Biomedical subjects

M E Stiles

Publications and source records attributed to M E Stiles.

At least 37 records · Page 2Linked to original sources

PubMed1996

Expression of the antimicrobial peptide carnobacteriocin B2 by a signal peptide-dependent general secretory pathway.

Carnobacteriocin B2 is a well-characterized class II bacteriocin produced by a 61-kb plasmid from Carnobacterium piscicola LV17. Export of this bacteriocin is dependent on specific ABC (ATP-binding cassette) secretion proteins. Divergicin A is a strongly hydrophobic narrow-spectrum bacteriocin produced by a 3.4-kb plasmid from Carnobacterium divergens LV13. Predivergicin A contains a signal peptide and utilizes the general secretary pathway for export (R. W. Worobo, M. J. van Belkum, M. Sailer, K. L. Roy, J. C. Vederas, and M. E. Stiles, J. Bacteriol. 177:3143-3149, 1995). Fusion of the carnobacteriocin B2 structural gene (devoid of its natural leader peptide) behind the signal peptide of divergicin A in the expression vector pMG36e permitted production and export of active carnobacteriocin B2 in the absence of the specific secretion genes. N-terminal sequencing of purified carnobacteriocin B2 established that correct processing of the prebacteriocin occurred beyond the Ala-Ser-Ala cleavage site of the signal peptide. Carnobacteriocin B2 was produced by the wild-type strain of C. divergens, LV13, and in C. piscicola LV17C, the nonbacteriocinogenic, plasmidless variant of the original carnobacteriocin B2 producer strain. The corresponding immunity gene was included immediately downstream of the structural gene. Both of the host strains are sensitive to the bacteriocin, and both acquired immunity when they were transformed with the construct. C. divergens LV13 containing the divergicin A signal peptide-carnobacteriocin B2 fusion construct produces both divergicin A and carnobacteriocin B2 and demonstrates the first example of multiple-bacteriocin expression via the general secretory pathway. The small amount of genetic material required for independent bacteriocin expression has implications for the development of a food-grade multiple-bacteriocin expression vector for use in lactic acid bacteria.

ATP-Binding Cassette Transporters↗

PubMed1996

Control of beef spoilage by a sulfide-producing Lactobacillus sake strain with bacteriocinogenic Leuconostoc gelidum UAL187 during anaerobic storage at 2 degrees C.

Chill-stored, vacuum-packaged beef inoculated with sulfide-producing Lactobacillus sake 1218 developed a distinct sulfide odor within 3 weeks of storage at 2 degrees C, at which time the bacteria had reached maximum numbers of 10(6) CFU cm(-2). Coinoculation of the meat with the wild-type, bacteriocinogenic (Bac+) strain of Leuconostoc gelidum UAL187 delayed the spoilage by L. sake 1218 for up to 8 weeks of storage. Coinoculation of meat samples with an isogenic, slowly growing Bac+ variant, UAL187-22, or with the Bac- variant UAL187-13 did not delay the onset of spoilage by L. sake 1218. The study showed that spoilage of chill-stored, vacuum-packaged beef by a susceptible target organism could be dramatically delayed by the Bac+ wild-type strain of L. gelidum UAL187. Inoculation with L. sake 1218 can be used as a model system to determine the efficacy of biopreservation of vacuum-packaged meats.

Anaerobiosis↗

PubMed1995

Utilization of the leucocin A export system in Leuconostoc gelidum for production of a Lactobacillus bacteriocin.

The lactacin F complex, composed of LafA and LafX peptides, is produced by Lactobacillus johnsonii VPI 11088 (ATCC 11506) and is active against various lactobacilli and Enterococcus faecalis. The genetic determinants encoding the lactacin F peptides, LafA and LafX, are organized in a chromosomal operon comprised of genes lafA, lafX, and ORFZ. The lactacin F operon was introduced into Leuconostoc (Lc.) gelidum UAL187-22 which produces leucocin A. Leucocin A, a plasmid-encoded bacteriocin, inhibits E. faecalis, Listeria monocytogenes, and other lactic acid bacteria. The culture supernatant of the Leuconostoc transformant containing the lactacin F operon inhibited both lactacin F-and leucocin A-sensitive indicators. Concurrent expression of both bacteriocins did not alter the production of native leucocin A. Additive inhibitory effects due to the presence of both bacteriocins were not observed. An isogenic derivative of UAL187-22, which has lost the leucocin-encoding plasmid, was unable to produce active lactacin F when transformed with the appropriate recombinant plasmid. The ability of Lc. gelidum UAL187-22 to produce lactacin F demonstrates that the export system for leucocin A is capable of producing both bacteriocins simultaneously.

Amino Acid Sequence↗

PubMed1995

Effect of growth of selected lactic acid bacteria on storage life of beef stored under vacuum and in air.

The effect of growth of different types of lactic acid bacteria (LAB) on the storage life of normal pH beef was determined anaerobically (under vacuum) and aerobically. Four LAB from meat were inoculated separately onto sterile slices of lean beef. Inoculated samples were stored anaerobically at 2 degrees C for 10 weeks or stored aerobically in an oxygen permeable film at 7 degrees C for 10 days, with and without previous storage under vacuum at 2 degrees C. The LAB strains used were Carnobacterium maltaromicus (previously C. piscicola) LV17 and UAL26, Leuconostoc gelidum UAL187-22 and Lactobacillus sake Lb706. Storage life was determined by sensory panel evaluation of colour and odour. Under anaerobic conditions Lb. sake Lb706, inoculated at log 2 CFU/cm2, grew rapidly to reach maximum population within three weeks of storage. L. gelidum UAL187-22 also grew on the meat but at a slower rate. In contrast, growth of C. maltaromicus LV17 and UAL26 was unpredictable, achieving maximum population after 2 to 8 weeks. None of the test strains caused spoilage of the meat within the 10-week storage period under vacuum. When the test organisms were inoculated at an initial level of log 4 CFU/cm2, C. maltaromicus LV17 and UAL26 produced off-odours after 8 weeks of storage under vacuum at 2 degrees C. Under aerobic conditions at 7 degrees C, all four of the strains grew well on the beef samples. C. maltaromicus LV17 and UAL26 and Lb. sake Lb706 all caused off-odours and discoloration. The rate of aerobic deterioration in meat quality was faster with increased time of storage under vacuum. L. gelidum UAL187-22 could be a suitable antagonistic strain with the potential to extend the storage life of beef, stored anaerobically and packaged aerobically for retail sale, without producing undesirable sensory changes.

Aerobiosis↗

PubMed1995

Molecular characterization of genes involved in the production of the bacteriocin leucocin A from Leuconostoc gelidum.

Leucocin A is a small heat-stable bacteriocin produced by Leuconostoc gelidum UAL187. A 2.9-kb fragment of plasmid DNA that contains the leucocin structural gene and a second open reading frame (ORF) in an operon was previously cloned (J. W. Hastings, M. Sailer, K. Johnson, K. L. Roy, J. C. Vederas, and M. E. Stiles, J. Bacteriol. 173:7491-7500, 1991). When a 1-kb DraI-HpaI fragment containing this operon was introduced into a bacteriocin-negative variant (UAL187-13), immunity but no leucocin production was detected. Leucocin production was observed when an 8-kb SacI-HindIII fragment of the leucocin plasmid was introduced into L. gelidum UAL187-13 and Lactococcus lactis IL1403. Nucleotide sequence analysis of this 8-kb fragment revealed the presence of three ORFs in an operon upstream of and on the strand opposite from the leucocin structural gene. The first ORF (lcaE) encodes a putative protein of 149 amino acids with no apparent function in leucocin A production. The second ORF (lcaC) contains 717 codons that encode a protein homologous to members of the HlyB family of ATP-binding cassette transporters. The third ORF (lcaD) contains 457 codons that encode a protein with marked similarity to LcnD, a protein essential for the expression of the lactococcal bacteriocin lactococcin A. Deletion mutations in lcaC and lcaD resulted in loss of leucocin production, indicating that LcaC and LcaD are involved in production and translocation of leucocin A. The secretion apparatus for lactococcin A did not complement mutations in the lcaCD genes to express leucocin A in L. lactis.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP-Binding Cassette Transporters↗

PubMed1995

Heterologous expression of the lactacin F peptides by Carnobacterium piscicola LV17.

The lactacin F complex, composed of LafA and LafX peptides, is produced by Lactobacillus johnsonii VPI 11088 and is active against five other Lactobacillus species and Enterococcus faecalis. The genetic determinants encoding the lactacin F complex are organized in a 1-kb polycistronic operon which comprises three genes, lafA, lafX, and ORFZ (encoding the putative immunity protein). The lafA and lafX genes encode the bacteriocin precursors with N-terminal extensions characterized by a Gly-Gly-1*Xaa+1 cleavage site (*). The Gly-Gly motif is conserved in several other bacteriocins, including carnobacteriocins A, BM1, and B2. Carnobacterium piscicola LV17 produces carnobacteriocins which are active against Listeria monocytogenes and other lactic acid bacteria. In this study, the lactacin F operon was introduced into C. piscicola LV17. The transformants produced lactacin F concurrently with the carnobacteriocins. When the lafA and lafX genes were separated and cloned individually into LV17, production of either LafA or LafX by C. piscicola LV17 was detected by complementation with L. johnsonii clones producing LafX or LafA, respectively. Transformants of C. piscicola LV17 which produced lactacin F, LafA, or LafX, in combination with the carnobacteriocins, were assayed for an increased and expanded inhibitory spectrum. The recombinant organisms were only active against lactacin F- and carnobacteriocin-sensitive strains. A plasmidless derivative of LV17 which does not produce the carnobacteriocins failed to produce lactacin F, LafA, or LafX when transformed with the appropriate recombinant plasmids. The ability of C. piscicola LV17 to produce lactacin F demonstrates that the machinery for the carnobacteriocins is capable of processing and exporting bacteriocins from both systems.

Amino Acid Sequence↗

PubMed1995

A signal peptide secretion-dependent bacteriocin from Carnobacterium divergens.

Divergicin A is a strongly hydrophobic, narrow-spectrum, nonlantibiotic bacteriocin produced by Carnobacterium divergens LV13. This strain of C. divergens contains a 3.4-kb plasmid that mediates production of, and immunity to, the bacteriocin. N-terminal amino acid sequencing of the purified divergicin A was used to locate the structural gene (dvnA). The structural gene encodes a prepeptide of 75 amino acids consisting of a 29-amino-acid N-terminal extension and a mature peptide of 46 amino acids. Directly downstream of dvnA there is a second open reading frame that encodes the immunity protein for divergicin A. Divergicin A has a calculated molecular mass of 4,223.89 Da. The molecular mass determined by mass spectrometry is 4,223.9 Da, indicating that there is no posttranslational modification of the peptide. The N-terminal extension of divergicin A has an Ala-Ser-Ala (positions -3 to -1) cleavage site and acts as a signal peptide that accesses the general export system of the cell (such as the sec pathway in Escherichia coli). This is the first bacteriocin of lactic acid bacteria to be reported that does not have dedicated maturation and secretion genes. Production of divergicin A was observed in heterologous hosts containing only the two genes associated with divergicin A production and immunity. Fusing alkaline phosphatase behind the signal peptide for divergicin resulted in the secretion of this enzyme in the periplasmic space and supernatant of E. coli.

Amino Acid Sequence↗

PubMed1995

Characterization of the protein conferring immunity to the antimicrobial peptide carnobacteriocin B2 and expression of carnobacteriocins B2 and BM1.

Cloning of a 16-kb DNA fragment from the 61-kb plasmid of Carnobacterium piscicola LV17B into plasmidless C. piscicola LV17C restores the production of the plasmid-encoded carnobacteriocin B2 and the chromosomally-encoded carnobacteriocin BM1 and restores the immune phenotype. This fragment also has sufficient genetic information to allow the expression of carnobacteriocin B2 and its immunity in a heterologous host. The gene locus (cbiB2) responsible for immunity to carnobacteriocin B2 is located downstream of the structural gene for carnobacteriocin B2 and encodes a protein of 111 amino acids (CbiB2). CbiB2 was expressed in Escherichia coli as a fusion of the maltose-binding protein and CbiB2. The fusion protein was purified on an amylose column and cleaved with factor Xa, and pure CbiB2 was isolated by high-performance liquid chromatography. The N-terminal amino acid sequence and mass spectrometry (molecular weight [mean +/- standard error], 12,662.2 +/- 3.4) of the purified protein agree with the information deduced from the nucleotide sequence of cbiB2. Western blot (immunoblot) analysis indicates that the majority of the intracellular pool of this immunity protein is in the cytoplasm and that a smaller proportion is associated with the membrane. CbiB2 confers immunity to carnobacteriocin B2, but not to carnobacteriocin BM1, when it is expressed in homologous or heterologous hosts. No protective effect is observed for sensitive cells growing in the presence of the bacteriocin when the immunity protein is added to the medium. The purified immunity protein does not show significant binding to microtiter plates coated with carnobacteriocin B2 and is not able to inactivate the bacteriocin in solution.

Amino Acid Sequence↗

PubMed1994

Chemical and genetic characterization of bacteriocins produced by Carnobacterium piscicola LV17B.

Carnobacteriocins BM1 and B2 are thermostable class II bacteriocins produced by Carnobacterium piscicola LV17B. These bacteriocins were purified by a three-step procedure that included hydrophobic interaction, size exclusion, and reversed-phase high performance liquid chromatography. The purified peptides and fragments derived by enzymatic digestion were analyzed by Edman degradation, amino acid analysis, and mass spectrometry. An oxidized form of carnobacteriocin BM1 (carnobacteriocin B1) was also purified and characterized. Probes synthesized using information from the N-terminal amino acid sequences for the purified bacteriocins were used to locate structural genes for the carnobacteriocins. A 1.9-kilobase (kb) HindIII fragment from a 61-kb plasmid (pCP40) containing the carnobacteriocin B2 structural gene and a 4.0-kb EcoRI-PstI genomic fragment containing the carnobacteriocin BM1 structural gene were cloned and fully or partially sequenced, respectively. Expression of the chromosomal bacteriocin and its immunity function requires the presence of the 61-kb plasmid. The results indicate that both bacteriocins are synthesized as prebacteriocins. Post-translational cleavage of an 18-amino acid N-terminal extension at a Gly-Gly (positions -2 and -1) site takes place in each prepeptide to yield the mature 43-amino acid carnobacteriocin BM1 (molecular mass 4524.6) and the mature 48-amino acid carnobacteriocin B2 (molecular mass 4969.9). These two peptides showed significant amino acid homology to each other and with those class II bacteriocins which contain the YGNGV amino acid motif near the N terminus.

Amino Acid Sequence↗

PubMed1994

Bacteriocins of leuconostocs isolated from meat.

Several bacteriocin-producing Leuconostoc strains have been isolated from meat and identified as Leuconostoc gelidum UAL 187, Leuconostoc paramesenteroides-La7a, Leuconostoc carnosum-Ta11a and Leuconostoc carnosum-La54a. All strains produce bacteriocins that are active against Listeria monocytogenes and other lactic acid bacteria of concern in meat spoilage. All of the bacteriocins studied are heat stable in acidic environments and are inactivated by a range of proteolytic enzymes but not by catalase or lysozyme. Most are detected early in the growth cycle and are produced at refrigeration temperatures and in a pH range of 4.0-7.0. Leucocin A-UAL187, produced by Leuconostoc gelidium UAL 187, is a small peptide (MW 3930) translated as a 61 amino acid prepeptide consisting of a 24 amino acid leader region and 37 amino acid active bacteriocin that is secreted. A probe designed from a region of the leucocin gene has been used to locate the bacteriocin genes in the other strains (La7a, La54a and Ta11a). Strong hybridization signals were detected from 8.9 MDa, 32 MDa and 8.9 MDa plasmids in strains La7a, La54a and Ta11a, respectively. The bacteriocin structural gene from Leuconostoc carnosum-Ta11a (leucocin B-Ta11a) has been cloned and sequenced and the bacteriocin shows 100% homology to leucocin A-UAL187; however, the prepeptide differs in six residues. The mature extracellular bacteriocin from strain UAL 187 was purified and characterized by precipitation, gel filtration, hydrophobic interaction chromatography followed by RP-HPLC and amino-terminal sequencing, whilst those of the other strains are in the process of being purified and characterized using similar techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Characteristics and genetic determinant of a hydrophobic peptide bacteriocin, carnobacteriocin A, produced by Carnobacterium piscicola LV17A.

Carnobacteriocin A is a hydrophobic nonlantibiotic bacteriocin that is detected early in the growth cycle of Carnobacterium piscicola LV17A and encoded by a 49 MDa plasmid. The bacteriocin was purified using hydrophobic interaction and gel filtration chromatography, and reversed-phase HPLC. Three different active peaks (A1, A2 and A3) were detected, but the purified samples had identical N-terminal amino acid sequences for the first 15 amino acids as determined by Edman degradation analysis. Only a 2.4 kb fragment of the EcoRI digest of the plasmid pCP49 hybridized with a 23-mer oligonucleotide probe derived from amino acids 5 to 13 of the amino acid sequence. The structural gene for carnobacteriocin A is located 600 base pairs into the 2.4 kb EcoRI fragment, but no other genetic information was detected on this unit. The structural gene includes an 18 amino acid N-terminal extension of the bacteriocin, ending with Gly-Gly residues in the -2, -1 positions with respect to the cleavage site. The bacteriocin consists of 53 amino acids that differ markedly from the majority of hydrophobic peptide bacteriocins characterized to date. Based on the amino acid sequence derived from the nucleotide sequence a molecular mass of 5052.85 Da was calculated. Mass spectrometric analysis showed that the molecular mass of the major component (A3) was 2 Da lower, thereby indicating the presence of a disulphide bridge between Cys 22 and Cys 51. Carnobacteriocin A2 has a similar structure except that Met 52 is oxidized to a sulphoxide, whereas A1 appears to be a mixture of peptides derived proteolytically from A3 or A2.

Amino Acid Sequence↗

PubMed1994

Bacteriocins produced by Leuconostoc species.

Leuconostoc spp. are lactic acid bacteria that are commonly associated with foods and that are used as starter bacteria in some dairy fermentations. Lactic acid bacteria are inhibitory to other bacteria because of pH, organic acids, hydrogen peroxide, and other chemicals produced during their growth, including bacteriocins. Bacteriocin production by Leuconostoc spp. was first observed in the 1950s, but only since 1984, when antagonistic activity of Leuconostoc spp. was reported, have more extensive studies of bacteriocins produced by Leuconostoc spp. been conducted, including mesentericin Y105, produced by Leuconostoc mesenteroides spp. mesenteroides; leucocin A-UAL 187, produced by Leuconostoc gelidum; carnosin 44A, produced by Leuconostoc carnosum; and leuconocin S, produced by Leuconostoc paramesenteroides. Bacteriocins produced by leuconostocs may or may not be active against other lactic acid bacteria, but all include Listeria in their activity spectra. Mesentericin Y105 is reported to be exclusively active against Listeria spp. The amino acid sequences for leucocin A and mesentericin Y105 have been determined. Despite considerable differences in antibacterial spectra, only two amino acids differ between these bacteriocins. The prevalence of leuconostocs in many adventitious fermentations of food and the use of leuconostocs as starter bacteria in controlled fermentations make the bacteriocins produced by these bacteria of interest as possible food preservatives by addition of the bacteriocin or its producer organism to foods.

Amino Acid Sequence↗

PubMed1993

Bacteriocin production by Carnobacterium piscicola LV 61.

Carnobacterium piscicola LV 61 produces a bacteriocin designated piscicolin 61, that is heat resistant, active over a wide pH range and inactivated by alpha-chymotrypsin, pepsin, trypsin and papain. It is effective against strains of the genera Carnobacterium, Enterococcus and Listeria. Other lactic acid bacteria tested were less sensitive or resistant to piscicolin. It is produced at temperatures from 1 to 30 degrees C. Maximum bacteriocin activity was detected after the culture had entered the stationary phase of growth and when the culture was grown in a medium with an initial pH between 8.0 and 9.0. The same high amounts of bacteriocin could be obtained in a culture at a constant pH of 6.5. No bacteriocin was produced at pH 5.0. Peptone in the growth medium promotes bacteriocin production, whereas meat and yeast extract did not influence the amounts of bacteriocin produced. Bacteriocin production and immunity are probably encoded by a 22 kb plasmid.

Animals↗

PubMed1993

15N- and 13C-labeled media from Anabaena sp. for universal isotopic labeling of bacteriocins: NMR resonance assignments of leucocin A from Leuconostoc gelidum and nisin A from Lactococcus lactis.

A procedure for universal 13C and/or 15N labeling of microbial peptides which are produced by fermentation in complex media and its application to two food-preserving bacteriocins from lactic acid bacteria are described. Isotopic enrichment of nisin A (from Lactococcus lactis) and of leucocin A (from Leuconostoc gelidum) is readily achieved using a soluble peptone derived from enzymatic hydrolysis (pepsin and chymopapain) of Anabaena sp. ATCC 27899 cells grown on sodium [13C]bicarbonate and/or sodium [15N]nitrate as sole carbon and nitrogen sources. Combustion of this peptone followed by mass spectrometric analysis indicates that 45% of the labeled carbon and 65% of the labeled nitrogen added to the Anabaena culture are utilized in the amino acids of the peptone and that the isotopic purity for both 13C and 15N remains essentially unchanged provided that the cells are grown under argon atmosphere to avoid nitrogen fixation. NMR analyses of [13C,15N]nisin A using H[13C]MQC, H[13C]MBC, 2D INADEQUATE, and H[15N]MQC techniques confirmed 1H spectral assignments previously reported for unlabeled material and readily provided carbon and nitrogen assignments. The results show that universal but not uniform 13C labeling occurs unless the nutrient source is completely isotopically enriched at high level (> or = 98%) because of differential levels of de novo amino acid synthesis. Application of NMR techniques such as TOCSY, DQF-COSY, NOESY, and H[13C]MQC to unlabeled and [13C]leucocin A afforded the complete 1H and 13C assignment. Leucocin A does not possess clearly defined conformational structure in DMSO or aqueous solutions.

Amino Acid Sequence↗

PubMed1993

Microbial ecology of fresh pork stored under modified atmosphere at -1, 4.4 and 10 degrees C.

The prevalent bacteria on fresh pork packaged in modified atmosphere with elevated CO2 were determined by selection of representative colonies from the greatest dilution of meat samples. The pork samples were stored in two packaging films of different oxygen permeability at three storage temperatures. Strains were classified and those identified as lactic acid bacteria were screened for production of inhibitory substances. The types of bacteria isolated from samples stored in the two packaging films were similar. Storage temperature influenced the type of bacteria that dominated the microbial population. At 10 degrees C the prevalent microflora consisted of aeromonads, Enterobacteriaceae and lactic acid bacteria but at 4.4 and -1 degrees C, aeromonads, Brochothrix thermosphacta and lactic acid bacteria dominated. Listeriae were detected as part of the prevalent microflora on samples stored at -1 degree C, but not on samples stored at 4.4 or 10 degrees C. Species of lactic acid bacteria dominating the microflora were influenced by growth medium. The majority of isolates taken from Plate Count agar were carnobacteria whereas those from Lactobacilli MRS agar were homofermentative lactic acid bacteria. Of the 538 lactic acid bacteria isolates screened for production of inhibitory substances, 162 strains showed deferred inhibition toward a range of lactic acid bacteria and nonlactic acid bacteria indicator strains.

Animals↗

PubMed1992

Mobilization and location of the genetic determinant of chloramphenicol resistance from Lactobacillus plantarum caTC2R.

The mobilization of a nonconjugative plasmid (pCaT) that mediates chloramphenicol resistance in Lactobacillus plantarum caTC2R was achieved by comobilization with the conjugative plasmid pAM beta 1. The conjugation studies confirmed that the 8.5-kb pCaT in L. plantarum caTC2R contains the gene responsible for chloramphenicol resistance and that the plasmid has several unique restriction sites which make it useful for genetic studies in Carnobacterium spp. Cloning studies showed that the gene responsible for chloramphenicol resistance is located in the 2.6-kb EcoRV-SalI region of pCaT. This was confirmed by probing the 3.0-kb BglII fragment of pCaT with a biotin-labeled 1.6-kb BstEII-HpaII fragment from the streptococcal-derived plasmid pVA797(Cmr). Expression of chloramphenicol resistance in Carnobacterium as well as in other Lactobacillus species was achieved by electrotransformation using donor DNA from pCaT.

Bacterial Proteins↗

PubMed1991

Antibiosis of Leuconostoc gelidum isolated from meat.

A heterofermentative lactic acid bacterium isolated from meat packaged under elevated CO2 levels was identified as Leuconostoc gelidum, based on the description of this new species by Shaw & Harding (1989). It grows well at refrigeration temperatures but not at 35 degrees C. The organism produces an inhibitory substance that is inactivated by protease and trypsin, but not by catalase or by heating at 62 degrees C, for 30 min. The bacteriocin-like inhibitory substance is produced early in the growth cycle, at 1, 5 and 25 degrees C. The inhibitory substance is active against a large number of closely related lactic acid bacteria, as well as a strain of Enterococcus faecalis and Listeria monocytogenes. There is initial evidence that the genetic information determining production of, and resistance to, the bacteriocin-like substance is plasmid mediated. Of the three plasmids found in this organism, loss of the 7.6 MDa plasmid resulted in loss of inhibitor production and resistance to the inhibitory substance. Loss of the 5.0 MDa plasmid did not result in a detectable phenotypic change in the organism.

Animals↗

PubMed1991

Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelidum.

Leucocin A-UAL 187 is a bacteriocin produced by Leuconostoc gelidum UAL 187, a lactic acid bacterium isolated from vacuum-packaged meat. The bacteriocin was purified by ammonium sulfate or acid (pH 2.5) precipitation, hydrophobic interaction chromatography, gel filtration, and reversed-phase high-performance liquid chromatography with a yield of 58% of the original activity. Leucocin A is stable at low pH and heat resistant, and the activity of the pure form is enhanced by the addition of bovine serum albumin. It is inactivated by a range of proteolytic enzymes. The molecular weight was determined by mass spectrometry to be 3,930.3 +/- 0.4. Leucocin A-UAL 187 contains 37 amino acids with a calculated molecular weight of 3,932.3. A mixed oligonucleotide (24-mer) homologous to the sequence of the already known N terminus of the bacteriocin hybridized to a 2.9-kb HpaII fragment of a 7.6-MDa plasmid from the producer strain. The fragment was cloned into pUC118 and then subcloned into a lactococcal shuttle vector, pNZ19. DNA sequencing revealed an operon consisting of a putative upstream promoter, a downstream terminator, and two open reading frames flanked by a putative upstream promoter and a downstream terminator. The first open reading frame downstream of the promoter contains 61 amino acids and is identified as the leucocin structural gene, consisting of a 37-amino-acid bacteriocin and a 24-residue N-terminal extension. No phenotypic expression of the bacteriocin was evident in several lactic acid bacteria that were electrotransformed with pNZ19 containing the 2.9-kb cloned fragment of the leucocin A plasmid.

Amino Acid Sequence↗