Classification and mode of action of membrane-active bacteriocins produced by gram-positive bacteria

Bacteriocins are ribosomally synthesized antimicrobial peptides produced by microorganisms belonging to different eubacterial taxonomic branches. Most of them are small cationic membrane-active compounds that form pores in the target cells, disrupting membrane potentials and causing cell death. The production of small cationic peptides with antibacterial activity is a defense strategy found not only in bacteria, but also in plants and animals. Bacteriocins are classified according to different criteria by different authors; in this review, we will summarize the principal bacteriocin classifications, highlight their main physical and chemical characteristics, and describe the mechanism of some selected bacteriocins that act at the membrane level.     

Production of two bacteriocins in various growth conditions produced by gram-positive bacteria isolated from chicken cecum

Lactobacillus plantarum CLP29 and Enterococcus faecium CLE34 isolated from the cecal contents of young broiler chicks were identified based on physiological and biochemical characteristics, and identification was confirmed by 16S rRNA sequencing. Both bacteria showed a broad range of inhibitory action against bacteria such as Salmonella and Escherichia coli and produced two peptides, plantaricin CLP29 and enterocin CLE34. Treatment with proteinase K, trypase, or benase resulted in the loss of activity of the two peptides, confirming their proteinaceous nature. The highest activity levels for both bacteria were recorded in de Man?- Rogosa?- Sharpe agar at pH?5.0, 6.0, and 7.0, at 37?°C. Carbon and nitrogen sources affected the antibacterial activities of the two bacteriocins in different combinations, which suggested that the antibacterial abilities of different bacteriocins produced in nutrient sources were various.     

Production, recovery and purification of bacteriocins from lactic acid bacteria

Bacteriocins produced by lactic acid bacteria are a heterogeneous group of peptide inhibitors which include lantibiotics (class I, e.g. nisin), small heat-stable peptides (class II, e.g. pediocin AcH/PA1) and large heat-labile proteins (class III, e.g. helveticin J). Many bacteriocins belonging to the first two groups can be successfully used to inhibit undesirable microorganisms in foods, but only nisin is produced industrially and is licensed for use as a food preservative in a partially purified form. This review focuses on the production and purification of class I and class II bacteriocins from lactic acid bacteria. Bacteriocin production is growth associated but the yield of bacteriocin per unit biomass is affected by several factors, including the producing strain, media (carbohydrate and nitrogen sources, cations, etc.) and fermentation conditions (pH, temperature, agitation, aeration and dilution rate in continuous fermentations). Continuous fermentation processes with cell recycle or immobilized cells can result in a dramatic improvement in productivity over batch fermentations. Several simple recovery processes, based on adsorbing bacteriocin on resins or silica compounds, have been developed and can be used to build integrated production processes. Received: 29 December 1998 / Received revision: 23 April 1999 / Accepted: 23 April 1999     

Native and heterologous production of bacteriocins from gram-positive microorganisms

In nature, microorganisms can present several mechanisms for setting intercommunication and defense. One of these mechanisms is related to the production of bacteriocins, which are peptides with antimicrobial activity. Bacteriocins can be found in Gram-positive and Gram-negative bacteria. Nevertheless, bacteriocins produced by Gram-positive bacteria are of particular interest due to the industrial use of several strains that belong to this group, especially lactic acid bacteria (LAB), which have the status of generally recognized as safe (GRAS) microorganisms. In this work, we will review recent tendencies in the field of invention and state of art related to bacteriocin production by Gram-positive microorganism. Hundred-eight patents related to Gram-positive bacteriocin producers have been disclosed since 1965, from which 57% are related bacteriocins derived from Lactococcus, Lactobacillus, Streptococcus, and Pediococcus strains. Surprisingly, patents regarding heterologous bacteriocins production were mainly presented just in the last decade. Although the major application of bacteriocins is concerned to food industry to control spoilage and foodborne bacteria, during the last years bacteriocin applications have been displacing to the diagnosis and treatment of cancer, and plant disease resistance and growth promotion.     

益生菌产生的细菌素及其功能机制

细菌素是细菌核糖体合成的具有抑菌活性的小肽.细菌素的产生是益生菌重要的益生特性,它们天然无毒,不仅对食品腐败菌和人体致病菌有很好的抑菌活性,还具有有助益生菌定殖和调节肠道菌群等益生特性.本文综述了益生菌产生的细菌素的种类、条件性合成、益生功能及其作用机理等,以期为深入认识益生菌的益生功能及其作用方式,研究开发对人体有益...     

Regulatory mechanisms differ in UMP kinases from gram-negative and gram-positive bacteria

In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5-10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K(m) for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Gram-positive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.     

Two-peptide bacteriocins produced by lactic acid bacteria

Bacteriocins from lactic acid bacteria are ribosomally produced peptides (usually 30-60 amino acids) that display potent antimicrobial activity against certain other Gram-positive organisms. They function by disruption of the membrane of their targets, mediated in at least some cases by interaction of the peptide with a chiral receptor molecule (e.g., lipid II or sugar PTS proteins). Some bacteriocins are unmodified (except for disulfide bridges), whereas others (i.e. lantibiotics) possess extensive post-translational modifications which include multiple monosulfide (lanthionine) bridges and dehydro amino acids as well as possible keto amide residues at the N-terminus. Most known bacteriocins are biologically active as single peptides. However, there is a growing class of two peptide systems, both unmodified and lantibiotic, which are fully active only when both partners are present (usually 1:1). In some cases, neither peptide has activity by itself, whereas in others, the activity of one is enhanced by the other. This review discusses the classification, structure, production, regulation, biological activity, and potential applications of such two-peptide bacteriocins.     

Staphylococcin 1580 is identical to the lantibiotic epidermin: implications for the nature of bacteriocins from gram-positive bacteria.

Staphylococcin 1580 was purified to homogeneity from culture supernatants of Staphylococcus epidermidis 1580 by means of adsorption to XAD 2, cation exchange chromatography, and high-performance liquid chromatography on reversed-phase C18. The purified active substance migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent M(r) of approximately 2,000. Amino acid analysis, mass determination (2,165 Da) and N-terminal sequencing (Ile-Ala-Xaa-Lys-Phe-Ile-Xaa-Xaa-Pro-Gly-Xaa-Ala-Lys-block) demonstrated that staphylococcin 1580 is identical to epidermin, a lanthionine-containing antibiotic peptide (lantibiotic).     

Production of bacteriocins by different enterococcal isolates

Bacteriocins produced by ruminal as well as environmental isolates were found to be heat-stable antimicrobial substances with a broad inhibitory spectrum (including Gramnegative species and sanitary-important species). The exponential phase of growth and pH 4–7 were optimal for their production. After purification, some of them were sequenced and specified.     

Two different primary oxidation mechanisms during biotransformation of thymol by gram-positive bacteria of the genera Nocardia and Mycobacterium

Thymol has antibacterial, antifungal, insecticidal, and antioxidative properties which are the basis for the wide use of this compound in the cosmetic, food, and pharmaceutical industries. Although thymol is a ubiquitously occurring substance in the environment, data about its degradation and detoxification by bacteria are sparse. Here, we show the existence of two different pathways for the biotransformation of thymol by Nocardia cyriacigeorgica and Mycobacterium neoaurum which were described for the first time for gram-positive bacteria. The first pathway starts with hydroxylation of thymol to thymohydroquinone (2-isopropyl-5-methylbenzene-1,4-diol) with subsequent oxidation to thymobenzoquinone (2-isopropyl-5-methyl-1,4-benzoquinone). The second pathway involves hydroxylation of the methyl group followed by oxidation to 3-hydroxy-4-isopropylbenzoic acid, possibly via the aldehyde 3-hydroxy-4-isopropylbenzaldehyde. It is noteworthy that the branched side chain of thymol was not oxidized. Similarities and differences of these oxidation processes with those of the gram-negative bacterium Pseudomonas putida, fungi, and plants are discussed and, in addition, the toxicity of thymol towards N. cyriacigeorgica and M. neoaurum was tested. The experiments showed a temporary growth inhibition with 0.025 % thymol. This was explained by degradation of thymol and the formation of products which are less toxic than thymol itself.     

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. This revised version was published online in August 2006 with corrections to the Cover Date.     

CRISPR-Cas系统转录调控机制的研究进展

原核生物可利用由CRISPR-Cas系统(clustered regularly interspaced short palindromic repeats-CRISPR associated)介导的适应性免疫机制防御外源核酸入侵.在适应性免疫过程中,原核生物将外源核酸部分片段整合至自身CRISPR阵列中,表达并加工的...     

Production of bacteriocins and siderophore-like activity by Azospirillum brasilense

Sixty Azospirillum strains were tested for their bacteriocin production ability; twenty-seven (45%) were able to produce bacteriocins and inhibited the growth of one or more indicator strains in solid medium. Mitomycin C treatment enhanced the proportion to 80%. Sometimes large growth inhibition zones were formed, but not when FeCl3 was added in the medium. These inhibition zones probably result from the activity of siderophores. Partially purified bacteriocins produced by four strains were inactivated at pH 4, but were very stable between pH 5 to 10; bacteriocins produced by three strains lost their activity between 55 and 80 degrees C. Loss or decrease in the bacteriocin activity was observed with pronase E treatment; trypsin, lysozyme and alpha-amylase did not have an effect on bacteriocin activity. These findings show that the antagonism among azospirilla was due principally to the bacteriocins and sometimes probably due to siderophores, but not to bacteriophages or other substances.     

Genetic features of circular bacteriocins produced by Gram-positive bacteria

This review highlights the main genetic features of circular bacteriocins, which require the co-ordinated expression of several genetic determinants. In general terms, it has been demonstrated that the expression of such structural genes must be combined with the activity of proteins involved in maturation (cleavage/circularization) and secretion outside the cell via different transporter systems, as well as multifaceted immunity mechanisms essential to ensuring the bacteria's self-protection against such strong inhibitors. Several circular antibacterial peptides produced by Gram-positive bacteria have been described to date, including enterocin AS-48, from Enterococcus faecalis S-48 (the first one characterized), gassericin A, from Lactobacillus gasseri LA39, and a similar one, reutericin 6, from Lactobacillus reuteri LA6, butyrivibriocin AR10, from the ruminal anaerobe Butyrivibrio fibrisolvens AR10, uberolysin, from Streptococcus uberis, circularin A, from Clostridium beijerinckii ATCC 25752, and subtilosin A, from Bacillus subtilis. We summarize here the progress made in the understanding of their principal genetic features over the last few years, during which the functional roles of circular proteins with wide biological activity have become clearer.     

Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope.

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins.     

Inhibition of food-borne pathogenic bacteria by bacteriocins from Lactobacillus gasseri

Seventy-three strains of the Lactobacillus acidophilus group and a Lact. reuteri isolated from human faeces were examined for production of antimicrobial agents against 16 strains of six species of food-borne enteric pathogenic bacteria. Several strains of Lact. gasseri showed wide inhibitory activity against the tested bacteria. Gassericin A produced by Lact. gasseri LA39 was one of the most widely active bacteriocins. It was bactericidal without causing cell lysis.     

Structural analysis and characterization of lacticin Q, a novel bacteriocin belonging to a new family of unmodified bacteriocins of gram-positive bacteria

Lactococcus lactis QU 5 isolated from corn produces a novel bacteriocin, termed lacticin Q. By acetone precipitation, cation-exchange chromatography, and reverse-phase high-performance liquid chromatography, lacticin Q was purified from the culture supernatant of this organism, and its molecular mass was determined to be 5,926.50 Da by mass spectrometry. Subsequent analyses of amino acid and DNA sequences revealed that lacticin Q comprised 53 amino acid residues and that its N-terminal methionine residue was formylated. In contrast to most bacteriocins produced by gram-positive bacteria, lacticin Q had no N-terminal extensions such as leader or signal sequences. It showed 66% and 48% identity to AucA, a hypothetical protein from Corynebacterium jeikeium plasmid pA501, and aureocin A53, a bacteriocin from Staphylococcus aureus A53, respectively. The characteristics of lacticin Q were determined and compared to those of nisin A. Similar to nisin A, lacticin Q exhibited antibacterial activity against various gram-positive bacteria. Lacticin Q was very stable against heat treatment and changes in pH; in particular, it was stable at alkaline pH values, while nisin A was inactivated. Moreover, lacticin Q induced ATP efflux from a Listeria sp. strain in a shorter time and at a lower concentration than nisin A, indicating that the former affected indicator cells in a different manner from that of the latter. The results described here clarified the fact that lacticin Q belongs to a new family of class II bacteriocins and that it can be employed as an alternative to or in combination with nisin A.