Bacteriocin diversity in Streptococcus and Enterococcus.
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Bacteriocins↗
Biomedical subjects
Dzung B Diep
Publications and source records attributed to Dzung B Diep.
Improved expression and purification of the correctly folded response regulator PlnC from lactobacilli.
The response regulator PlnC is part of the signal transduction system that plays a key role in the regulation of bacteriocin production in Lactobacillus plantarum C11. In this study, we wanted to express high levels of the response regulator PlnC in a soluble and native form for purification and further studies. The protein was expressed as a fusion protein (fPlnC) containing an N-terminal Flag-tag to facilitate detection and purification. When the fusion gene, fplnC, was expressed in Escherichia coli BL21, nearly all (99%) of the recombinant protein ended up inside inclusion bodies as an incorrectly folded protein. By utilizing two different Gram-positive expression systems (SIP and NICE) in L. plantarum NC8 and Lactobacillus sakei Lb790, the expression of the soluble fPlnC was significantly increased, being 20-40 times more than that in E. coli BL21. Using the N-terminal tag, the expressed protein was purified by immunoprecipitation. By DNA-binding study (EMSA), we demonstrated that the fusion protein purified from the soluble pool was correctly folded as judged by its ability to bind specifically on regulated promoters. Using our approach, we estimate that about 1 mg of fPlnC can be purified from 11 of the bacterial culture.
Bacteriocins↗
Data mining and characterization of a novel pediocin-like bacteriocin system from the genome of Pediococcus pentosaceus ATCC 25745.
The genome of Pediococcus pentosaceus ATCC 25745 contains a gene cluster that resembles a regulated bacteriocin system. The gene cluster has an operon-like structure consisting of a putative pediocin-like bacteriocin gene (termed penA) and a potential immunity gene (termed peiA). Genetic determinants involved in bacteriocin transport and regulation are also found in proximity to penA and peiA but the so-called accessory gene involved in transport and the inducer gene involved in regulation are missing. Consequently, this bacterium is a poor bacteriocin producer. To analyse the potency of the putative bacteriocin operon, the two genes penA-peiA were heterologously expressed in a Lactobacillus sakei host that contains the complete apparatus for gene activation, maturation and externalization of bacteriocins. It was demonstrated that the heterologous host expressing penA and peiA produced a strong bacteriocin activity; in addition, the host became immune to its own bacteriocin, identifying the gene pair penA-peiA as a potent bacteriocin system. The novel pediocin-like bacteriocin, termed penocin A, has an isotopic mass [M+H]+ of 4684.6 Da as determined by mass spectrometry; this value corresponds well to the expected size of the mature 42 aa peptide containing a disulfide bridge. The bacteriocin is heat-stable but protease-sensitive and has a calculated pI of 9.45. Penocin A has a relatively broad inhibition spectrum, including pathogenic Listeria and Clostridium species. Immediately upstream of the regulatory genes reside some features that resemble remnants of a disrupted inducer gene. This degenerate gene was restored and shown to encode a double-glycine leader-containing peptide. Furthermore, expression of the restored gene triggered high bacteriocin production in P. pentosaceus ATCC 25745, thus confirming its role as an inducer in the pen regulon.
Amino Acid Sequence↗
Complete sequence of the enterocin Q-encoding plasmid pCIZ2 from the multiple bacteriocin producer Enterococcus faecium L50 and genetic characterization of enterocin Q production and immunity.
The locations of the genetic determinants for enterocin L50 (EntL50A and EntL50B), enterocin Q (EntQ), and enterocin P (EntP) in the multiple bacteriocin producer Enterococcus faecium strain L50 were determined. These bacteriocin genes occur at different locations; entL50AB (encoding EntL50A and EntL50B) are on the 50-kb plasmid pCIZ1, entqA (encoding EntQ) is on the 7.4-kb plasmid pCIZ2, and entP (encoding EntP) is on the chromosome. The complete nucleotide sequence of pCIZ2 was determined to be 7,383 bp long and contains 10 putative open reading frames (ORFs) organized in three distinct regions. The first region contains three ORFs: entqA preceded by two divergently oriented genes, entqB and entqC. EntqB shows high levels of similarity to bacterial ATP-binding cassette (ABC) transporters, while EntqC displays no significant similarity to any known protein. The second region encompasses four ORFs (orf4 to orf7), and ORF4 and ORF5 display high levels of similarity to mobilization proteins from E. faecium and Enterococcus faecalis. In addition, features resembling a transfer origin region (oriT) were found in the promoter area of orf4. The third region contains three ORFs (orf8 to orf10), and ORF8 and ORF9 exhibit similarity to the replication initiator protein RepE from E. faecalis and to RepB proteins, respectively. To clarify the minimum requirement for EntQ synthesis, we subcloned and heterologously expressed a 2,371-bp fragment from pCIZ2 that encompasses only the entqA, entqB, and entqC genes in Lactobacillus sakei, and we demonstrated that this fragment is sufficient for EntQ production. Moreover, we also obtained experimental results indicating that EntqB is involved in ABC transporter-mediated EntQ secretion, while EntqC confers immunity to this bacteriocin.
Amino Acid Sequence↗
Characterisation of the Wnt antagonists and their response to conditionally activated Wnt signalling in the developing mouse forebrain.
In the present work, the expression patterns of the Wnt antagonists of the Dickkopf (Dkk) family were characterized in the developing mouse forebrain. In situ hybridisation on sections from E12 embryos showed an expression of dkk2 in the thalamus and dkk3 in the cortical hem and thalamus. At later developmental stages (E15.5, E17.5, and P0), little or no expression of dkk1, dkk2, and dkk4 was found in the forebrain, while dkk3 expression was detected in the ventricular zone (VZ) of the lateral and III ventricles, cortical neurons, migrating cells of the primary and secondary dentate migration, and the neuroblastic layer of the eye. In the adult forebrain, dkk3 expression was detected in the lateral VZ, pyramidal neurons of the hippocampus, and cortical neurons. We also provide evidence indicating that only dkk1 and dkk4, along with two other Wnt antagonists axin2 and wif1, but not dkk2 and dkk3, are involved in a feedback mechanism to restrain Wnt signalling in transgenic mice carrying a conditional augmentation of beta-catenin in the forebrain.
Adaptor Proteins, Signal Transducing↗
Inducible bacteriocin production in Lactobacillus is regulated by differential expression of the pln operons and by two antagonizing response regulators, the activity of which is enhanced upon phosphorylation.
Expression of the five (pln) operons involved in the bacteriocin production of Lactobacillus plantarum C11 is regulated by a so-called pheromone-based signal-transducing network, in which the peptide pheromone (PlnA) induces bacteriocin production through the action of a histidine protein kinase (PlnB) and two antagonizing response regulators (PlnC as an activator and PlnD as a negative regulator). All pln-regulated promoters contain a conserved pair of direct repeats that serve as binding sites for PlnC and PlnD. In the present work, we show that the five PlnA-responsive operons are differentially expressed with regard to both timing and strength, and that the pheromone triggers a strong autoactivating loop of the regulatory unit (plnABCD) during an early stage of induction that gradually leads to enhanced activation of the other operons. The transport operon (plnGHSTUV), which is involved in the secretion of the pheromone and bacteriocins, is also expressed relatively early upon induction, but is quickly turned off soon after peak expression. Further investigation of the various promoters revealed that, although subtle differences within the promoter regions could account for the observed differential regulation, the presence of a downstream promoter-proximal sequence in one promoter was found to cause delayed peak activity. How phosphorylation regulates the activity of the pln response regulators was also accessed by direct mutagenesis at their phosphorylation sites. It was found that the two response regulators exert activity at two different levels: a low level when they are not phosphorylated and an elevated level when they are phosphorylated. The present data demonstrate that bacteriocin production in L. plantarum C11 is a highly regulated process, in which different regulatory mechanisms are applied to fine tune the timing and strength of expression of the five pln operons.
Amino Acid Sequence↗
Structural analysis of the peptide pheromone receptor PlnB, a histidine protein kinase from Lactobacillus plantarum.
Intercellular communication plays a key role in the regulation of several physiological processes in gram-positive bacteria. Cell-cell communication is often mediated by secreted inducer peptide pheromones (IPs), which upon reaching a threshold concentration in the environment specifically activate a cognate membrane-localized histidine protein kinase (HPK). Interestingly, the majority of IP-activated HPKs fall into one distinct subfamily (HPK(10)). As part of an effort to study the mechanism underlying pheromone-mediated activation of the HPK(10) subfamily, the present work investigated the membrane topology of PlnB from Lactobacillus plantarum. Gene fusion experiments with Escherichia coli and Lactobacillus sakei, using alkaline phosphatase, beta-lactamase, and beta-galactosidase reporter fusions, suggested that PlnB is anchored to the cytoplasmic membrane via seven transmembrane segments. By domain switching between HPK(10) members, it was demonstrated that the determinants for pheromone binding and specificity are contained within the transmembrane domain. The results also indicate that the mechanism of signal transduction, in which the final transmembrane segment apparently plays a key role, is conserved between members of the HPK(10) subfamily.
Amino Acid Sequence↗
Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication.
Lactic acid bacteria (LAB) fight competing Gram-positive microorganisms by secreting anti-microbial peptides called bacteriocins. Peptide bacteriocins are usually divided into lantibiotics (class I) and non-lantibiotics (class II), the latter being the main topic of this review. During the past decade many of these bacteriocins have been isolated and characterized, and elements of the genetic mechanisms behind bacteriocin production have been unravelled. Bacteriocins often have a narrow inhibitory spectrum, and are normally most active towards closely related bacteria likely to occur in the same ecological niche. Lactic acid bacteria seem to compensate for these narrow inhibitory spectra by producing several bacteriocins belonging to different classes and having different inhibitory spectra. The latter may also help in counteracting the possible development of resistance mechanisms in target organisms. In many strains, bacteriocin production is controlled in a cell-density dependent manner, using a secreted peptide-pheromone for quorum-sensing. The sensing of its own growth, which is likely to be comparable to that of related species, enables the producing organism to switch on bacteriocin production at times when competition for nutrients is likely to become more severe. Although today a lot is known about LAB bacteriocins and the regulation of their production, several fundamental questions remain to be solved. These include questions regarding mechanisms of immunity and resistance, as well as the molecular basis of target-cell specificity.
Amino Acid Sequence↗
Ribosomally synthesized antibacterial peptides in Gram positive bacteria.
The emergence of multidrug-resistant pathogens that has caused a serious problem in hospitals worldwide, has intensified the search for novel drugs, in order to replace or to be used in complement with the existing antibiotics. In this connection much interest has been focused on a group of antimicrobial peptides, so-called bacteriocins. These antagonising peptides, which are gene-encoded in contrast to those made by multi-enzyme-complexes, share some common physico-chemical properties, such as being small, cationic, amphiphilic and often being membrane active. However, they differ greatly from each other in their primary sequence and exhibit an impressively large inhibitory spectrum which covers almost all bacterial genera, including many important pathogens and food-spoilage bacteria. Many of these peptides are produced by lactic acid bacteria, organisms which have been used by man from ancient time in diverse fermentation processes, to improve and/or prolong self-life of many food and feed products. Numerous bacteriocins have been purified and characterised in great detail, both at biochemical and genetic levels. Still, novel bacteriocins with new properties are reported in an increasing number in recent years. In this review we will give a brief status quo of the present knowledge on bacteriocin research; thus different aspects such as their diversity in nature, biochemical properties, modes of action, biosynthesis and genetics will be treated.
Animals↗
The synthesis of the bacteriocin sakacin A is a temperature-sensitive process regulated by a pheromone peptide through a three-component regulatory system.
Sakacin A is a bacteriocin produced by Lactobacillus sakei Lb706. The gene cluster (sap) encompasses a regulatory unit composed of three consecutive genes, orf4 and sapKR. sapKR encode a histidine protein kinase and a response regulator, while orf4 encodes the putative precursor of a 23-amino-acid cationic peptide (termed Sap-Ph). The authors show that Sap-Ph serves as a pheromone regulating bacteriocin production. Lb706 produced bacteriocin when the growth temperature was kept at 25 or 30 degrees C, but production was reduced or absent at higher temperatures (33.5-35 degrees C). Production was restored by lowering the growth temperature to 30 degrees C, but at temperatures of 33-34 degrees C also by adding exogenous Sap-Ph to the growth medium. A knock-out mutation in orf4 abolished sakacin A production. Exogenously added Sap-Ph complemented this mutation, unambiguously showing the essential role of this peptide for bacteriocin production. Another sakacin A producer, Lactobacillus curvatus LTH1174, had a similar response to temperature and exogenously added Sap-Ph.
Amino Acid Sequence↗