Molecular characterization of Lactobacillus curvatus and Lact. sake isolated from sauerkraut and their application in sausage fermentations

Lactobacillus curvatus and Lact. sake are best adapted to meat fermentations and dominate the flora during the whole process. In fermenting sauerkraut, Leuconostoc mesenteroides subsp. mesenteroides is the major organism only during the early phase. In this environment Lact. curvatus and Lact. sake provide up to 50% of the microbial flora especially of the later phase, depending on the process conditions. Strains of Lact. curvatus and Lact. sake isolated from fermenting sauerkraut were identified by hybridization with species specific 23S rRNA-targeted oligonucleotide probes and further characterized. In 59 of 72 strains, plasmid DNA was detected. Small cryptic plasmids of 20 strains were found to be homologous with pLc2, a 2·6 kb plasmid from Lact. curvatus LTH683, which was originally isolated from meat. The ability to compete was investigated in fermenting sausages of two strains each of Lact. curvatus and Lact. sake isolated from sauerkraut. One strain each of Lact. curvatus and Lact sake was found to outnumber the meat-borne flora and govern the process.     

Characterization and molecular cloning of cryptic plasmids isolated from Lactobacillus casei

Four small cryptic plasmids were isolated from Lactobacillus casei strains, and restriction endonuclease maps of these plasmids were constructed. Three of the small plasmids (pLZ18C, pLZ19E, and pLZ19F1; 6.4, 4.9, and 4.8 kilobase pairs, respectively) were cloned into Escherichia coli K-12 by using pBR322, pACYC184, and pUC8 as vectors. Two of the plasmids, pLZ18C and pLZ19E, were also cloned into Streptococcus sanguis by using pVA1 as the vector. Hybridization by using nick-translated cloned 32P-labeled L. casei plasmid DNA as the probe revealed that none of the cryptic plasmids had appreciable DNA-DNA homology with the large lactose plasmids found in the L. casei strains, with chromosomal DNAs isolated from these strains. Partial homology was detected among several plasmids isolated from different strains, but not among cryptic plasmids isolated from the same strain.     

Characterization and molecular cloning of cryptic plasmids isolated from Lactobacillus casei.

Four small cryptic plasmids were isolated from Lactobacillus casei strains, and restriction endonuclease maps of these plasmids were constructed. Three of the small plasmids (pLZ18C, pLZ19E, and pLZ19F1; 6.4, 4.9, and 4.8 kilobase pairs, respectively) were cloned into Escherichia coli K-12 by using pBR322, pACYC184, and pUC8 as vectors. Two of the plasmids, pLZ18C and pLZ19E, were also cloned into Streptococcus sanguis by using pVA1 as the vector. Hybridization by using nick-translated cloned 32P-labeled L. casei plasmid DNA as the probe revealed that none of the cryptic plasmids had appreciable DNA-DNA homology with the large lactose plasmids found in the L. casei strains, with chromosomal DNAs isolated from these strains. Partial homology was detected among several plasmids isolated from different strains, but not among cryptic plasmids isolated from the same strain.     

Lactose metabolism in Lactobacillus curvatus and Lactobacillus sake

Abstract The lactose metabolism was investigated in five strains of Lactobacillus curvatus and 14 strains of L. sake isolated from meat or meat-derived products. Strains with the ability to ferment lactose were found in both species. They exhibited either phospho-β-galactosidase (P-β-gal) or β-galactosidase (β-gal) activity, or both. P-β-gal activity of L. curvatus and L. sake was induced and detected only in the presence of lactose or galactose. Furthermore, catabolite repression by glucose was demonstrated. The immunological properties of the P-β-gal enzymes of these organisms resemble those of Lactococcus lactis . Several strains of L. sake but none of L. curvatus exhibited β-gal activity which was constitutive. In hybridisation experiments, the β-gal genes of L. sake and L. casei ATCC393 showed over 60% DNA-homology. The presence of β-gal genes in L. sake was demonstrated in both β-gal-producing and non-producing strains. This observations is consistent with a genetic potential of lactic acid bacteria exceeding their physiological capabilities.     

Plasmid-linked maltose utilization in Lactobacillus ssp

Five strains of Lactobacillus plantarum and 4 strains of Lactobacillus ssp. isolated from fresh meat contained between 1 and 5 plasmids ranging in Mr from 1.3 to 49 MDa. Plasmid-curing studies suggested that maltose utilization is associated with a 49 MDa plasmid (pML291) in Lactobacillus sp. DB29 and 34.5 MDa plasmids in Lactobacillus ssp. DB27, DB28 and DB31. Restriction digestion of pML291 and a putative plasmid deletion derivative, pML292, isolated from a maltose negative mutant of DB29, generated common restriction fragments. Southern blot DNA-DNA hybridization using pML 291 as a probe indicated that there is strong homology between putative maltose plasmids.     

Antibacterial activity of Lactobacillus sake isolated from meat.

A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.     

Antibacterial activity of Lactobacillus sake isolated from meat

A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.     

Conventional taxonomy of lactobacilli surviving radurization of meat

All of the 113 catalase-negative, Gram-positive, rod-shaped strains isolated from radurized minced beef (5 kGy) were homofermentative, non-thermophilic, and belonged to the sub-genus Streptobacterium. The majority of the strains (100) were identified as Lactobacillus sake. These were divided into four sub-groups based on their sugar fermentation pattern: group IA1 (melibiose (+), maltose (-), amygdalin (-), 76 strains); group IA2 (melibiose (+), maltose (-), amygdalin (+), 14 strains); group IB1 (melibiose (+), maltose (+), amygdalin (+), four strains); group IB2 (melibiose (+), maltose (+), amygdalin (-), six strains). Of the remaining strains, two produced L(+)-lactic acid and were identified as L. farciminis, three were identified as L. curvatus and eight showed characteristics of both L. sake and L. curvatus and were designated 'L. sake/curvatus.' With one exception, all strains were aciduric and relatively insensitive to the chemical preservatives tested. Most L. sake strains produced significant amounts of H2O2. Electron microscopy confirmed a possible relationship between the thickness of the cells and radiation resistance. The problems and limitations of this type of taxonomic study and possible reasons for the predominance of L. sake species in radurized meat are discussed.     

Conventional taxonomy of lactobacilli surviving radurization of meat

All of the 113 catalasc-negative, Gram-positive, rod-shaped strains isolated from radurized minced beef (5 kGy) were homofermentative, non-thermophilic, and belonged to the sub-genus Streptobacterium. The majority of the strains (100) were identified as Lactobacillus sake. These were divided into four sub-groups based on their sugar fermentation pattern: group IA₁ (melibiose (+), maltose (—), amygdalin (—), 76 strains); group IA₂ (melibiose (+), maltose (—), amygdalin (+), 14 strains); group IB₁ (melibiose (+), maltose (+), amygdalin (+), four strains); group IB₂ (melibiose (+), maltose (+), amygdalin (—), six strains). Of the remaining strains, two produced L(+)-lactic acid and were identified as L. farciminis , three were identified as L. curvatus and eight showed characteristics of both 'L. sake and L. curvatus and were designated 'L. sake/curvatus.' With one exception, all strains were aciduric and relatively insensitive to the chemical preservatives tested. Most L. sake strains produced significant amounts of H₂O₂. Electron microscopy confirmed a possible relationship between the thickness of the cells and radiation resistance. The problems and limitations of this type of taxonomic study and possible reasons for the predominance of L. sake species in radurized meat are discussed.     

Characterization, cloning, curing, and distribution in lactic acid bacteria of pLP1, a plasmid from Lactobacillus plantarum CCM 1904 and its use in shuttle vector construction

A small 2.1-kb plasmid called pLP1 was extracted from Lactobacillus plantarum CCM 1904 (ATCC 8014) and cloned into the Escherichia coli pUC19 plasmid. As determined by DNA-DNA Southern hybridization with a pLP1-radioactively labeled probe, other lactic acid bacteria such as L. curvatus, L. sake, Carnobacterium, and Leuconostoc mesenteroides harbor pLP1-related plasmids. Shuttle vectors based on the pLP1 replicon were constructed by inserting the erythromycin-resistance gene from pVA891 into the various pUC19-pLP1 constructions. pLP1-based shuttle vector transformation efficiencies (TE) by electroporation were compared to TE of a broad-host-range plasmid pGK12 in different lactobacilli strains. Expression of the pUC19-pLP1 plasmids in Escherichia coli maxicells showed that pLP1 encodes for a 37,000 MW protein which can act in trans allowing the replication of plasmids in which this protein is truncated. The pLP1-based shuttle vectors producing this protein replicate in lactobacilli and also in Bacillus subtilis. A pLP1-free strain was obtained by incompatibility with a pLP1-based shuttle vector introduced in L. plantarum CCM 1904 by electroporation. The absence of pLP1 has no incidence on the strain phenotype suggesting that pLP1 is not essential for the strain in our laboratory conditions.     

Radiation resistance of lactobacilli isolated from radurized meat relative to growth and environment.

Of 113 lactobacilli isolated from radurized (5 kGy) minced meat, 7 Lactobacillus sake strains, 1 L. curvatus strain, and 1 L. farciminis strain were used for radiation resistance studies in a semisynthetic substrate (i.e., modified MRS broth). Five reference Lactobacillus spp., one Staphylococcus aureus strain, and one Salmonella typhimurium strain were used for comparative purposes. All L. sake isolates exhibited the phenomenon of being more resistant to gamma-irradiation in the exponential (log) phase than in the stationary phase of their growth cycles by a factor of 28%. Four references strains also exhibited this phenomenon, with L. sake (DSM 20017) showing a 68% increase in resistance in the log phase over the stationary phase. This phenomenon was not common to all bacteria tested and is not common to all strains with high radiation resistance. Four L. sake isolates and three reference strains were used in radiation sensitivity testing in a natural food system (i.e., meat). The bacteria were irradiated in minced meat and packaged under four different conditions (air, vacuum, CO2, and N2). Organisms exhibited the highest death rate (lowest D10 values [doses required to reduce the logarithm of the bacterial population by 1] ) under CO2 packaging conditions, but resistance to irradiation was increased under N2. The D10 values of the isolates were generally greater than those of the reference strains. The D10 values were also higher (approximately two times) in meat than in semisynthetic growth medium.     

Radiation resistance of lactobacilli isolated from radurized meat relative to growth and environment

Of 113 lactobacilli isolated from radurized (5 kGy) minced meat, 7 Lactobacillus sake strains, 1 L. curvatus strain, and 1 L. farciminis strain were used for radiation resistance studies in a semisynthetic substrate (i.e., modified MRS broth). Five reference Lactobacillus spp., one Staphylococcus aureus strain, and one Salmonella typhimurium strain were used for comparative purposes. All L. sake isolates exhibited the phenomenon of being more resistant to gamma-irradiation in the exponential (log) phase than in the stationary phase of their growth cycles by a factor of 28%. Four references strains also exhibited this phenomenon, with L. sake (DSM 20017) showing a 68% increase in resistance in the log phase over the stationary phase. This phenomenon was not common to all bacteria tested and is not common to all strains with high radiation resistance. Four L. sake isolates and three reference strains were used in radiation sensitivity testing in a natural food system (i.e., meat). The bacteria were irradiated in minced meat and packaged under four different conditions (air, vacuum, CO2, and N2). Organisms exhibited the highest death rate (lowest D10 values [doses required to reduce the logarithm of the bacterial population by 1] ) under CO2 packaging conditions, but resistance to irradiation was increased under N2. The D10 values of the isolates were generally greater than those of the reference strains. The D10 values were also higher (approximately two times) in meat than in semisynthetic growth medium.     

Complete Sequence of Plasmid pLH1 from Lactobacillus helveticus ATCC15009: Analysis Reveals the Presence of Regions Homologous to Other Native Plasmids from the Host Strain

The complete sequence for plasmid pLH1 from Lactobacillus helveticus ATCC15009 has been determined. Analysis of the 19,360-bp primary sequence revealed a putative replication origin and initiation protein, information that could provide the basis for the construction of cloning vectors for L. helveticus. Evidence that pLH1 is theta-replicating could be deduced from the plasmid size, from the homology to the replication protein of the Bacillus natto theta-replicating plasmid pLS32, and from the identification of a putative resolvase gene (orf-195). Although 14 open reading frames capable of encoding polypeptides longer than 100 amino acids were identified, none, on the basis of homology with known sequences, appeared to encode a well-characterized trait relevant to milk fermentation. Plasmid pLH1 revealed regions of identity with the smaller cryptic plasmids (pLH2 and pLH3) from the same strain and with other tracts of DNA, including insertion sequence elements, from a variety of other lactic acid bacteria. The presence of such regions provides a basis for developing an explanation of the phenotypic variability observed in these bacteria. The plasmid also appears to possess a number of genetic elements present in other lactic acid bacterial plasmids, conservation of which would be consistent with an important functional or evolutionary role. It could be argued that the plasmid complement of L. helveticus ATCC15009 consists of parasitic entities concerned only with their own replication and survival.     

Characterization of a cryptic plasmid pM4 from Lactobacillus plantarum M4

A cryptic plasmid from Lactobacillus plantarum M4 isolated from fresh milk, designated as pM4, was sequenced and characterized. It was 3320 bp in length with a G+C content of 38.73 mol%. The plasmid pM4 was predicted to encode three putative ORFs, in which ORF1 shared 99% and 98% homology, respectively, with the Rep proteins of reported plasmids pWCFS101 and pF8801, members of the rolling circle replication (RCR) pC194 family. Sequence analysis revealed a typical pC194 family double strand origin (dso) and a putative single strand origin (sso) located upstream of the rep gene. Mung bean nuclease analysis and Southern hybridization confirmed the presence of single-stranded DNA (ssDNA) intermediates, suggesting that pM4 belongs to the RCR pC194 family. Accumulation of ssDNA in rifampicin-treated strains implied that the host-encoded RNA polymerase was involved in the conversion of ssDNA to double-stranded DNA. Furthermore, the relative copy number of pM4 was estimated to be about 25 in each cell by real-time PCR. The new RCR plasmid would be valuable in constructing cloning vectors for application in the food industry.     

Cryptic plasmids from Lactobacillus helveticus and their evolutionary relationship

Abstract Three different cryptic plasmids from Lactobacillus helveticus have been identified and their DNA sequences determined. Analyses and comparisons of their primary structures revealed stretches of DNA with considerable homology. Thus, large portions of the plasmid non-coding sequences were conserved at 80–90% identity between the different plasmids identified so far in L. helveticus . Nevertheless, different plasmids found in a same host strain utilise different genes of replication, probably acquired during evolution from different replicons from Gram-positive bacterial origins. A remnant structure of such a possible genetic integration of a foreign replication gene into one of the plasmids of L. helveticus was identified.     

Integration and partial excision of a cryptic plasmid in Pseudomonas syringae pv. phaseolicola.

A virulent strain of Pseudomonas syringae pv. phaseolicola, a pathogen of the common bean Phaseolus vulgaris (L.), was shown to harbor a 98-megadalton cryptic plasmid, pMC7105. After exposure of this strain to the plasmid-curing agent mitomycin C, a colony was isolated which had no detectable extrachromosomal DNA. Hybridization of labeled pMC7105 probe to nitrocellulose filters containing Southern-blotted BamHI cleavage products of cellular DNA revealed that pMC7105 was integrated into the chromosome rather than cured from this strain. Imprecise excision of pMC7105 resulted in the formation of three smaller plasmids of 34, 50, and 58 megadaltons. BamHI and EcoRI fingerprint analyses revealed that these plasmids were excised from a common region of pMC7105. The BamHI fragments of pMC7105 which were not present in the excision plasmids remained integrated and could be detected by hybridization of pMC7105 probe to Southern-blotted cellular DNA from these strains. Certain chromosomal fragments also had homology with the pMC7105 probe. The excision plasmids were stably maintained and neither integration nor excision altered the pathogenicity of these strains.     

Characterization of two nisin-producing Lactococcus lactis subsp. lactis strains isolated from a commercial sauerkraut fermentation.

Two Lactococcus lactis subsp. lactis strains, NCK400 and LJH80, isolated from a commercial sauerkraut fermentation were shown to produce nisin. LJH80 was morphologically unstable and gave rise to two stable, nisin-producing (Nip+) derivatives, NCK318-2 and NCK318-3. NCK400 and derivatives of LJH80 exhibited identical morphological and metabolic characteristics, but could be distinguished on the basis of plasmid profiles and genomic hybridization patterns to a DNA probe specific for the iso-ISS1 element, IS946. NCK318-2 and NCK318-3 harbored two and three plasmids, respectively, which hybridized with IS946. Plasmid DNA was not detected in NCK400, and DNA from this strain failed to hybridize with IS946. Despite the absence of detectable plasmid DNA in NCK400, nisin-negative derivatives (NCK402 and NCK403) were isolated after repeated transfer in broth at 37 degrees C. Nisin-negative derivatives concurrently lost the ability to ferment sucrose and became sensitive to nisin. A 4-kbp HindIII fragment containing the structural gene for nisin (spaN), cloned from L. lactis subsp. lactis ATCC 11454, was used to probe genomic DNA of NCK318-2, NCK318-3, NCK400, and NCK402 digested with EcoRI or HindIII. The spaN probe hybridized to an 8.8-kbp EcoRI fragment and a 10-kbp HindIII fragment in the Nip+ sauerkraut isolates, but did not hybridize to the Nip- derivative, NCK402. A different hybridization pattern was observed when the same probe was used against Nip+ L. lactis subsp. lactis ATCC 11454 and ATCC 7962. These phenotypic and genetic data confirmed that unique Nip+ L. lactis subsp. lactis strains were isolated from fermenting sauerkraut.     

Characterization of two nisin-producing Lactococcus lactis subsp. lactis strains isolated from a commercial sauerkraut fermentation.

Two Lactococcus lactis subsp. lactis strains, NCK400 and LJH80, isolated from a commercial sauerkraut fermentation were shown to produce nisin. LJH80 was morphologically unstable and gave rise to two stable, nisin-producing (Nip+) derivatives, NCK318-2 and NCK318-3. NCK400 and derivatives of LJH80 exhibited identical morphological and metabolic characteristics, but could be distinguished on the basis of plasmid profiles and genomic hybridization patterns to a DNA probe specific for the iso-ISS1 element, IS946. NCK318-2 and NCK318-3 harbored two and three plasmids, respectively, which hybridized with IS946. Plasmid DNA was not detected in NCK400, and DNA from this strain failed to hybridize with IS946. Despite the absence of detectable plasmid DNA in NCK400, nisin-negative derivatives (NCK402 and NCK403) were isolated after repeated transfer in broth at 37 degrees C. Nisin-negative derivatives concurrently lost the ability to ferment sucrose and became sensitive to nisin. A 4-kbp HindIII fragment containing the structural gene for nisin (spaN), cloned from L. lactis subsp. lactis ATCC 11454, was used to probe genomic DNA of NCK318-2, NCK318-3, NCK400, and NCK402 digested with EcoRI or HindIII. The spaN probe hybridized to an 8.8-kbp EcoRI fragment and a 10-kbp HindIII fragment in the Nip+ sauerkraut isolates, but did not hybridize to the Nip- derivative, NCK402. A different hybridization pattern was observed when the same probe was used against Nip+ L. lactis subsp. lactis ATCC 11454 and ATCC 7962. These phenotypic and genetic data confirmed that unique Nip+ L. lactis subsp. lactis strains were isolated from fermenting sauerkraut.     

Inhibition of Listeria innocua and Brochothrix thermosphacta in vacuum-packaged meat by addition of bacteriocinogenic Lactobacillus curvatus CRL705 and its bacteriocins

AIMS: To evaluate the inhibition effectiveness of Lactobacillus curvatus CRL705 used as a bioprotective culture and of its bacteriocins, lactocin 705 and lactocin AL705, against Listeria innocua, Brochothrix thermosphacta and indigenous lactic acid bacteria (LAB) in vacuum-packaged meat stored at 2 degrees C. METHODS AND RESULTS: The live culture of Lact. curvatus CRL705 as well as synthetic lactocin 705 and purified lactocin AL705 were shown to be similarly effective in preventing the growth of B. thermosphacta and L. innocua in meat discs in contrast to control samples in which these micro-organisms grew rapidly, their numbers increasing by 3.0- and 2.1-log cycles respectively. In addition, indigenous LAB population showed a lower growth rate in the presence of lactocin 705. Bacteriocin activity was detected in the meat discs during 36 days at 2 degrees C irrespective of the biopreservation strategy applied. Changes in pH were not significantly different in meat discs treated with the protective culture when compared with control samples. CONCLUSIONS: Lactobacillus curvatus CRL705 and the produced bacteriocins, lactocin 705 and lactocin AL 705, were effective in inhibiting L. innocua and B. thermosphacta. The use of the bioprotective culture in refrigerated vacuum-packaged fresh meat would be more feasible from an economic and legal point of view. SIGNIFICANCE AND IMPACT OF THE STUDY: Establishment of biopreservation as a method to ensure the microbiological safety of vacuum-packaged fresh meat at 2 degrees C.     

Analysis of pYC2, a cryptic plasmid in <Emphasis Type="Italic">Lactobacillus sakei</Emphasis> BM5 isolated from kimchi

Analysis of the structural properties of pYC2, a cryptic plasmid from Lactobacillus sakei BM5 isolated from kimchi, determined its length as 1,970 bp with a G + C content of 34%. The double-strand origin (dso) and single-strand origin (sso) of rolling-circle replicating plasmids were found in the nucleotide sequence of the pYC2 plasmid. Sequence analysis of pYC2 revealed that ORF 1 and ORF 2 showed high homology with the Cop and Rep proteins encoded by the pMV158 family of plasmids replicating via the rolling-circle mechanism. pYC2 also replicates by this mechanism, as confirmed by Southern hybridization analysis.