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PubMed2002

Characterization of a cryptic plasmid from Lactobacillus fermentum KC5b and its use for constructing a stable Lactobacillus cloning vector.

Lactobacillus fermentum KC5b, a strain originally isolated from the human vagina, contains a cryptic plasmid pKC5b. The sequence and genetic organization of the 4392-bp plasmid were determined. It contains two convergently oriented replicons, which are homologous to each other and to the stable replicon of the Enterococcus faecium plasmid pMBB1. The two replicons of pKC5b were used either individually or together to construct Lactobacillus-Escherichia coli shuttle plasmids. Only the plasmid pSP1 that carried both replicons transformed lactobacilli, suggesting a complementary function between the two replicons. Since the replicons had a high homology to those of other plasmids that replicate via a theta-like mechanism and no detectable single-stranded intermediates were found for the plasmid, it is possible that pKC5b may replicate via a theta-like mechanism. The new shuttle plasmid pSP1 has been transformed and stably maintained in several Lactobacillus strains. As an initial application, pSP1 was used to clone the S-layer protein gene (slpA) of Lactobacillus acidophilus ATCC 4356 into a heterologous vaginal Lactobacillus strain and achieved surface-bound expression of the protein.

Bacterial Proteins↗
PubMed1997

High level heterologous protein production in Lactococcus and Lactobacillus using a new secretion system based on the Lactobacillus brevis S-layer signals.

A secretion cassette, based on the expression and secretion signals of a S-layer protein (SlpA) from Lactobacillus brevis, was constructed. E. coli beta-lactamase (Bla) was used as the reporter protein to determine the functionality of the S-layer signals for heterologous expression and secretion in Lactococcus lactis, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus gasseri and Lactobacillus casei using a low-copy-number plasmid derived from pGK12. In all hosts tested, the bla gene was expressed under the slpA signals and all Bla activity was secreted to the culture medium. The Lb. brevis S-layer promoters were very efficiently recognized in L. lactis, Lb. brevis and Lb. plantarum, whereas in Lb. gasseri the slpA promoter region appeared to be recognized at a lower level and in Lb. casei the level of transcripts was below the detection limit. The production of Bla was mainly restricted to the exponential phase of growth. The highest yield of Bla was obtained with L. lactis and Lb. brevis. Without pH control, substantial degradation of Bla occurred during prolonged cultivations with all lactic acid bacteria (LAB) tested. When growing L. lactis and Lb. brevis under pH control, the Bla activity could be stabilized also at the stationary phase. L. lactis produced up to 80 mg/l of Bla which to our knowledge represents the highest amount of a heterologous protein secreted by LAB so far. The short production phase implied a very high rate of secretion with a calculated value of 5 x 10(5) Bla molecules/cell per h. Such a high rate was also observed with Lb. plantarum, whereas in Lb. brevis the competition between the wild type slpA gene and the secretion construct probably lowered the rate of Bla production. The results obtained indicate wide applicability of the Lb. brevis slpA signals for efficient protein production and secretion in LAB.

Base Sequence↗
PubMed2002

Lactobacillus bacteremia during a rapid increase in probiotic use of Lactobacillus rhamnosus GG in Finland.

Lactobacilli supposedly have low pathogenicity; they are seldom detected in blood culture. Lactobacillus rhamnosus GG, which originates indigenously in the human intestine, became available for use as a probiotic in 1990 in Finland. We evaluated the possible effects of the increased probiotic use of L. rhamnosus GG on the occurrence of bacteremia due to lactobacilli. Lactobacilli were isolated in 0.02% of all blood cultures and 0.2% of all blood cultures with positive results in Helsinki University Central Hospital and in Finland as a whole, and no trends were seen that suggested an increase in Lactobacillus bacteremia. The average incidence was 0.3 cases/100,000 inhabitants/year in 1995-2000 in Finland. Identification to the species level was done for 66 cases of Lactobacillus bacteremia, and 48 isolates were confirmed to be Lactobacillus strains. Twenty-six of these strains were L. rhamnosus, and 11 isolates were identical to L. rhamnosus GG. The results indicate that increased probiotic use of L. rhamnosus GG has not led to an increase in Lactobacillus bacteremia.

Bacteremia↗
PubMed2006

Lactobacillus cypricasei Lawson et al. 2001 is a later heterotypic synonym of Lactobacillus acidipiscis Tanasupawat et al. 2000.

The applicability of a multilocus sequence analysis (MLSA)-based identification system for lactobacilli was evaluated. Two housekeeping genes that code for the phenylalanyl-tRNA synthase alpha-subunit (pheS) and RNA polymerase alpha-subunit (rpoA) were sequenced and analysed for members of the Lactobacillus salivarius species group. The type strains of Lactobacillus acidipiscis and Lactobacillus cypricasei were investigated further using a third gene that encodes the alpha-subunit of ATP synthase (atpA). The MLSA data revealed close relatedness between L. acidipiscis and L. cypricasei, with 99.8-100 % pheS, rpoA and atpA gene sequence similarities. Comparison of the 16S rRNA gene sequences of the type strains of the two species confirmed the close relatedness (99.8 % gene sequence similarity) between the two taxa. Similar phenotypes and high DNA-DNA binding values in the range of 84 to 97.5 % confirmed that L. acidipiscis and L. cypricasei are synonymous species. On the basis of the present study, it is proposed that Lactobacillus cypricasei is a later heterotypic synonym of Lactobacillus acidipiscis.

Bacterial Proteins↗
PubMed1991

A human Lactobacillus strain (Lactobacillus casei sp strain GG) promotes recovery from acute diarrhea in children.

To determine the effect of a human Lactobacillus strain (Lactobacillus casei sp strain GG, Gefilac) on recovery from acute diarrhea (82% rotavirus), 71 well-nourished children between 4 and 45 months of age were studied. After oral rehydration, the patients randomly received either Lactobacillus GG-fermented milk product, 125 g (10(10-11) colony-forming units) twice daily (group 1); Lactobacillus GG freeze-dried powder, one dose (10(10-11) colony-forming units) twice daily (group 2); or a placebo, a pasteurized yogurt (group 3) 125 g twice daily; each diet was given for 5 days, in addition to normal full diet otherwise free of fermented dairy products. The mean (SD) duration of diarrhea after commencing the therapy was significantly shorter in group 1 (1.4 [0.8] days) and in group 2 (1.4 [0.8] days) than in group 3 (2.4 [1.1] days); F = 8.70, P less than 0.001. After rehydration, each dietary group maintained a positive weight trend. The urinary lactulose-mannitol recovery ratios (means [95% confidence intervals]) on admission were 0.09 (0.03, 0.24) in group 1, 0.12 (0.07, 0.22) in group 2, and 0.08 (0.04, 0.18) in group 3; no significant alterations in intestinal permeability were observed at retesting after 2 days of realimentation. The result indicates that early nutritional repletion after rehydration causes no mucosal disruption and is beneficial for recovery from diarrhea. It is further suggested that Lactobacillus GG in the form of fermented milk or freeze-dried powder is effective in shortening the course of acute diarrhea.

Acute Disease↗
PubMed2006

Lactobacillus suntoryeus Cachat and Priest 2005 is a later synonym of Lactobacillus helveticus (Orla-Jensen 1919) Bergey et al. 1925 (Approved Lists 1980).

Strain R0052, isolated from a North American dairy starter culture, was initially identified as Lactobacillus acidophilus based on phenotypic analyses. However, upon sequencing the 16S rRNA gene, it became clear that the isolate was very highly related to Lactobacillus suntoryeus, Lactobacillus helveticus and Lactobacillus gallinarum, as similarities ranging from 99.3 to 99.8 % were observed. As an initial screening test to investigate the relatedness of strain R0052 and reference strains of L. suntoryeus, L. helveticus and L. gallinarum, the partial sequences for the genes encoding the alpha subunit of ATP synthase (atpA), RNA polymerase alpha subunit (rpoA), phenylalanyl-tRNA synthase alpha subunit (pheS), the translational elongation factor Tu (tuf), a surface-layer protein (slp) and the Hsp60 chaperonins (groEL) were determined and they revealed high relatedness between all of the strains. The determination of the 16S-23S rRNA internally transcribed spacer (ITS) sequences revealed 98.3-100% similarity between L. suntoryeus and L. helveticus strains. SDS-PAGE of whole-cell proteins did not distinguish between these species. Fluorescent amplified fragment length polymorphism (FAFLP) could distinguish between these taxa, but they still constituted a single cluster within the L. acidophilus group. Finally, DNA-DNA hybridization experiments between strain R0052 and the type strains of L. helveticus and L. suntoryeus yielded reassociation values above 70% and confirmed that these names are synonyms.

Bacterial Proteins↗
PubMed2001

Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination.

An efficient method is described for the generation of site-specific chromosomal integrations in Lactobacillus acidophilus and Lactobacillus gasseri. The strategy is an adaptation of the lactococcal pORI system (K. Leenhouts, G. Venema, and J. Kok, Methods Cell Sci. 20:35-50, 1998) and relies on the simultaneous use of two plasmids. The functionality of the integration strategy was demonstated by the insertional inactivation of the Lactobacillus acidophilus NCFM lacL gene encoding beta-galactosidase and of the Lactobacillus gasseri ADH gusA gene encoding beta-glucuronidase.

Biotechnology↗
PubMed1991

Molecular cloning and deoxyribonucleic acid polymorphisms in Lactobacillus acidophilus and Lactobacillus gasseri.

Lactobacillus strain ADH is a bile-resistant, bacteriocin-producing human isolate that was phenotypically classified within the Lactobacillus acidophilus group. Total DNA and phage DNA extracted from strain ADH were separately digested with BclI and ligated with BclI-digested pGK12. Following electroporation of these ligation mixtures directly into strain ADH, electrotransformants were recovered at frequencies of 1.5 x 10(3) and 2.0 x 10(4)/micrograms of pGK12 for preparations of pGK12::phage DNA and pGK12::total DNA, respectively. Among the electrotransformants screened, 6 and 22% contained passenger DNA of either phage DNA or chromosomal origin, respectively, as determined by restriction-enzyme analyses and hybridization assays. Derivatives of pGK12 containing passenger DNA of chromosomal (pTRK120) or phage (pTRK121) origin and pTRK15 (native cryptic plasmid) were evaluated for use as species-specific probes. The strain ADH-derived probes hybridized primarily to members of the B-1 and B-2 lactobacilli homology groups and demonstrated strain-specific polymorphisms within these groups. Identical hybridization patterns were established for strain ADH and Lactobacillus gasseri VPI 6033 (ATCC 19992). Identification of DNA probes and establishment of a host-vector cloning system have facilitated our efforts to characterize the Lactobacillus chromosome and to distinguish between closely related species thought to be important inhabitants of the gastrointestinal tract.

Chromosomes, Bacterial↗
PubMedDate not supplied

Expression of Bacillus subtilis levanase gene in Lactobacillus plantarum and Lactobacillus casei.

Two Lactobacillus-Escherichia coli shuttle vectors, harbouring the levanase gene from Bacillus subtilis under the control of its own promoter (pLPEW1) or behind the E. coli tac promoter (pESIEW2), were constructed. Lactobacillus plantarum showed the same growth characteristics on selective plates and in liquid media containing inulin, after transformation with either pLPEW1 or pESIEW2. L. plantarum transformed with pLPEW1 could be selected on inulin plates, indicating that levanase expression can be used as a food-grade selection system for Lactobacillus. Lactobacillus casei grew faster in inulin-containing medium than L. plantarum after transformation with pESIEW2, but did not grow when harbouring pLPEW1. Inulin-degrading activities of 90 mU/ml were found in culture medium of L. plantarum containing pLPEW1 or pESIEW2, and of 500 mU/ml in medium of L. casei (pESIEW2). Addition of 1 mM isopropyl beta-D-thiogalactoside to the culture medium had no effect on growth and levanase expression in L. plantarum (pESIEW2) and L. casei (pESIEW2) strains. Levanase produced by L. casei (pESIEW2) has a size of 75 kDa and 72 kDa, corresponding to that of unprocessed and mature B. subtilis levanase, respectively, suggesting that the protein produced is recognized and processed by a signal peptidase.

Bacillus subtilis↗
PubMed2005

The interaction of the non-bacteriocinogenic Lactobacillus sakei 10A and lactocin S producing Lactobacillus sakei 148 towards Listeria monocytogenes on a model cooked ham.

Two lactic acid bacteria, Lactobacillus sakei subsp. carnosus (10A) and lactocin S producing Lactobacillus sakei 148 (LS5), were examined for their usefulness as protective culture in the biopreservation of cooked meat products. Co-culture experiments on a model cooked ham (MCH) between 10A or LS5 and a cocktail of three Listeria monocytogenes strains were performed to examine the influence of inoculum level (10(5) vs. 10(6)cfu/g), storage temperature (4 vs. 7 degrees C) and packaging type (vacuum-packaging vs. modified atmosphere-packaging). At 7 degrees C, applying Lactobacillus sakei 10A at 10(6) cfu/g limited the growth of Listeria monocytogenes to <1 log(10) cfu/g during 27 days, whilst an application level of 10(5) cfu/g failed to prevent growth to unacceptable levels. Lactobacillus sakei LS5 did not demonstrate an antagonistic effect towards Listeria monocytogenes. Lowering the temperature to 4 degrees C or switching from vacuum-packaging to modified atmosphere packaging (MAP) did not influence the ability of strain 10A to grow on the MCH, as its dominance did not change. A combination of strain 10A and 4 degrees C or a MAP containing 50% CO(2) completely inhibited the growth of Listeria monocytogenes. Sensory assessments and pH measurements confirmed that 10A, even when present at a high level for prolonged storage times, did not acidify the cooked ham to a point of sensory rejection.

Animals↗
PubMed2004

Lactobacillus curvatus subsp. melibiosus is a later synonym of Lactobacillus sakei subsp. carnosus.

On the basis of phenotypic and DNA-DNA reassociation studies, strain CCUG 34545T has been considered to represent a distinct Lactobacillus curvatus subspecies, Lactobacillus curvatus subsp. melibiosus. However, in several independent studies dealing with Lactobacillus sakei and L. curvatus strains, the subspecies division of L. curvatus has been found to be controversial. The original study distinguishing the two subspecies within both L. curvatus and L. sakei also lacked 16S rRNA gene sequence analyses. Therefore, the taxonomic position of L. curvatus subsp. melibiosus CCUG 34545T was re-evaluated in a polyphasic taxonomy study that included 16S rRNA gene sequence analysis, DNA-DNA reassociation, DNA G + C content determination, numerical analysis of ribotypes and whole-cell protein patterns and the examination of some fundamental phenotypic properties. The results obtained indicate that strain CCUG 34545T and its duplicate, CCUG 41580T, are Lactobacillus sakei subsp. carnosus strains and that L. curvatus subsp. melibiosus is a later synonym of L. sakei subsp. carnosus.

Bacterial Proteins↗
PubMed2006

Reclassification of Lactobacillus thermotolerans Niamsup et al. 2003 as a later synonym of Lactobacillus ingluviei Baele et al. 2003.

The relatedness of the species Lactobacillus ingluviei and Lactobacillus thermotolerans was investigated by comparing partial sequences of the 16S rRNA gene (99.9 % similarity over 1504 bp), the hsp60 gene (98.8 % similarity over 954 bp) and the recA gene (98.5 % similarity over 452 bp) and by determining DNA-DNA binding levels (79+/-3 %) and genomic DNA G+C contents (50 and 49 mol%, respectively). These data, in addition to their similar biochemical characteristics, suggest that the two taxa constitute a single species. According to Rules 38 and 42 of the Bacteriological Code, they should be united under the name Lactobacillus ingluviei, with the name Lactobacillus thermotolerans as a later heterotypic synonym.

Lactobacillus↗
PubMed2006

Reclassification of Lactobacillus brevis strains LMG 11494 and LMG 11984 as Lactobacillus parabrevis sp. nov.

A polyphasic study revealed taxonomic heterogeneity among reference strains of the species Lactobacillus brevis. Representative strains of L. brevis and related taxa were investigated by partial sequence analysis of the housekeeping gene encoding the alpha-subunit of phenylalanyl-tRNA synthase (pheS). Species-specific clusters were delineated for all taxa studied except for two L. brevis strains, LMG 11494 and LMG 11984, respectively isolated from cheese and wheat, which occupied a distinct position. Their phylogenetic affiliation was determined using 16S rRNA gene sequence analysis and it was found that both strains (with 99.9 % gene sequence similarity between them) belonged to the Lactobacillus buchneri group, with nearest neighbours Lactobacillus hammesii and L. brevis (gene sequence similarities of 99.2 and 98.1 %, respectively). Further genotypic and phenotypic studies, including fluorescent amplified fragment length polymorphism, DNA-DNA hybridization and DNA G+C content, clearly demonstrated that the two strains represent a single novel taxon for which the name Lactobacillus parabrevis sp. nov. is proposed (type strain LMG 11984(T)=ATCC 53295(T)).

Bacterial Proteins↗
PubMed2006

Lactobacillus durianis Leisner et al. 2002 is a later heterotypic synonym of Lactobacillus vaccinostercus Kozaki and Okada 1983.

The taxonomic status of the species Lactobacillus durianis and Lactobacillus vaccinostercus is briefly summarized and experimental evidence concerning their similarity is presented. Highly similar 16S rRNA gene sequences (99.8 % similarity over 1,523 bp), partial recA gene sequences (99.5 % similarity over 600 bp) and partial hsp60 gene sequences (99.1 % similarity over 924 bp) suggest that the two species are closely related. Moreover, a high DNA-DNA binding level (87 %) and similar genomic DNA G+C contents (41-44 mol% for both species) as well as similar biochemical characteristics support the evidence that they constitute a single species. Consequently, according to Rules 38 and 42 of the Bacteriological Code, the name Lactobacillus vaccinostercus, the oldest legitimate name, must be maintained and the name Lactobacillus durianis should be considered a later heterotypic synonym.

Bacterial Proteins↗
PubMed2006

Polyphasic analysis indicates that Lactobacillus salivarius subsp. salivarius and Lactobacillus salivarius subsp. salicinius do not merit separate subspecies status.

Lactobacillus salivarius Rogosa et al. 1953 was described as a homofermentative lactobacillus with two varieties: salivarius, typified inter alia by the ability to ferment rhamnose, and salicinius, characterized by the ability to ferment the glucoside salicin. These varieties have become accepted as subspecies divisions. We have examined the relatedness of 32 L. salivarius strains by a polyphasic approach. Carbohydrate fermentation profile analysis did not support clear distinction of the two subspecies. L. salivarius UCC118 was shown to be facultatively heterofermentative, confirming in silico genome analysis. 16S rRNA gene sequences and 16S-23S rRNA intergenic spacer region sequences provided no discrimination between any of the strains or subspecies. Broad subdivisions were distinguishable by pulsed-field gel genomic digest patterns, but they did not allow subspecific or phenotypic distinctions. A phylogeny based upon groEL gene sequences was discordant with rhamnose or salicin fermentation data for many taxa, and no reliable phenotypic correlations could be established. In the absence of meaningful taxonomic criteria, we therefore propose that Lactobacillus salivarius comprises a single species with no infraspecific taxa. Based on the present study and literature data, an emended description of the species Lactobacillus salivarius is provided.

Aged↗
PubMed2006

Reclassification of Lactobacillus amylophilus LMG 11400 and NRRL B-4435 as Lactobacillus amylotrophicus sp. nov.

The taxonomic position of six Lactobacillus amylophilus strains isolated from swine waste-corn fermentations was reinvestigated. All strains were included in a multilocus sequence analysis (MLSA) study for species identification of Lactobacillus using the genes encoding the phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA). Partial pheS and rpoA gene sequences showed that strains LMG 11400 and NRRL B-4435 represent a separate lineage that is distantly related to the type strain of L. amylophilus, LMG 6900T, and to three other strains of the species. The MLSA data showed that the two strains LMG 11400 and NRRL B-4435 constituted a distinct cluster, sharing 100% pheS and rpoA gene sequence similarity. The other reference strains clustered together with the type strain of L. amylophilus, LMG 6900T, and were clearly differentiated from strains LMG 11400 and NRRL B-4435 (80 and 89% pheS and rpoA gene sequence similarity, respectively). The 16S rRNA gene sequences of the latter two strains are 100% identical, with the nearest phylogenetic neighbour L. amylophilus LMG 6900T showing only 97.2% 16S rRNA gene sequence similarity. Further polyphasic taxonomic study based on whole-cell protein fingerprinting, DNA-DNA hybridization and biochemical features demonstrated that the two strains represent a single, novel Lactobacillus species, for which the name Lactobacillus amylotrophicus sp. nov. is proposed. The type strain is LMG 11400T (=NRRL B-4436T=DSM 20534T).

Animal Husbandry↗
PubMed2006

Adhesion of Lactobacillus plantarum 423 and Lactobacillus salivarius 241 to the intestinal tract of piglets, as recorded with fluorescent in situ hybridization (FISH), and production of plantaricin 423 by cells colonized to the ileum.

AIMS: To determine which intestinal section of pre and postweaned piglets are colonized by Lactobacillus plantarum 423 and Lactobacillus salivarius 241, and follow production of plantaricin 423 in a gastro-intestinal model. METHODS AND RESULTS: Lactobacillus plantarum 423 and Lact. salivarius 241, single or in combination, were administered to 1-, 14- and 28-day-old (postweaned) piglets. According to results obtained by fluorescent in situ hybridization (FISH), Lact. plantarum 423 adhered strongly to the ileum and posterior colon and Lact. salivarius 241 to the duodenum in preweaned piglets. High numbers of strain 241 were recorded in the duodenum and posterior colon of postweaned piglets, whereas strain 423 remained localized to the ileum. Lowering in Enterococcus faecalis cell numbers were recorded when preweaned piglets were challenged with strain 241. Plantaricin 423 was produced for 96 h in the ileum section of a gastro-intestinal model. CONCLUSIONS: Lactobacillus plantarum 423 and Lact. salivarius 241 adhere to different sections of the intestinal tract, depending on the piglet's age. Ent. faecalis were inhibited in vivo, probably by plantaricin 423. SIGNIFICANCE AND IMPACT OF THE STUDY: Fluorescent in situ hybridization proved valuable in the detection of probiotic bacteria adhered to the intestine. This is the first report of bacteriocin production in a model simulating the porcine gastro-intestinal tract.

Animals↗
PubMed1980

Factors affecting the quaternary structure of the allosteric L-lactate dehydrogenase from Lactobacillus casei and Lactobacillus curvatus as investigated by hybridization and ultracentrifugation.

The allosteric L-lactate dehydrogenases of Lactobacillus curvatus and Lactobacillus casei exist in the tetrameric from (molecular weight about 145 000) at pH 5.0--5.5 even in the absence of the effectors Mn2+ and Fru(1,6)P2 (fructose 1,6-bisphosphate), but undergo reversible dissociation to monomers (molecular weight about 35 000) at higher pH values or in the presence of urea. In the range between pH 5.5 (tetrameric state) and pH 7.4 (monomeric state) the L. curvatus L-lactate dehydrogenase exists in a dissociation-association equilibrium comprising tetramers, dimers and monomers as indicated by the Sc20,w values and the results of hybridization experiments. The simultaneous addition of both effectors [Mn2+ and Fru(1,6)P2] at pH 7.4, however, resulted in the stabilization of the tetrameric form. The addition of Fru(1,6)P2 alone at pH 7.4 had almost no influence on the quarternary structure, whereas the addition of Mn2+, as well as that of NADH, largely prevented dissociation. The L-lactate dehydrogenase of L. casei showed similar properties, although the enzyme dissociates only at about pH greater than or equal to 7.8. As in the case of the L. curvatus enzyme, Fru(1,6)P2 has no influence on the pH-dependent dissociation of the L. casei enzyme, whereas Mn2+ stabilizes the tetrameric structure. Reconstitution of a mixture of the two dissociated enzymes results in the formation of all statistically possible, enzymatically active hybrids. No hybridization between the allosteric enzymes from L. casei and L. curvatus and the non-allosteric ones of Lactobacillus plantarum and Lactobacillus acidophilus was observed.

Allosteric Regulation↗