Conjugative Plasmid from Lactobacillus gasseri LA39 That Carries Genes for Production of and Immunity to the Circular Bacteriocin Gassericin A

Gassericin A is a circular bacteriocin produced by Lactobacillus gasseri strain LA39. We found a 33,333-bp plasmid, designated pLgLA39, in this strain. pLgLA39 contained 44 open reading frames, including seven genes related to gassericin A production/immunity (gaa), as well as genes for replication, plasmid maintenance, and conjugative transfer. pLgLA39 was transferred from LA39 to the type strain of L. gasseri (JCM 1131) by filter mating. The transconjugant exhibited >30-fold-higher more resistance to gassericin A and produced antibacterial activity. Lactobacillus reuteri LA6, the producer of reutericin 6, was proved to harbor a plasmid indistinguishable from pLgLA39 and carrying seven genes 100% identical to gaa. This suggests that pLgLA39 might have been transferred naturally between L. gasseri LA39 and L. reuteri LA6. The seven gaa genes of pLgLA39 were cloned into a plasmid vector to construct pGAA. JCM 1131^T transformed with pGAA expressed antibacterial activity and resistance to gassericin A. pGAA was segregationally more stable than a pGAA derivative plasmid from which gaaA was deleted and even was more stable than the vector. This suggests the occurrence of postsegregational host killing by the gaa genes. pLgLA39 carried a pemIK homolog, and segregational stabilization of a plasmid by the pLgLA39-type pemIK genes was also confirmed. Thus, pLgLA39 was proved to carry the genes for at least two plasmid maintenance mechanisms, i.e., gaa and pemIK. Plasmids containing a repA gene similar to pLgLA39 repA were distributed in several L. gasseri strains.Lactobacillus species are normal inhabitants of the human gastrointestinal tract, and Lactobacillus gasseri is one of the most commonly detected of these species (37, 47). Health-promoting effects of this species, such as immunomodulation (35), suppression of Helicobacter pylori-induced interleukin-8 production (44), and improvement of intestinal conditions (34), have been reported, and some L. gasseri strains are used in commercial probiotic products.Bacteriocins are antimicrobial peptides, proteins, or protein complexes produced by bacteria and active mainly against related bacterial species (38). Several bacteriocins also inhibit the growth of food-borne pathogens, such as Listeria, Bacillus cereus, and Clostridium perfringens. Production of bacteriocin is thought to be a desired feature for probiotic strains, since bacteriocin is believed to provide an advantage for survival in the ecological niche and to prevent the growth of pathogens. Several L. gasseri strains are known to produce bacteriocins (18). The classification of bacteriocins remains controversial. We use the definition proposed by Maqueda et al. (30), where bacteriocins are classified into class I (lantibiotics), class II (nonlantibiotics), class III (large heat-labile bacteriocins), and class IV (circular bacteriocins linked at the N- and C-terminal ends). Among these, the class IV circular bacteriocins have attracted increasing attention, since they are the simplest prokaryotic representatives of the ubiquitous circular peptides with various physiological activities (6). Enterocin AS-48 from Enterococcus faecalis strain S-48 is the first and most vigorously characterized member of the class IV bacteriocins (30). L. gasseri strain LA39 (JCM 11657) produces a 58-amino-acid (aa) circular bacteriocin, gassericin A (18). Gassericin A is a representative of the non-AS-48-like circular bacteriocin group including butyrivibriocin AR10 from Butyrivibrio fibrisolvens AR10 (15) and carnocyclin A from Carnobacterium maltaromaticum UAL307 (32), as well as reutericin 6 from Lactobacillus reuteri LA6 (17) and acidocin B from Lactobacillus acidophilus M46 (26). The last two bacteriocins have nearly identical amino acid sequences to that of gassericin A. Though the number of reported circular bacteriocins has been increasing, their primary sequences and the genes responsible for production of and immunity to them are diversified (for a review, see reference 31). Recently, we isolated and sequenced seven genes (gaaBCADITE) from LA39 deduced to be responsible for production of and immunity to gassericin A (20). The gaa genes add new information to the complex world of the class IV bacteriocin genes.The structural gene of gassericin A, gaaA, was reported to be located on the chromosome of LA39 (19). However, the high amino acid sequence identity of gassericin A to reutericin 6 (100%) and to acidocin B (98%) suggests recent horizontal gene transfers of the relevant bacteriocin genes, possibly via mobile elements. In fact, the acidocin B genes were reported to be located on a plasmid, namely, pCV461 (26). Many Lactobacillus strains are known to harbor one or more plasmids of various sizes, and several Lactobacillus plasmids have been reported to contain genes for production of bacteriocins (48). To our knowledge, however, only three have been sequenced entirely: these are pLA103 from Lactobacillus acidophilus TK8912 (16), pRC18 from Lactobacillus curvatus (previously known as Lactobacillus casei) CRL705 (7), and pMP118 from Lactobacillus salivarius subsp. salivarius UCC118 (5). Thus, genetic information about bacteriocin-producing Lactobacillus plasmids is still limited. Furthermore, little has been known about plasmids of L. gasseri, even though the existence of plasmids in a few strains has been reported, including a 26.5-kb anonymous plasmid in strain ADH (27) and pK7 in strain K7 (28).Here we describe a 33.3-kb plasmid, designated pLgLA39, from L. gasseri LA39. The gaa genes are located on this plasmid. pLgLA39 carries a set of genes for conjugative transfer and was shown to be transmitted to another L. gasseri strain. L. reuteri LA6 also harbors a plasmid almost identical to pLgLA39. We demonstrated that production of gassericin A increased the apparent segregational stability of a plasmid carrying the gaa genes. A pemIK homolog in pLgLA39 was also functional as a plasmid-stabilizing mechanism. This is the first report describing the entire nucleotide sequence and detailed genetic analysis of an L. gasseri plasmid, which contains functional genes for circular bacteriocin production, conjugation, and plasmid maintenance.     

Gassericin A: a circular bacteriocin produced by Lactic acid bacteria Lactobacillus gasseri

During the recent years extensive efforts have been made to find out bacteriocins from lactic acid bacteria (LAB) active against various food spoilage and pathogenic bacteria, and superior stabilities against heat treatments and pH variations. Bacteriocins isolated from LAB have been grouped into four classes. Circular bacteriocins which were earlier grouped among the four groups of bacteriocins, have recently been proposed to be classified into a different class, making it class V bacteriocins. Circular bacteriocins are special molecules, whose precursors must be post translationally modified to join the N to C termini with a head-to-tail peptide bond. Cyclization appears to make them less susceptible to proteolytic cleavage, high temperature and pH, and, therefore, provides enhanced stability as compared to linear bacteriocins. The advantages of circularization are also reflected by the fact that a significant number of macrocyclic natural products have found pharmaceutical applications. Circular bacteriocins were unknown two decades ago, and even to date, only a few circular bacteriocins from a diverse group of Gram positive organisms have been reported. The first example of a circular bacteriocin was enterocin AS-48, produced by Enterococcus faecalis AS-48. Gassereccin A, produced by Lactobacillus gasseri LA39, Reutericin 6 produced by Lactobacillus reuteri LA6 and Circularin A, produced by Clostridium beijerinickii ATCC 25,752, are further examples of this group of antimicrobial peptides. In the present scenario, Gassericin A can be an important tool in the food preservation owing to its properties of high pH and temperature tolerance and the fact that it is produced by LAB L. gasseri, whose many strains are proven probiotic.     

Solution Structure of Acidocin B,a Circular Bacteriocin Produced by Lactobacillus acidophilus M46

Acidocin B, a bacteriocin produced by Lactobacillus acidophilus M46, was originally reported to be a linear peptide composed of 59 amino acid residues. However, its high sequence similarity to gassericin A, a circular bacteriocin from Lactobacillus gasseri LA39, suggested that acidocin B might be circular as well. Acidocin B was purified from culture supernatant by a series of hydrophobic interaction chromatographic steps. Its circular nature was ascertained by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry and tandem mass spectrometry (MS/MS) sequencing. The peptide sequence was found to consist of 58 amino acids with a molecular mass of 5,621.5 Da. The sequence of the acidocin B biosynthetic gene cluster was also determined and showed high nucleotide sequence similarity to that of gassericin A. The nuclear magnetic resonance (NMR) solution structure of acidocin B in sodium dodecyl sulfate micelles was elucidated, revealing that it is composed of four α-helices of similar length that are folded to form a compact, globular bundle with a central pore. This is a three-dimensional structure for a member of subgroup II circular bacteriocins, which are classified based on their isoelectric points of ∼7 or lower. Comparison of acidocin B with carnocyclin A, a subgroup I circular bacteriocin with four α-helices and a pI of 10, revealed differences in the overall folding. The observed variations could be attributed to inherent diversity in their physical properties, which also required the use of different solvent systems for three-dimensional structural elucidation.     

Amylocyclicin,a Novel Circular Bacteriocin Produced by Bacillus amyloliquefaciens FZB42

Bacillus amyloliquefaciens FZB42 is a Gram-positive plant growth-promoting bacterium with an impressive capacity to synthesize nonribosomal secondary metabolites with antimicrobial activity. Here we report on a novel circular bacteriocin which is ribosomally synthesized by FZB42. The compound displayed high antibacterial activity against closely related Gram-positive bacteria. Transposon mutagenesis and subsequent site-specific mutagenesis combined with matrix-assisted laser desorption ionization–time of flight mass spectroscopy revealed that a cluster of six genes covering 4,490 bp was responsible for the production, modification, and export of and immunity to an antibacterial compound, here designated amylocyclicin, with a molecular mass of 6,381 Da. Peptide sequencing of the fragments obtained after tryptic digestion of the purified peptide revealed posttranslational cleavage of an N-terminal extension and head-to-tail circularization of the novel bacteriocin. Homology to other putative circular bacteriocins in related bacteria let us assume that this type of peptide is widespread among the Bacillus/Paenibacillus taxon.     

Isolation and Characterization of Carnocyclin A, a Novel Circular Bacteriocin Produced by Carnobacterium maltaromaticum UAL307

Carnobacterium maltaromaticum UAL307, isolated from fresh pork, exhibits potent activity against a number of gram-positive organisms, including numerous Listeria species. Three bacteriocins were isolated from culture supernatant, and using matrix-assisted laser desorption ionization-time of flight mass spectrometry and Edman sequencing, two of these bacteriocins were identified as piscicolin 126 and carnobacteriocin BM1, both of which have previously been described. The remaining bacteriocin, with a molecular mass of 5,862 Da, could not be sequenced by traditional methods, suggesting that the peptide was either cyclic or N-terminally blocked. This bacteriocin showed remarkable stability over a wide temperature and pH range and was unaffected by a variety of proteases. After digestion with trypsin and α-chymotrypsin, the peptide was de novo sequenced by tandem mass spectrometry and a linear sequence deduced, consisting of 60 amino acids. Based on this sequence, the molecular mass was predicted to be 5,880 Da, 18 units higher than the observed molecular mass, which suggested that the peptide has a cyclic structure. Identification of the genetic sequence revealed that this peptide is circular, formed by a covalent linkage between the N and C termini following cleavage of a 4-residue peptide leader sequence. The results of structural studies suggest that the peptide is highly structured in aqueous conditions. This bacteriocin, named carnocyclin A, is the first reported example of a circular bacteriocin produced by Carnobacterium spp.     

Plasmid-associated Bacteriocin Production by and Immunity of Lactobacillus acidophilus TK8912

Lactobacillus acidophilus TK8912 produces an antibacterial substance, designated acidocin 8912, which is active against strains of Lactobacillus and Lactococcus. Of all conditions tested, the production of acidocin 8912 was maximum at 30°C in MRS broth. Acidocin 8912 was stable to heat treatment (120°C for 20min), but completely inactivated by protease treatment. Curing a plasmid pLA103 resulted in the loss of both acidocin 8912 production (Acd⁺) and host immunity (Acd^r). A plasmid-cured strain, TK1–4 (Acd^- Acd⁸), was transformed to Acd⁺Acd^r with the pLA103 plasmid. These results provided the first direct evidence in lactobacilli for involvement of this plasmid in bacteriocin production and immunity.     

Development of a Homologous Expression System for and Systematic Site-Directed Mutagenesis Analysis of Thurincin H,a Bacteriocin Produced by Bacillus thuringiensis SF361

Thurincin H is an antimicrobial peptide produced by Bacillus thuringiensis SF361. With a helical back bone, the 31 amino acids of thurincin H form a hairpin structure maintained by four pairs of very unique sulfur-to-α-carbon thioether bonds. The production of thurincin H depends on a putative gene cluster containing 10 open reading frames. The gene cluster includes three tandem structural genes (thnA1, thnA2, and thnA3) encoding three identical 40-amino-acid thurincin H prepeptides and seven other genes putatively responsible for prepeptide processing, regulation, modification, exportation, and self-immunity. A homologous thurincin H expression system was developed by transforming a thurincin H-deficient host with a novel expression vector, pGW133. The host, designated B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3, was constructed by deletion of the three tandem structural genes from the chromosome of the natural thurincin H producer. The thurincin H expression vector pGW133 was constructed by cloning the thurincin H native promoter, thnA1, and a Cry protein terminator into the Escherichia coli-B. thuringiensis shuttle vector pHT315. Thirty-three different pGW133 variants, each containing a different point mutation in the thnA1 gene, were generated and separately transformed into B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3. Those site-directed mutants contained either a single radical or conservative amino acid substitution on the thioether linkage-forming positions or a radical substitution on all other nonalanine amino acids. The bacteriocin activities of B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3 carrying different pGW133 variants against three different indicator strains were subsequently compared.     

Purification and Some Properties of Acidocin 8912, a Novel Bacteriocin Produced by Lactobacillus acidophilus TK8912

Acidocin 8912, a bacteriocin produced by Lactobacillus acidophilus TK8912, was purified by ammonium sulfate fractionation and successive chromatographies on CM-cellulose, Sephadex G-50, Sephadex G-25, and reversed-phase HPLC on Aquapore RP-300. The purified acidocin 8912 migrated as a single band on SDS–PAGE. The molecular weight was estimated to be 5200 by SDS–PAGE, and 5400 by HPLC gel filtration on TSKgel G3000PW_XL. Both the amino acid composition and the N-terminal amino acid sequence analysis indicated that acidocin 8912 was a peptide composed of presumably 50 amino acids containing a Lys residue at the N-terminus. The purified acidocin 8912 showed a bactericidal effect on sensitive cells but not a bacteriolytic effect.     

Purification and Characterization of Plantaricin ZJ5, a New Bacteriocin Produced by Lactobacillus plantarum ZJ5

The aim of this study is to investigate the antimicrobial potential of Lactobacillus plantarum ZJ5, a strain isolated from fermented mustard with a broad range of inhibitory activity against both Gram-positive and Gram-negative bacteria. Here we present the peptide plantaricin ZJ5 (PZJ5), which is an extreme pH and heat-stable. However, it can be digested by pepsin and proteinase K. This peptide has strong activity against Staphylococcus aureus. PZJ5 has been purified using a multi-step process, including ammonium sulfate precipitation, cation-exchange chromatography, hydrophobic interactions and reverse-phase chromatography. The molecular mass of the peptide was found to be 2572.9 Da using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The primary structure of this peptide was determined using amino acid sequencing and DNA sequencing, and these analyses revealed that the DNA sequence translated as a 44-residue precursor containing a 22-amino-acid N-terminal extension that was of the double-glycine type. The bacteriocin sequence exhibited no homology with known bacteriocins when compared with those available in the database, indicating that it was a new class IId bacteriocin. PZJ5 from a food-borne strain may be useful as a promising probiotic candidate.     

Purification and Partial Amino Acid Sequence of Brevicin 27, a Bacteriocin Produced by Lactobacillus brevis SB27

Brevicin 27, a bacteriocin produced by Lactobacillus brevis SB27, is inhibitory mainly against closely related Lactobacillus brevis and Lactobacillus büchneri strains. It was purified from the culture supernatant by a four-step purification procedure including ammonium sulfate precipitation, cation exchange, hydrophobic interaction, and reverse-phase, high performance liquid chromatographies. The purified bacteriocin was subjected to mass spectrometry, amino acid composition analysis, and sequencing by Edman degradation. It was shown to be an about 5200-Da basic protein containing a high proportion of lysine and of hydrophobic amino acids. The partial N-terminal amino acid sequence (25 residues) was unique when compared with the Protein Data Bank (PDB), Swiss Prot, and Protein Information Resource (PIR) data banks and to the translated Gen Bank. Received: 24 July 1996 / Accepted: 10 September 1996     

Evaluation of random amplified polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus acidophilus,Lactobacillus crispatus,Lactobacillus amylovorus,Lactobacillus gallinarum,Lactobacillus gasseri,and Lactobacillus johnsonii

The technique random amplified polymorphic DNA (RAPD)-PCR was evaluated as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, and Lactobacillus johnsonii. Representative strains, including the type of each species, were selected from different clusters obtained by numerical analysis of total soluble cell protein patterns. Results obtained by RAPD-PCR corresponded well with results obtained by numerical analysis of total soluble cell protein patterns. The type strains of each species displayed different RAPD profiles. Strains with identical L(+)- nicotinamide adenine dinucleotide-dependent lactic dehydrogenase (nLDH) electrophoretic profiles could be distinguished on the basis of their RAPD profiles.     

Sodium Chloride Reduces Production of Curvacin A, a Bacteriocin Produced by Lactobacillus curvatus Strain LTH 1174, Originating from Fermented Sausage

Lactobacillus curvatus LTH 1174, a strain originating in fermented sausage, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of salt (sodium chloride) were investigated in vitro during laboratory fermentations using modified MRS medium. A model was set up to describe the effects of different NaCl concentrations on microbial behavior. Both cell growth and bacteriocin activity were affected by changes in the salt concentration. Sodium chloride clearly slowed down the growth of L. curvatus LTH 1174, but more importantly, it had a detrimental effect on specific curvacin A production (k_B) and hence on overall bacteriocin activity. Even a low salt concentration (2%, wt/vol) decreased bacteriocin production, while growth was unaffected at this concentration. The inhibitory effect of NaCl was mainly due to its role as an a_w-lowering agent. Further, it was clear that salt interfered with bacteriocin induction. Additionally, when 6% (wt/vol) sodium chloride was added, the minimum biomass concentration necessary to start the production of curvacin A (X_B) was 0.90 g (cell dry mass) per liter. Addition of the cell-free culture supernatant or a protein solution as a source of induction factor resulted in a decrease in X_B, an increase in k_B, and hence an increase in the maximum attainable bacteriocin activity.     

Characterization of a New Bacteriocin Operon in Sakacin P-Producing Lactobacillus sakei, Showing Strong Translational Coupling between the Bacteriocin and Immunity Genes

Previous studies of genes involved in the production of sakacin P by Lactobacillus sakei Lb674 revealed the presence of an inducible promoter downstream of the known spp gene clusters. We show here that this promoter drives the expression of an operon consisting of a bacteriocin gene (sppQ), a cognate immunity gene (spiQ), another gene with an unknown function (orf4), and a pseudoimmunity gene containing a frameshift mutation (orf5). The leader peptide of the new one-peptide bacteriocin sakacin Q contains consensus elements that are typical for so-called “double-glycine” leader peptides. The mature bacteriocin shows weak similarity to the BrcA peptide of the two-peptide bacteriocin brochocin C. Sakacin Q has an antimicrobial spectrum that differs from that of sakacin P, thus expanding the antimicrobial properties of the producer strain. The genes encoding sakacin Q and its cognate immunity protein showed strong translational coupling, which was investigated in detail by analyzing the properties of a series of β-glucuronidase fusions. Our results provide experimental evidence that production of the bacteriocin and production of the cognate immunity protein are tightly coregulated at the translational level.     

Peptide Pheromone Plantaricin A Produced by Lactobacillus plantarum Permeabilizes Liver and Kidney Cells

Certain antimicrobial peptides from multicellular animals kill a variety of tumor cells at concentrations not affecting normal eukaryotic cells. Recently, it was reported that also plantaricin A (PlnA), which is a peptide pheromone with strain-specific antibacterial activity produced by Lactobacillus plantarum, permeabilizes cancerous rat pituitary cells (GH₄ cells), whereas normal rat anterior pituitary cells are resistant to the peptide. To examine whether the preferential permeabilization of cancerous cells is a general feature of PlnA, we studied its effect on primary cultures of cells from rat liver (hepatocytes, endothelial, and Kupffer cells) and rat kidney cortex, as well as two epithelial cell lines of primate kidney origin (Vero cells from green monkey and human Caki-2 cells). The Vero cell line is derived from normal cells, whereas the Caki-2 cell line is derived from a cancerous tumor. The membrane effects were studied by patch clamp recordings and microfluorometric (fura-2) monitoring of the cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) and fluorophore. In all the tested cell types except Kupffer cells, exposure to 10–100 μM PlnA induced a nearly instant permeabilization of the membrane, indicated by the following criteria: increased membrane conductance, membrane depolarization, increased [Ca²⁺]_i, and diffusional loss of fluorophore from the cytosol. At a concentration of 5 μM, PlnA had no effect on any of the cell types. The Kupffer cells were permeabilized by 500 μM PlnA. We conclude that the permeabilizing effect of PlnA is not restricted to cancerous cells.     

Lactocin 160, a Bacteriocin Produced by Vaginal Lactobacillus rhamnosus, Targets Cytoplasmic Membranes of the Vaginal Pathogen, Gardnerella vaginalis

Bacterial vaginosis (BV) is a commonly occurring vaginal infection that is associated with a variety of serious risks related to the reproductive health of women. Conventional antibiotic treatment for this condition is frequently ineffective because the antibiotics tend to inhibit healthy vaginal microflora along with the pathogens. Lactocin 160, a bacteriocin produced by healthy vaginal lactobacilli, is a promising alternative to antibiotics; this compound specifically inhibits the BV-associated vaginal pathogens such as Gardnerella vaginalis and Prevotella bivia without affecting the healthy microflora. This study investigates the molecular mechanism of action for lactocin 160 and reveals that this compound targets the cytoplasmic membrane of G. vaginalis, causing the efflux of ATP molecules and dissipation of the proton motive force.     

Characterization,Cytotoxic Analysis and Action Mechanism of Antilisterial Bacteriocin Produced by Lactobacillus plantarum Isolated from Cheddar Cheese

International Journal of Peptide Research and Therapeutics - Emergence of food borne pathogens and multidrug resistant organisms has enforced the screening of natural antimicrobial compounds....     

gly Gene Cloning and Expression and Purification of Glycinecin A, a Bacteriocin Produced by Xanthomonas campestris pv. glycines 8ra

Glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines, inhibits the growth of X. campestris pv. vesicatoria. We have cloned and expressed the genes encoding glycinecin A in Escherichia coli. Recombinant glycinecin A was purified from cell extracts by ammonium sulfate precipitation followed by chromatography on Q-Sepharose, Mono Q (ion exchange), and size exclusion columns. Purified glycinecin A is composed of two polypeptides, is active over a wide pH range (6 to 9), and is stable at temperatures up to 60°C. Glycinecin A is a heterodimer consisting of 39- and 14-kDa subunits, as revealed through size exclusion chromatography and cross-linking analysis. Two genes, glyA and glyB, encoding the 39- and 14-kDa subunits, respectively, were identified based on the N-terminal sequences of the subunits. From the nucleotide sequences of glyA and glyB, we conclude that both genes are translated as bacteriocin precursors that include N-terminal leader sequences. When expressed in E. coli, recombinant glycinecin A was found primarily in cell extracts. In contrast, most glycinecin A from Xanthomonas was found in the culture media. E. coli transformed with either glyA or glyB separately did not show the bacteriocin activity.     

Primary amino acid and DNA sequences of gassericin T, a lactacin F-family bacteriocin produced by Lactobacillus gasseri SBT2055

A broad-spectral bacteriocin, named gassericin T, produced by Lactobacillus gasseri SBT 2055 (from human feces) was isolated to homogeneity from the culture supernatant by hydrophobic chromatography. By SDS-PAGE and in situ activity assay, the purified gassericin T migrated as a single band with bacteriocin activity and molecular size of 5,400. A 2.9-kbp HindIII-HindIII fragment of chromosome DNA was hybridized with the oligonucleotide probe designed from the partial N-terminal amino acid sequence of gassericin T and was cloned. Six ORFs including the structural gene of gassericin T were deduced by computer analysis and the data bases. The structural gene of gassericin T (gatA) was identified as the fourth ORF, which encoded a protein composed of 75 amino acids that included the GG motif of the cleavage site. Chemical sequencing analysis of the complete amino acid sequence showed that gassericin T (57 amino acids) had a disulfide bond in the molecule and no modified amino acid residues, making it a class II bacteriocin. The gassericin T had 60% sequence similarity to mature LafA (57 amino acids, lactacin F, bacteriocins produced by L. johnsonii VPI11088), and the sequences around the processing site and C-terminal area were well conserved. The fifth ORF was designated as gatX, encoded as a peptide composed of 65 amino acids containing the GG motif of the putative cleavage site, however mature GatX and its antibacterial activity were not detected in the culture supernatant. GatX has higher similarity with LafX than with lactobin A (50 amino acids) belonging to the first lactacin F-family. These results indicated that gassericin T belongs to the hydrophobic class II bacteriocins and the most vicinal lactacin F-family.     

Growth-Regulated Expression of a Bacteriocin,Produced by the Sweet Potato Pathogen Streptomyces ipomoeae,That Exhibits Interstrain Inhibition

Certain strains of the bacterial sweet potato pathogen Streptomyces ipomoeae produce the bacteriocin ipomicin, which inhibits other sensitive strains of the same species. Within the signal-sequence-encoding portion of the ipomicin structural gene ipoA exists a single rare TTA codon, which is recognized in Streptomyces bacteria by the temporally accumulating bldA leucyl tRNA. In this study, ipomicin was shown to stably accumulate in culture supernatants of S. ipomoeae in a growth-regulated manner that did not coincide with the pattern of ipoA expression. Similar growth-regulated production of ipomicin in Streptomyces coelicolor containing the cloned ipoA gene was found to be directly dependent on translation of the ipoA TTA codon by the bldA leucyl tRNA. The results here suggest that bldA-dependent translation of the S. ipomoeae ipoA gene leads to growth-regulated production of the ipomicin precursor, which upon processing to the mature form and secretion stably accumulates in the extracellular environment. To our knowledge, this is the first example of bldA regulation of a bacteriocin in the streptomycetes.Streptomyces bacteria are gram-positive spore-forming soil bacteria which display a complex life cycle (4, 6). Spore germination leads to vegetative growth as tangled masses of substrate mycelia on solid surfaces. Nutrient limitation triggers production of secondary metabolites as well as growth of vertically extending aerial hyphae, which are morphologically distinct filaments that derive nutrients from the dying substrate cell layer. Eventually, individual aerial hyphae differentiate into chains of spores. Streptomyces mycelia growing in liquid media display a multiphasic growth curve similar to that of unicellular bacteria. As the culture enters stationary phase, Streptomyces bacteria produce secondary metabolites but do not typically differentiate morphologically (2).Streptomyces ipomoeae is the causative agent of soil rot, the widespread and destructive disease of sweet potatoes. The disease results in decay of fibrous feeder roots and development of necrotic lesions on the edible storage roots (9). Prevention of soil rot currently relies solely on development of resistant sweet potato cultivars; however, alternative approaches, including the use of biocontrol methods, are now beginning to be investigated.Previously, 36 strains of S. ipomoeae were divided into three groups based on interstrain inhibition during their cocultivation on agar plates (7). The group III strains were found to produce a diffusible 10-kDa bacteriocin-like protein (ipomicin), which is inhibitory to group I and II members and which is stable in culture supernatants for at least 1 h at 40°C (in a pH range of 6 to 10) (26). Sequence analyses of the ipomicin structural gene (ipoA) and its protein product revealed that ipomicin is initially expressed in precursor form, which upon processing of an N-terminal signal sequence becomes the 10-kDa mature form (26).Given its potential as a biocontrol agent, we were interested in elucidating the expression characteristics of ipomicin. It was noted previously that TTA, the rarest codon found within the GC-rich Streptomyces genus, is present once in ipoA within the region that designates the ipomicin signal sequence (26). TTA codons in Streptomyces bacteria are recognized by a single leucyl tRNA species encoded by the gene bldA, which is required for normal morphological and physiological differentiation in these organisms (5, 14). The latter effect is due in part to the fact that regulatory genes of antibiotic clusters and aerial hyphae development also contain TTA codons (5). In the model streptomycete, Streptomyces coelicolor, bldA has been shown to be an effective growth phase regulator because it is expressed initially in liquid and surface cultures as an inactive precursor and, upon processing, begins to temporally accumulate as a mature tRNA, at least under certain physiological conditions (15, 24). Interestingly, TTA-containing genes show variation in their dependence on bldA for expression, with some genes demonstrating strong dependence while others show only partial or no apparent dependence (14, 23).Here, stable ipomicin protein is shown to be temporally produced during S. ipomoeae growth in a manner inconsistent with ipoA mRNA levels. Similar contrary results for ipomicin protein and ipoA mRNA concentrations were observed during growth of the heterologous host S. coelicolor expressing a cloned version of the S. ipomoeae ipoA gene, and this effect was shown to be directly dependent on translation of the ipoA TTA codon by the bldA leucyl tRNA. The data here suggest that regulation by bldA leads to temporal production of the ipomicin precursor, which when processed to the mature form is secreted to the external environment, where it accumulates.