RESUMEN
BACKGROUND: Xenorhabdus spp. live in close symbiosis with nematodes of the Steinernema genus. Steinernema nematodes infect an insect larva and release their symbionts into the haemocoel of the insect. Once released into the haemocoel, the bacteria produce bioactive compounds to create a semi-exclusive environment by inhibiting the growth of bacteria, yeasts and molds. The antimicrobial compounds thus far identified are xenocoumacins, xenortides, xenorhabdins, indole derivatives, xenoamicins, bicornutin and a number of antimicrobial peptides. The latter may be linear peptides such as the bacteriocins xenocin and xenorhabdicin, rhabdopeptides and cabanillasin, or cyclic, such as PAX lipopeptides, taxlllaids, xenobactin and szentiamide. Thus far, production of antimicrobial compounds have been reported for Xenorhabdus nematophila, Xenorhabdus budapestensis, Xenorhabdus cabanillasii, Xenorhabdus kozodoii, Xenorhabdus szentirmaii, Xenorhabdus doucetiae, Xenorhabdus mauleonii, Xenorhabdus indica and Xenorhabdus bovienii. Here we describe, for the first time, PAX lipopeptides and xenocoumacin 2 produced by Xenorhabdus khoisanae. These compounds were identified using ultraperformance liquid chromatography, linked to high resolution electrospray ionisation mass spectrometry and tandem mass spectrometry. RESULTS: Cell-free supernatants of X. khoisanae SB10 were heat stable and active against Bacillus subtilis subsp. subtilis, Escherichia coli and Candida albicans. Five lysine-rich lipopeptides from the PAX group were identified in HPLC fractions, with PAX1' and PAX7 present in the highest concentrations. Three novel PAX7 peptides with putative enoyl modifications and two linear analogues of PAX1' were also detected. A small antibiotic compound, yellow in colour and λmax of 314 nm, was recovered from the HPLC fractions and identified as xenocoumacin 2. The PAX lipopeptides and xenocoumacin 2 correlated with the genes and gene clusters in the genome of X. khoisanae SB10. CONCLUSION: With UPLC-MS and MSe analyses of compounds in the antimicrobial complex of X. khoisanae SB10, a number of PAX peptides and a xenocoumacin were identified. The combination of pure PAX1' peptide with xenocoumacin 2 resulted in high antimicrobial activity. Many of the fractions did, however, contain labile compounds and some fractions were difficult to resolve. It is thus possible that strain SB10 may produce more antimicrobial compounds than reported here, as suggested by the APE Ec biosynthetic complex. Further research is required to develop these broad-spectrum antimicrobial compounds into drugs that may be used in the fight against microbial infections.
Asunto(s)
Antiinfecciosos/farmacología , Benzopiranos/farmacología , Lipopéptidos/metabolismo , Xenorhabdus/fisiología , Antiinfecciosos/metabolismo , Bacillus subtilis/efectos de los fármacos , Proteínas Bacterianas , Benzopiranos/metabolismo , Vías Biosintéticas , Candida albicans/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Escherichia coli/efectos de los fármacos , Espectrometría de Masa por Ionización de Electrospray , Simbiosis , Espectrometría de Masas en Tándem , Xenorhabdus/genética , Xenorhabdus/metabolismoRESUMEN
Probiotics play an important role in maintaining a healthy and stable intestinal microbiota, primarily by preventing infection. Probiotic lactic acid bacteria (LAB) are known to be inhibitory to many bacterial enteric pathogens, including antibiotic-resistant strains. Whilst the positive role that probiotics have on human physiology, specifically in the treatment or prevention of specific infectious diseases of the gastro-intestinal tract (GIT) is known, the precise mechanistic basis of these effects remains a major research goal. In this study, molecular evidence to underpin the protective and anti-listerial effect of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA against orally administered Listeria monocytogenes EGDe in the GIT of mice is provided. Bacteriocins plantaricin 423 and mundticin ST4SA, produced by L. plantarum 423 and E. mundtii ST4SA, respectively, inhibited the growth of L. monocytogenes in vitro and in vivo. Bacteriocin-negative mutants of L. plantarum 423 and E. mundtii ST4SA failed to exclude L. monocytogenes EGDe from the gastrointestinal tract (GIT) of mice. Furthermore, L. plantarum 423 and E. mundtii ST4SA failed to inhibit recombinant strains of L. monocytogenes EGDe in vivo that expressed the immunity proteins of the two bacteriocins. These results confirmed that bacteriocins plantaricin 423 and mundticin ST4SA acted as anti-infective mediators in vivo. Compared to wild type strains, mutants of L. plantarum 423 and E. mundtii ST4SA, in which the adhesion genes were knocked out, were less effective in the exclusion of L. monocytogenes EGDe from the GIT of mice. This work demonstrates the importance of bacteriocin and adhesion genes as probiotic anti-infective mechanisms.
Asunto(s)
Antibacterianos/metabolismo , Adhesión Bacteriana/fisiología , Bacteriocinas/metabolismo , Enterococcus/química , Lactobacillus plantarum/química , Listeria monocytogenes/crecimiento & desarrollo , Animales , Antibacterianos/farmacología , Antibiosis , Adhesión Bacteriana/genética , Bacteriocinas/genética , Bacteriocinas/farmacología , Enterococcus/genética , Femenino , Tracto Gastrointestinal/microbiología , Lactobacillus plantarum/genética , Listeria monocytogenes/efectos de los fármacos , Ratones Endogámicos BALB C , Mutación , ProbióticosRESUMEN
Listeria monocytogenes is an opportunistic food-borne pathogen and is life-threatening to individuals with a weakened immune system. The aim of this study was to determine if Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA could prevent colonisation of L. monocytogenes in the gastro-intestinal tract (GIT). Mice were gavaged with L. plantarum 423, E. mundtii ST4SA, and a combination of the two strains, for 6 consecutive days and orally infected with a bioluminescent strain of L. monocytogenes (strain EGDe) on the last day of treatment. 30 min after infection, high cell numbers of L. plantarum 423, E. mundtii ST4SA and L. monocytogenes EGDe were isolated from faeces. L. monocytogenes EGDe cells were absent from the small intestine of L. plantarum 423-treated mice 4 h after infection and from the large intestine 2 h later. No bioluminescent, and thus metabolically active, cells of L. monocytogenes EGDe were recorded in the GIT of mice treated with E. mundtii ST4SA, suggesting that their growth was repressed. L. plantarum 423 and E. mundtii ST4SA colonised the colon the strongest. These strains may be considered for the competitive exclusion of L. monocytogenes from the GIT.
Asunto(s)
Enterococcus/fisiología , Tracto Gastrointestinal/microbiología , Lactobacillus plantarum/fisiología , Listeria monocytogenes/crecimiento & desarrollo , Probióticos/administración & dosificación , Animales , Antibiosis , Enterococcus/química , Heces/microbiología , Femenino , Tracto Gastrointestinal/química , Listeria monocytogenes/fisiología , Mediciones Luminiscentes , Ratones , Ratones Endogámicos BALB CRESUMEN
Two large cryptic plasmids (59.2 and 65.9 kb) from isolates of Sulfobacillus thermotolerans from Yellowstone National Park (United States) and the Caribbean island of Montserrat were isolated and sequenced. This analysis revealed a common "backbone" region coding for a potential plasmid stability system plus a nonpheromone conjugation system containing homologues of both type IV and type II (tight adherence, or Tad-like) secretion systems.
Asunto(s)
Microbiología Ambiental , Bacterias Grampositivas/genética , Plásmidos/aislamiento & purificación , Región del Caribe , Conjugación Genética , ADN Bacteriano/química , ADN Bacteriano/genética , Genes Bacterianos , Bacterias Grampositivas/aislamiento & purificación , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Estados UnidosRESUMEN
A moderately thermophilic (45 to 50 degrees C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic Acidithiobacillus caldus strain, f, was isolated from a biooxidation process used to treat nickel ore. Trans-alternating field electrophoresis analysis of total DNA from the A. caldus cells revealed two plasmids of approximately 14 and 45 kb. The 14-kb plasmid, designated pTC-F14, was cloned and shown by replacement of the cloning vector with a kanamycin resistance gene to be capable of autonomous replication in Escherichia coli. Autonomous replication was also demonstrated in Pseudomonas putida and Agrobacterium tumefaciens LBA 4404, which suggested that pTC-F14 is a broad-host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five open reading frames and a replicon organization like that of the broad-host-range IncQ plasmids. Three of the open reading frames encoded replication proteins which were most closely related to those of IncQ-like plasmid pTF-FC2 (amino acid sequence identities: RepA, 81%; RepB, 78%; RepC, 74%). However, the two plasmids were fully compatible and pTC-F14 represents a new IncQ-like plasmid replicon. Surprisingly, asymmetrical incompatibility was found with the less closely related IncQ plasmid R300B derivative pKE462 and the IncQ-like plasmid derivative pIE1108. Analysis of the pTC-F14 oriV region revealed five direct repeats consisting of three perfectly conserved 22-bp iterons flanked by iterons of 23 and 21 bp. Plasmid pTC-F14 had a copy number of 12 to 16 copies per chromosome in both E. coli, and A. caldus. The rep gene products of pTC-F14 and pTF-FC2 were unable to functionally complement each other's oriV regions, but replication occurred when the genes for each plasmid's own RepA, RepB, and RepC proteins were provided in trans. Two smaller open reading frames were found between the repB and repA genes of pTC-F14, which encode proteins with high amino acid sequence identity (PasA, 81%; PasB, 72%) to the plasmid addiction system of pTF-FC2. This is the second time a plasmid stability system of this type has been found on an IncQ-like plasmid.
Asunto(s)
Plásmidos/genética , Plásmidos/aislamiento & purificación , Replicón/genética , Thiobacillus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Dosificación de Gen , Datos de Secuencia Molecular , Origen de Réplica , Análisis de Secuencia de ADNRESUMEN
The chromosomal arsenic resistance genes of the acidophilic, chemolithoautotrophic, biomining bacterium Thiobacillus ferrooxidans were cloned and sequenced. Homologues of four arsenic resistance genes, arsB, arsC, arsH, and a putative arsR gene, were identified. The T. ferrooxidans arsB (arsenite export) and arsC (arsenate reductase) gene products were functional when they were cloned in an Escherichia coli ars deletion mutant and conferred increased resistance to arsenite, arsenate, and antimony. Therefore, despite the fact that the ars genes originated from an obligately acidophilic bacterium, they were functional in E. coli. Although T. ferrooxidans is gram negative, its ArsC was more closely related to the ArsC molecules of gram-positive bacteria. Furthermore, a functional trxA (thioredoxin) gene was required for ArsC-mediated arsenate resistance in E. coli; this finding confirmed the gram-positive ArsC-like status of this resistance and indicated that the division of ArsC molecules based on Gram staining results is artificial. Although arsH was expressed in an E. coli-derived in vitro transcription-translation system, ArsH was not required for and did not enhance arsenic resistance in E. coli. The T. ferrooxidans ars genes were arranged in an unusual manner, and the putative arsR and arsC genes and the arsBH genes were translated in opposite directions. This divergent orientation was conserved in the four T. ferrooxidans strains investigated.
Asunto(s)
Acidithiobacillus thiooxidans/genética , Adenosina Trifosfatasas/genética , Antimonio/farmacología , Arsénico/farmacología , Farmacorresistencia Microbiana/genética , Escherichia coli/genética , Bombas Iónicas , Complejos Multienzimáticos , Acidithiobacillus thiooxidans/clasificación , Acidithiobacillus thiooxidans/efectos de los fármacos , ATPasas Transportadoras de Arsenitos , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Cinética , Proteínas de la Membrana/genética , Filogenia , Plásmidos , Mapeo Restrictivo , Tiorredoxinas/metabolismoRESUMEN
A recombinant plasmid which contains the gltD gene coding for the glutamate synthase (GOGAT) small subunit was isolated from a Thiobacillus ferrooxidans ATCC33020 gene bank by complementation of an Escherichia coli gltD mutant. The sequence of gltD was determined. The deduced amino acid sequence shows strong similarity to the two other prokaryote gltD sequences available, namely those of E. coli and A. brasilense (53% and 45% identity, respectively). A cosmid containing the gltBD region was isolated from a T. ferrooxidans cosmid gene bank, but was unable to complement an E. coli gltB mutant.
Asunto(s)
Escherichia coli/genética , Glutamato Sintasa/genética , Mutación , Thiobacillus/enzimología , Secuencia de Aminoácidos , Clonación Molecular , Genes Bacterianos , Prueba de Complementación Genética , Datos de Secuencia Molecular , Thiobacillus/genéticaRESUMEN
The Thiobacillus ferrooxidans thioredoxin gene, trxA, was isolated by its ability to complement an Escherichia coli gshA trxA mutant which was otherwise unable to grow on minimal medium lacking glutathione. The T. ferrooxidans thioredoxin also enabled the in vivo reduction by E. coli of methionine sulfoxide to methionine, as well as the in vitro reduction of insulin. When present in E. coli, the T. ferrooxidans thioredoxin supported the replication of phage T7, but not the growth of phage M13. The T. ferrooxidans trxA gene was sequenced and the thioredoxin was found to be most like that of E. coli (71% identity) and Chromatium vinosum (70% identity). As in the case of E. coli, the gene was located immediately upstream of the gene for the rho transcriptional terminator. DNA:RNA blot hybridization and primer-extension analysis of the trxA gene in T. ferrooxidans and the cloned gene in E. coli indicated that it was transcribed as an independent unit and that the major transcriptional start sites were the same in both organisms.
Asunto(s)
Genes Bacterianos , Thiobacillus/genética , Tiorredoxinas/genética , Bacteriófago M13/crecimiento & desarrollo , Bacteriófago T7/crecimiento & desarrollo , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Prueba de Complementación Genética , Insulina/metabolismo , Metionina/análogos & derivados , Metionina/metabolismo , Datos de Secuencia Molecular , Thiobacillus/metabolismoRESUMEN
The nucleotide sequence of the Vibrio alginolyticus alkaline serine exoprotease A (ProA) gene cloned in Escherichia coli was determined. The exoprotease A gene (proA) consisted of 1602 bp which encoded a protein of 534 amino acids (aa) with an Mr of 55,900. The region upstream from the gene was characterized by a putative promoter consensus region (-10 -35), a ribosome-binding site and ATG start codon. The proA gene encodes a typical 21-aa N-terminal signal sequence which, when fused to alkaline phosphatase by means of transposon TnphoA, was able to mediate transport of the alkaline phosphatase to the periplasm in E. coli. Deletions of up to 106 aa from the C terminus of ProA did not result in the loss of extracellular protease activity. Additional V. alginolyticus genes were not involved in the secretion into the medium of the cloned ProA in E. coli. The amino acid sequence of ProA showed low overall homology to a Serratia marcescens serine exoprotease but significant homology was detected with other subtilisin family exoproteases. The fungal proteinase K, another sodium dodecyl sulfate-resistant protease, had 44% aa homology with ProA.