonpathogenic strain as a biocontrol agent may improve soft rot disease control.
Assuntos
Antibiose/efeitos dos fármacos , Antibiose/efeitos da radiação , Bacteriocinas/metabolismo , Pectobacterium carotovorum/fisiologia , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Solanum tuberosum/microbiologia , Bacteriocinas/genética , Programas de Rastreamento , Mitomicina/metabolismo , Pectobacterium carotovorum/efeitos dos fármacos , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/efeitos da radiação , Controle Biológico de Vetores/métodos , Raios UltravioletaRESUMO
In this study, we evaluated the probiotic properties of Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus fermentum strains isolated from fermented radish. All the strains survived the simulated oro-gastrointestinal transit condition and showed significantly higher adherence to Caco-2 cells compared with the probiotic strain Lactobacillus rhamnosus GG. The strains showed broad-spectrum antimicrobial activity, autoaggregation, and coaggregation capacity with pathogens. Furthermore, the Lactobacillus strains inhibited the adherence of Yersinia enterocolitica subsp. enterocolitica, Shigella boydii, and Salmonella choleraesuis to the Caco-2 cell line. The strains possessed bile salt hydrolase activity and their cholesterol-lowering activity in vitro was above 50% in the presence of bile. Strains of L. plantarum and L. pentosus possessed the plantaricin-encoding plnEF gene. In addition, the Lactobacillus strains maintained about 80% cell viability after freeze-drying in the presence of a combination of 5% skim milk and 5% maltodextrin as cryoprotectant, and 70% recovery of cell viability was observed in the absence of any cryoprotectant.
Assuntos
Lactobacillus/metabolismo , Probióticos/química , Raphanus/microbiologia , Animais , Anti-Infecciosos/química , Aderência Bacteriana , Bacteriocinas/genética , Bile , Ácidos e Sais Biliares , Células CACO-2 , Meios de Cultura , Avaliação Pré-Clínica de Medicamentos , Fermentação , Liofilização , Humanos , Ácido Láctico/química , Limosilactobacillus fermentum , Lactobacillus plantarum , Testes de Sensibilidade Microbiana , Leite , Filogenia , Polissacarídeos/química , Precursores de Proteínas/genética , Especificidade da EspécieAssuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Bacteriocinas/genética , Colostro/microbiologia , Bactérias/genética , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana/métodos , DNA Bacteriano/análise , Feminino , Voluntários Saudáveis , Humanos , Microbiota , Gravidez , EslovêniaRESUMO
Microbial communities inhabiting the breast milk microenvironment are essential in supporting mammary gland health in lactating women and in providing gut-colonizing bacterial 'inoculum' for their infants' gastro-intestinal development. Bacterial DNA was extracted from colostrum samples of 45 healthy Slovenian mothers. Characteristics of the communities in the samples were assessed by polymerase chain reaction (PCR) coupled with denaturing gradient gel electrophoresis (DGGE) and by quantitative real-time PCR (qPCR). PCR screening for the prevalence of bacteriocin genes was performed on DNA of culturable and total colostrum bacteria. DGGE profiling revealed the presence of Staphylococcus and Gemella in most of the samples and exposed 4 clusters based on the abundance of 3 bands: Staphylococcus epidermidis/Gemella, Streptococcus oralis/pneumonia and Streptococcus salivarius. Bacilli represented the largest proportion of the communities. High prevalence in samples at relatively low quantities was confirmed by qPCR for enterobacteria (100%), Clostridia (95.6%), Bacteroides-Prevotella group (62.2%) and bifidobacteria (53.3%). Bacterial quantities (genome equivalents ml-1) varied greatly among the samples; Staphylococcus epidermidis and staphylococci varied in the range of 4 logs, streptococci and all bacteria varied in the range of 2 logs, and other researched groups varied in the range of 1 log. The quantity of most bacterial groups was correlated with the amount of all bacteria. The majority of the genus Staphylococcus was represented by the species Staphylococcus epidermidis (on average 61%), and their abundances were linearly correlated. Determinants of salivaricin A, salivaricin B, streptin and cytolysin were found in single samples. This work provides knowledge on the colostrum microbial community composition of healthy lactating Slovenian mothers and reports bacteriocin gene prevalence.
Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Bacteriocinas/genética , Colostro/microbiologia , Bactérias/genética , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana/métodos , DNA Bacteriano/análise , Feminino , Voluntários Saudáveis , Humanos , Microbiota , Gravidez , EslovêniaRESUMO
BACKGROUND: The present study was designed to investigate the effect of albusin B on lipid metabolism and antioxidant defense in broiler chickens by a proteomic approach. The bacteriocin, albusin B of Ruminococcus albus 7, expressed by yeast was applied in this study. Three dietary treatments, consisting of the basal diet (control), basal diet + albusin B (2.5 g kg⻹), and basal diet + nosiheptide (2.5 mg kg⻹) were randomly fed to 90 broiler chickens from 1 to 35 days of age, respectively. After 35 days of supplementation, the growth performance, lipid metabolism and antioxidant proteins in the jejunum and liver, intestinal protein profile, and plasma lipid profile were analyzed. RESULTS: Broilers with albusin B supplementation had greater body weight than the control broilers. Compared with the control broilers, lower triglyceride and higher high-density lipoprotein concentration in the blood were observed in both broilers with albusin B and nosiheptide supplementation. In addition, albusin B suppressed the mRNA expression of fatty acid binding protein 2 and ATP binding cassette transporter G 5 in the jejunum. In the jejunal protein profiles, four antioxidant proteins were upregulated by albusin B and nosiheptide treatments. The jejunal antioxidant gene expression had a concordant pattern. Hepatic genes related to lipid metabolism, 3-hydroxy-3-methyl-glutaryl CoA reductase, and superoxide dismutase were upregulated by albusin B supplementation. CONCLUSION: Albusin B supplementation modulated lipid metabolism and activated systemic antioxidant defense, which might partially contribute to the performance of broiler chickens.
Assuntos
Bacteriocinas/metabolismo , Galinhas/metabolismo , Dieta/veterinária , Indução Enzimática , Hidroximetilglutaril-CoA Redutases/biossíntese , Metabolismo dos Lipídeos , Superóxido Dismutase/biossíntese , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antioxidantes/metabolismo , Bacteriocinas/genética , Galinhas/sangue , Galinhas/crescimento & desenvolvimento , Suplementos Nutricionais , Regulação para Baixo , Proteínas de Ligação a Ácido Graxo/antagonistas & inibidores , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Perfilação da Expressão Gênica/veterinária , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Hipolipemiantes/metabolismo , Jejuno/enzimologia , Jejuno/crescimento & desenvolvimento , Jejuno/metabolismo , Fígado/enzimologia , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Masculino , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Tiazóis/metabolismo , Aumento de PesoRESUMO
Enterocin C (EntC), a class IIb bacteriocin was purified from culture supernatants of Enterococcus faecalis C901, a strain isolated from human colostrum. Enterocin C consists of two distinct peptides, named EntC1 and EntC2, whose complementary action is required for full antimicrobial activity. The structural genes entC1 and entC2 encoding enterocins EntC1 and EntC2, respectively, and that encoding the putative immunity protein (EntCI) are located in the 9-kb plasmid pEntC, harboured by E. faecalis C901. The N-terminal sequence of both antimicrobial peptides revealed that EntC1 (4284 Da) is identical to Ent1071A, one of the two peptides that form enterocin 1071 (Ent1071), a bacteriocin produced by E. faecalis BFE 1071. In contrast, EntC2 (3867 Da) presents the non-polar alanine residue at position 17 (Ala(17)) instead of the polar threonine residue (Thr(17)) in Ent1071B, the second peptide constituting Ent1071. In spite of peptide similarities, EntC differs from Ent1071 in major aspects, including the complementary activity among its constitutive peptides and its wider inhibitory spectrum of activity. Different amphiphilic alpha-helical conformations between EntC2 and Ent1071B could explain both, acquired complementary activity and increased antimicrobial spectrum.
Assuntos
Anti-Infecciosos/isolamento & purificação , Bacteriocinas/isolamento & purificação , Colostro/microbiologia , Enterococcus faecalis/metabolismo , Genes Bacterianos , Peptídeos/isolamento & purificação , Alanina/análise , Sequência de Aminoácidos , Plasmídeos de Bacteriocinas/genética , Bacteriocinas/genética , Sequência de Bases , Enterococcus faecalis/genética , Enterococcus faecalis/isolamento & purificação , Feminino , Humanos , Dados de Sequência Molecular , Gravidez , Treonina/análiseRESUMO
The lantibiotic nisin is produced by Lactococcus lactis. In the biosynthesis of nisin, the enzyme NisB dehydrates nisin precursor, and the enzyme NisC is needed for lanthionine formation. In this study, the nisA gene encoding the nisin precursor, and the genes nisB and nisC of the lantibiotic modification machinery were expressed together in vitro by the Rapid Translation System (RTS). Analysis of the RTS mixture showed that fully modified nisin precursor was formed. By treating the mixture with trypsin, active nisin was obtained. However, no nisin could be detected in the mixture without zinc supplementation, explained by the fact that NisC requires zinc for its function. The results revealed that the modification of nisin precursor, which is supposed to occur at the inner side of the membrane by an enzyme complex consisting of NisB, NisC, and the transporter NisT, can take place without membrane association and without NisT. This in vitro production system for nisin opens up the possibility to produce nisin variants that cannot be producedin vivo. Moreover, the system is a promising tool for utilizing the NisB and NisC enzymes for incorporation of thioether rings into medical peptides and hormones for increased stability.
Assuntos
Antibacterianos/metabolismo , Bacteriocinas/biossíntese , Microbiologia Industrial/métodos , Lactococcus lactis/enzimologia , Nisina/biossíntese , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Bacteriocinas/genética , Bacteriocinas/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Lactococcus lactis/genética , Proteínas de Membrana/genética , Nisina/genética , Nisina/farmacologia , Biossíntese de ProteínasRESUMO
Porphyromonas gingivalis infections cause problems in periodontal diseases and in certain systemic diseases. There is evidence that Lactobacillus spp. can control populations of P. gingivalis, but there are few data on the effects of purified bacteriocins from Lactobacillus paracasei HL32 on P. gingivalis. The objective of this study was to examine the antibacterial activity of a bacteriocin from L. paracasei HL32 and to relate this activity to its composition. A bacteriocin was purified from culture supernatants of Lactobacillus spp. using a dialysis technique followed by gel-permeation chromatography. Composition of the bacteriocin was characterised by ninhydrin tests, ultraviolet spectrophotometry, thin-layer chromatography, sodium-dodecyl sulphate-polyacrylamide gel electrophoresis, electrospray ionisation mass spectrometry and amino acid analysis. The amino acid sequence from the N-terminal of the bacteriocin was determined. Antibacterial activity was examined by the cylinder plate method, microtitre assay and scanning electron microscopy as compared with standard antibiotics. The bacteriocin had a molecular weight of approximately 56kDa, was comprised of 68% carbohydrate and 32% protein, and showed maximum peak absorbance at 214 and 254nm. The bacteriocin was found to be effective against P. gingivalis; it caused swelling and pore formation on the cell envelope at a minimum bactericidal concentration of 0.14mM, and caused death within 2h. Metronidazole killed P. gingivalis but did not affect the envelope, whereas tetracycline affected P. gingivalis with cell deformation. In conclusion, the bacteriocin from L. paracasei HL32 had the ability to kill P. gingivalis, suggesting that it could be a promising alternative chemotherapeutic agent for P. gingivalis infections.
Assuntos
Antibacterianos/uso terapêutico , Bacteriocinas/uso terapêutico , Infecções por Bacteroidaceae/tratamento farmacológico , Lactobacillus/metabolismo , Periodontite/tratamento farmacológico , Porphyromonas gingivalis/efeitos dos fármacos , Sequência de Aminoácidos , Antibacterianos/isolamento & purificação , Bacteriocinas/genética , Bacteriocinas/isolamento & purificação , Parede Celular/efeitos dos fármacos , Cromatografia em Camada Fina , Eletroforese em Gel de Poliacrilamida , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Peso Molecular , Porphyromonas gingivalis/ultraestrutura , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Using DNA microarray analysis, mRNA levels from wild-type Yersinia pestis cells treated with the iron chelator 2,2'-dipyridyl were compared with those supplemented with excessive iron, and subsequent to this, gene expression in the fur mutant was compared with that in the wild-type strain under iron rich conditions. The microarray analysis revealed many iron transport or storage systems that had been induced in response to the iron starvation, which is mediated by the Fur protein, using the iron as a co-repressor. The iron-Fur complex also affected some genes involved in various non-iron functions (ribonucleoside-diphosphate reductase, membrane proteins, electron transport and oxidative defense, etc.). The Fur protein still participated in the regulation of genes involved in broad cellular processes (virulence factors, pesticin activity, haemin storage and many proteins with unknown functions) that were not affected by iron depletion conditions. In addition to its classical negative regulatory activities, the Fur protein activates gene transcription. Using bioinformatics tools, we were able to predict the Y. pestis Fur box sequence that was clearly the over-presented motif in the promoter regions of members of the iron-Fur modulon.