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PubMed1997

The lac operon of Lactobacillus casei contains lacT, a gene coding for a protein of the Bg1G family of transcriptional antiterminators.

The 5' region of the lac operon of Lactobacillus casei has been investigated. An open reading frame of 293 codons, designated lacT, was identified upstream of lacE. The gene product encoded by lacT is related to the family of transcriptional antiterminator proteins, which includes BglG from Escherichia coli, ArbG from Erwinia chrysanthemi, SacT, SacY, and LicT from Bacillus subtilis, and BglR from Lactococcus lactis. Amino acid sequence identities range from 35 to 24%, while similarities range from 56 to 47%. The transcriptional start site of the lac operon was identified upstream of lacT. The corresponding mRNA would contain in the 5' region a sequence with high similarity to the consensus RNA binding site of transcriptional antiterminators overlapping a sequence capable of folding into a structure that resembles a rho-independent terminator. LacT was shown to be active as an antiterminator in a B. subtilis test system using the sacB target sequence. lacT directly precedes lacEGF, the genes coding for enzyme IICB, phospho-beta-galactosidase, and enzyme IIA, and these genes are followed by a sequence that appears to encode a second rho-independent transcription terminator-like structure. Northern hybridizations with probes against lacT, lacE, and lacF revealed transcripts of similar sizes for the lac mRNAs of several L. casei strains. Since the length of the lac mRNA is just sufficient to contain lacTEGF, we conclude that the lac operon of L. casei does not contain the genes of the accessory tagatose-6-phosphate pathway as occurs in the lac operons of Lactococcus lactis, Streptococcus mutans, or Staphylococcus aureus.

Amino Acid Sequence↗
PubMed2000

Evidence for horizontal gene transfer in evolution of elongation factor Tu in enterococci.

The elongation factor Tu, encoded by tuf genes, is a GTP binding protein that plays a central role in protein synthesis. One to three tuf genes per genome are present, depending on the bacterial species. Most low-G+C-content gram-positive bacteria carry only one tuf gene. We have designed degenerate PCR primers derived from consensus sequences of the tuf gene to amplify partial tuf sequences from 17 enterococcal species and other phylogenetically related species. The amplified DNA fragments were sequenced either by direct sequencing or by sequencing cloned inserts containing putative amplicons. Two different tuf genes (tufA and tufB) were found in 11 enterococcal species, including Enterococcus avium, Enterococcus casseliflavus, Enterococcus dispar, Enterococcus durans, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, and Enterococcus raffinosus. For the other six enterococcal species (Enterococcus cecorum, Enterococcus columbae, Enterococcus faecalis, Enterococcus sulfureus, Enterococcus saccharolyticus, and Enterococcus solitarius), only the tufA gene was present. Based on 16S rRNA gene sequence analysis, the 11 species having two tuf genes all have a common ancestor, while the six species having only one copy diverged from the enterococcal lineage before that common ancestor. The presence of one or two copies of the tuf gene in enterococci was confirmed by Southern hybridization. Phylogenetic analysis of tuf sequences demonstrated that the enterococcal tufA gene branches with the Bacillus, Listeria, and Staphylococcus genera, while the enterococcal tufB gene clusters with the genera Streptococcus and Lactococcus. Primary structure analysis showed that four amino acid residues encoded within the sequenced regions are conserved and unique to the enterococcal tufB genes and the tuf genes of streptococci and Lactococcus lactis. The data suggest that an ancestral streptococcus or a streptococcus-related species may have horizontally transferred a tuf gene to the common ancestor of the 11 enterococcal species which now carry two tuf genes.

Amino Acid Sequence↗
PubMed2001

Determination of Enterococcus faecalis groESL full-length sequence and application for species identification.

Amplification of the partial Cpn60 (or GroEL) gene segment has been used for identification of many bacteria, including Enterococcus species. To obtain more sequence data from groESL genes of Enterococcus faecalis, the full-length sequence of the E. faecalis groESL genes containing groES (285 bp), spacer (57 bp), and groEL (1,626 bp) was determined. A database search of GenBank revealed that the deduced E. faecalis GroES and GroEL proteins show significant homology to the GroES and GroEL proteins of other bacteria. The GroEL (groEL) of E. faecalis had the highest identity with Streptococcus pneumoniae (81.8% amino acid sequence identity and 73.0% nucleotide sequence identity), followed by Lactococcus zeae, while GroES (groES) had 60.2% (64.6%) identity with Lactobacillus zeae and 58.5% (66.2%) identity with Lactococcus lactis, followed by 57.0% (65.5%) identity with Bacillus subtilis. Based on the groES sequence, an E. faecalis-specific PCR assay was developed, and this PCR assay was positive for all the E. faecalis strains tested. Dot blot hybridization using either groES or groEL as the probe distinguished E. faecalis clearly from other species, indicating that both genes can be used as suitable targets for E. faecalis identification. Moreover, broad-range PCR-restriction fragment length polymorphism of groESL was designed to differentiate eight commonly encountered Enterococcus species. The Enterococcus species of reference strains could be easily differentiated on the basis of restriction patterns produced by HaeIII and RsaI. The DNA-based assays developed in this study provide an alternative to currently used methods of identification for clinically important enterococcal species.

Bacterial Proteins↗
PubMed2005

Monitoring the lactic acid bacterial diversity during shochu fermentation by PCR-denaturing gradient gel electrophoresis.

The presence of lactic acid bacteria (LAB) during shochu fermentation was monitored by PCR-denaturing gradient gel electrophoresis (DGGE) and by bacteriological culturing. No LAB were detected from fermented mashes by PCR-DGGE using a universal bacterial PCR primer set. However, PCR-DGGE using a new primer specific for the 16S rDNA of Lactococcus, Streptococcus, Tetragenococcus, Enterococcus, and Vagococcus and two primers specific for the 16S rDNA of Lactobacillus, Pediococcus, Leuconostoc, and Weissella revealed that Enterococcus faecium, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus nagelii, Lactobacillus plantarum, Lactococcus lactis, Leuconostoc citreum, Leuconostoc mesenteroides, and Weissella cibaria inhabited in shochu mashes. It was also found that the LAB community composition during shochu fermentation changed after the main ingredient and water were added during the fermentation process. Therefore, we confirmed that PCR-DGGE using all three primers specific for groups of LAB together was well suited to the study of the LAB diversity in shochu mashes. The results of DGGE profiles were similar to the results of bacteriological culturing. In conclusion, LAB are present during shochu fermentation but not dominant.

Bacteria, Aerobic↗
PubMed1991

The behavior of selected microorganisms during the manufacture of high moisture Jack cheeses from ultrafiltered milk.

Whole milk was pasteurized and concentrated two times by ultrafiltration. Starter cultures, Lactococcus lactis ssp. cremoris and Lactococcus lactis ssp. lactis, were propagated in either reconstituted skim milk, two times UF retentate, or UF permeate, or a direct vat system was used for the starter culture. The cheese milk was simultaneously inoculated with starter culture and Pseudomonas fragi 4973, Staphylococcus aureus 196E, and Salmonella typhimurium var. Hillfarm. Control whole milk, UF control milk, inoculated whole milk, and inoculated UF milk were made into Monterey Jack cheese using traditional procedures. The process of cheese manufacture was followed by determination of pH, titratable acidity, and microbial population levels. The cheeses were stored for 6 mo and analyzed every month for percentage solids and microbial population levels. Generally, numbers of contaminant microbes increased at a similar rate during manufacture in all cheeses. During the 6-mo ripening period, bacterial starter culture population levels remained high, psychrotrophs declined slowly, Staphylococcus levels remained stable, and Salmonella populations decreased. No Staphylococcus enterotoxin was detected by reverse passive latex agglutination assay.

Animals↗
PubMed1999

Study of the possible mechanisms involved in the mucosal immune system activation by lactic acid bacteria.

The induction of a mucosal immune response is not easy due to the development of oral tolerance, but under some conditions, bacteria can activate this immune system. Antigens administered orally can interact with M cells of Peyer's patches or bind to the epithelial cells. We have demonstrated that certain lactic acid bacteria are able to induce specific secretory immunity, and others will enhance the gut inflammatory immune response. The aim of this work was to establish the reason for these different behaviors and to define possible mechanisms involved in the interaction of lactic acid bacteria at the intestinal level. We studied IgA+ and IgM+ B cells comparatively in bronchus and intestine and CD4+ T cells and IgA anti-lactic acid bacteria antibodies in the intestinal fluid, induced by oral administration of Lactobacillus casei, Lb. delbrueckii ssp. bulgaricus, Lb. acidophilus, Lb. plantarum, Lb. rhamnosus, Lactococcus lactis, and Streptococcus salivarius ssp. thermophilus. The increase in the IgA+ B cells in the bronchus means that these lactic acid bacteria were able to induce the IgA cycle by interaction with M cells from Peyer's patches or intestinal epithelial cells. The IgM+ cells increased when the stimulus did not induce the switch from IgM+ to IgA+. The increase in the CD4+ cells suggests interaction of Peyer's patches and enhancement of the B- and T-cell migration. The anti-lactic acid bacteria antibody is related to the processing and presentation of the microorganisms to the immune cells. We demonstrated that Lb. casei and Lb. plantarum were able to interact with Peyer's patch cells and showed an increase in IgA-, CD4+ cells, and antibodies specific for the stimulating strain. Lactobacillus acidophilus induced gut mucosal activation by interaction with the epithelial cells without increase in the immune cells associated with the bronchus. Although Lb. rhamnosus and Strep. salivarius ssp. thermophilus interact with epithelial cells, they also induced an immune response against their epitopes. Lactococcus lactis and Lb. delbrueckii ssp. bulgaricus induced an increase of IgA+ cells entering the IgA cycle but not CD4+ cells; thus, these bacteria would have been bound to epithelial cells that activated B lymphocytes without processing and presenting of their epitopes. We did not determine specific antibodies against Lc. lactis or Lb. bulgaricus.

Animals↗
PubMed1998

Persistence of Escherichia coli O157:H7 in dairy fermentation systems.

We examined (i) the persistence of Escherichia coli O157:H7 as a postpasteurization contaminant in fermented dairy products; (ii) the ability of E. coli O157:H7 strains with and without the general stress regulatory protein, RpoS, to compete with commercial starter cultures in fermentation systems; and (iii) the survival of E. coli O157:H7 in the yogurt production process. In commercial products inoculated with 10(3) CFU/ml, E. coli O157:H7 was recovered for up to 12 days in yogurt (pH 4.0), 28 days in sour cream (pH 4.3), and at levels > 10(2) CFU/ml at 35 days in buttermilk (pH 4.1). For the starter culture competition trials, the relative inhibition of E. coli O157:H7 in the experimental fermentation systems was, in decreasing order, thermophilic culture mixture, Lactobacillus delbrueckii subsp. bulgaricus R110 alone, Lactococcus lactis subsp. lactis D280 alone, Lactococcus lactis subsp. cremoris D62 alone, and Streptococcus thermophilus C90 alone showing the least inhibition. Recovery of the rpoS mutant was lower than recovery of its wild-type parent by 72 h or earlier in the presence of individual starter cultures. No E. coli O157:H7 were recovered after the curd formation step in yogurt manufactured with milk inoculated with 10(5) CFU/ml. Our results show that (i) postprocessing entry of E. coli O157:H7 into fermented dairy products represents a potential health hazard; (ii) commercial starter cultures differ in their ability to reduce E. coli O157:H7 CFU numbers in fermentation systems; and (iii) the RpoS protein appears to most effectively contribute to bacterial survival in the presence of conditions that are moderately lethal to the cell.

Animals↗
PubMed2001

Characterization of the lactic acid bacteria in ewe's milk and cheese from northwest Argentina.

Indigenous lactic acid bacteria in ewe's milk and artisanal cheese were studied in four samples of fresh raw milk and four 1-month-old cheeses from the provinces of northwest Argentina. Mean growth counts on M17, MRS, and MSE agar media did not show significant differences (P < 0.05) in raw milk and cheeses. Isolates of lactic acid bacteria from milk were identified as Enterococcus (48%), lactococci (14%), leuconostocs (8%), and lactobacilli (30%). All lactococci were identified as Lactococcus lactis (subsp. lactis and subsp. cremoris). Lactobacilli were identified as Lactobacillus plantarum (92%) and Lactobacillus acidophilus (8%). Enterococci (59%) and lactobacilli (41%) were isolated from cheeses. L. plantarum (93%), L. acidophilus (5%), and Lactobacillus casei (2%) were most frequently isolated. L. lactis subsp. lactis biovar diacetylactis strains were considered as fast acid producers. L. lactis subsp. cremoris strains were slow acid producers. L. plantarum and L. casei strains identified from the cheeses showed slow acid production. The majority of the lactobacilli and Lactococcus lactis strains utilized citrate and produced diacetyl and acetoin in milk. Enzyme activities (API-ZYM tests) of lactococci were low, but activities of L. plantarum strains were considerably higher. The predominance of L. plantarum in artisanal cheese is probably important in the ripening of these cheeses due to their physiological and biochemical characteristics.

Animals↗
PubMed2004

Isolation, identification, and selection of lactic acid bacteria from alfalfa sprouts for competitive inhibition of foodborne pathogens.

Several studies have investigated the control of pathogens on alfalfa sprouts, and some treatments have been shown to be effective in reducing pathogen populations. However, control methods investigated thus far only provide pathogen control at a given point in the sprouting process and can affect germination. Competitive inhibition of pathogens with lactic acid bacteria might provide pathogen control throughout the sprouting process and up to consumption. The purpose of this study was to isolate and identify lactic acid bacteria from alfalfa sprouts to inhibit the growth of foodborne pathogens. Fifty-eight lactic acid bacteria isolates were obtained from alfalfa seeds and sprouts. These isolates were evaluated for inhibitory action against Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes by agar spot tests. All pathogens were inhibited by 32 (55%) of the isolates, S. enterica by 56 (97%), E. coli O157:H7 by 49 (84%), and L. monocytogenes by 41 (71%). The isolates were identified by the Analytical Profile Index evaluation of carbohydrate utilization. Isolates obtained from a sample of alfalfa seeds and identified as Lactococcus lactis subsp. lactis showed zones of inhibition of 4.0 mm or greater for all pathogens. One of these isolates, Lactococcus lactis subsp. lactis (L7), and an isolate previously obtained, Pediococcus acidilactici (D3), were evaluated for competitive inhibition of S. enterica, E. coli O157:H7, and L. monocytogenes in deMan Rogosa Sharpe agar and broth. Pathogen populations were significantly reduced by day 5. The selected isolates will be further evaluated in future studies for inhibitory action toward S. enterica, E. coli O157:H7, and L. monocytogenes during sprouting.

Antibiosis↗
PubMedDate not supplied

A non-dairy probiotic's (poi) influence on changing the gastrointestinal tract's microflora environment.

JUSTIFICATION: Yogurt has been historically used to restore gut microflora adversely affected by antibiotic treatment. Certain fermented dairy products are probiotics; "live microorganisms which when administered in adequate amounts confer a health benefit to the host." Microorganisms in foods may benefit certain health conditions such as diarrhea, gastroenteritis, irritable bowel syndrome, inflammatory bowel disease, and cancer. A potential new probiotic from a Polynesian traditional food is poi; a starchy paste made from the corm of taro plants. OBJECTIVE: The purpose of this study was to determine if consumption of poi, a potential non-dairy probiotic, altered the microflora in the gastrointestinal tract of healthy adults. METHODS: A cross-over clinical study included 18 subjects (19-64 years of age) divided into a poi group (n = 10) and control group (n = 8). The study duration of 14 weeks consisted of a 2-week washout, 4-week treatment or control, a subsequent 2-week washout, cross-over of 4-week treatment or control, and a final 2-week washout. Subjects thus served as their own controls. While receiving the poi treatment, participants consumed fresh poi (1-2 days old) three times a day (130 g/meal or approximately 1/2 cup/meal); the control group did not. Both groups filled out 3-day dietary records to ensure compliance. Measurable outcomes included pre- and post-treatment microbiological fecal culture analyses. RESULTS: We found no significant differences in total bacterial counts following a poi diet versus following a control diet, nor were significant differences found in counts of specific bacterial species. Lactococcus tends to be higher in poi when it is analyzed for specific bacteria, but the poi consumption in our study did not alter the mean concentration of individual bacterial species (log10 CFU/g wet feces) for Escherichia coli, Enterobacter, Klebsiella, Lactobacillus, Lactococcus, and Bifidobacterium. No significant differences in stool frequency or consistency were observed between the treatment and control group periods. CONCLUSION: Poi consumption did not significantly alter total or individual bacterial counts in the human gastrointestinal tract. Further research might determine if "sour poi" (3-4 days old) has a greater affect than "fresh poi" (1-2 days old) as a potential probiotic, and a larger trial with longer diet durations may detect more subtle effects of poi consumption on bacterial counts.

Adult↗
PubMed1989

Primary structure and organization of the gene for a procaryotic, cell envelope-located serine proteinase.

We have determined the complete nucleotide sequence of the gene for the cell envelope-located proteinase of Lactococcus lactis SK11. The gene contains a very AT-rich promoter region followed by the coding sequence of a protein of 1962 amino acids. Comparison of the NH2-terminal amino acid sequence of the mature proteinase and the expected primary translation product of the proteinase gene indicates that the enzyme is probably synthesized as a pre-pro-protein. This is confirmed by expression studies of the proteinase gene in Escherichia coli. The amino acid sequence of the proteinase shows significant homology to a number of serine proteinases of the subtilisin family. Compared with the related proteinase of L. lactis Wg2, the proteinase of L. lactis SK11 contains a 60-amino acids duplication and a total of 44-amino acid substitutions, some of which may account for the different cleavage specificity of both enzymes. Furthermore, a region was identified in the Lactococcus proteinase, which shows homology to the membrane-anchoring domains of a number of proteins from other Gram-positive bacteria.

Amino Acid Sequence↗
PubMed1997

Immunization with bacterial antigens: infections with streptococci and related organisms.

Streptococcal infections of fish have been reported from various parts of the world, including the Far East, the United States, South Africa, Australia, Israel and Europe. Classification of Gram-positive cocci (DNA-DNA hybridization studies coupled with 165 sequencing) has shown that at least five different defined species are pathogenic to fish, e.g. Streptococcus iniae (syn. S. shilot), Streptococcus difficile, Lactococcus garvieae (syn. Enterococcus seriolicida), Lactococcus piscium and Vagococcus salmoninarum. "Streptococcosis" of fish should therefore be regarded as a complex of similar diseases caused by different genera and species of Gram-positive cocci, each capable of inducing CNS damage, as well as various degrees of multisystem organ involvement. Panophthalmitis ("pop-eye") and meningitis/meningoencephalitis are the sole findings in trout infected by S. iniae and in tilapines infected by S. difficile. In contrast, L. garvieae-infected trout bear a systemic hyperacute infection with diffuse haemorrhages. Therapeutic measures are generally ineffective. Development of vaccines is therefore essential to control these diseases. In our studies, trout were vaccinated intraperitoneally with whole-cell formalin-inactivated S. iniae and L. garvieae and tilapines with whole-cell formalin-inactivated and acellular S. difficile extract. Under laboratory conditions, S. difficile-vaccinated tilapines were protected against a challenge of 100 LD50s. Protection was correlated with the development of specific agglutinins. Western blot analysis supported the hypothesis that only a few proteins act as protective antigens. S. iniae autovaccines were effective in preventing the disease in rainbow trout in Israel. Under field conditions, fish vaccinated at 50 g were protected for over four months. The qualitative analysis of the humoral response indicated that specific antibodies are directed against a few protein moieties. The fact that passive transfer of antibodies protected fish from experimental infection suggests that the basic mechanism of protection is antibody mediated. L. garvieae autovaccines developed for Italian trout farming were found to elicit a response similar to that of S. iniae. Despite the high virulence of L. garvieae (LD50 of 6 x 10(1) CFU/fish, compared with 3 x 10(4) CFU/fish of S. iniae), the protection against the experimentally induced disease lasted for five months under laboratory conditions, with survival rates of 80-90%. A single injection of the vaccine (0.1 ml/fish) resulted in specific antibody production detectable for six months. In the field, protection rates of 70-80% were obtained for a period of three months, in fish of 200-300 g reared at water temperatures of 18-21 degrees C.

Animals↗
PubMed1991

Specific and intraspecific molecular typing of lactococci based on polymorphism of DNA encoding rRNA.

The rRNA gene restriction patterns or species of the genus Lactococcus were determined. Chromosomal DNA was digested with endonucleases and probed with radiolabelled DNA complementary to rRNA synthesized by random oligonucleotide priming using reverse transcriptase. Highly discriminatory restriction patterns were obtained which served to distinguish the five currently recognized lactococcal species. In addition the observed variations in the patterns at intra-specific level indicate that rRNA gene restriction fingerprinting may be of value in distinguishing the individual strains for epidemiological studies, and monitoring and checking authenticity of starter strains.

Blotting, Southern↗
PubMed2005

Cheddar cheese cooking temperature induces differential lactococcal cell permeabilization and autolytic responses as detected by flow cytometry: implications for intracellular enzyme accessibility.

AIMS: To determine the influence of cheese cooking temperature on autolysis and permeabilization of two lactococcal starter strains in broth and in Cheddar cheese juice during ripening. METHODS AND RESULTS: Flow cytometry (FCM) was used to identify and enumerate intact and permeabilized cells in broth and in Cheddar cheese juice. Levels of intracellular enzyme activities were quantified concurrently. Permeabilized cell numbers increased for both strains in broth following a temperature shift from 32 to 38 degrees C and was accompanied by an increase in the level of accessible intracellular enzyme activities. The relative proportions of intact and permeabilized cell populations, as detected by FCM in cheese juice, changed during 42-day ripening. Permeabilized cell populations increased during ripening for both strains; however, an increase in accessible intracellular enzyme activity was observed only for the highly autolytic strain Lactococcus lactis AM2. CONCLUSIONS: Differences in the autolytic and permeabilization response induced by cooking temperature in two lactococcal strains affects intracellular enzyme accessibility in Cheddar cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the importance of the autolytic and permeabilization properties of lactic acid bacteria starter strains and their impact on cheese ripening.

Bacteriolysis↗
PubMed1995

Genomic organization of Lactococci.

The genus Lactococcus contains four species of which L. lactis is the most thoroughly studied. Its genome is A+T-rich and consists of a circular chromosome of 2.0 to 2.7 Mbp, a wide range of plasmids, and frequently one or more prophages. Insertion sequence elements are commonly present in both the chromosome and the plasmids, while one conjugative transposon of 68 kbp has been described. Genetic maps of the chromosomes of a number of different L. lactis strains have been determined and found to differ quite significantly. For example, the maps of two L. lactis subsp. cremoris strains, MG1363 and FG2, have an inversion of approximately 40% of the chromosome when compared with the maps of two L. lactis subsp. lactis strains, DL11 and IL1403. Other differences indicative of smaller scale translocations and inversions are also found. Chromosomal rearrangements have also been induced in the laboratory by incubation of L. lactis cultures with infecting lytic phage and by treatment with mutagens. These results are indicative of a great deal of plasticity in the lactococcal genome.

Bacteriophages↗
PubMed1999

Isolation and physical characterization of an exocellular polysaccharide.

The physical properties of a polysaccharide produced by the lactic acid bacterium Lactococcus lactis subsp. cremoris strain NIZO B40 were investigated. Separation of the polysaccharide from most low molar mass compounds in the culture broth was performed by filtration processes. Residual proteins and peptides were removed by washing with a mixture of formic acid, ethanol, and water. Gel permeation chromatography (GPC) was used to size fractionate the polysaccharide. Fractions were analyzed by multiangle static light scattering in aqueous 0.10 M NaNO3 solutions from which a number- (Mn) and weight-averaged (Mw) molar mass of (1.47 +/- 0.06).10(3) and (1.62 +/- 0.07).10(3) kg/mol, respectively, were calculated so that Mw/Mn approximately 1.13. The number-averaged radius of gyration was found to be 86 +/- 2 nm. From dynamic light scattering an apparent z-averaged diffusion coefficient was obtained. Upon correcting for the contributions from intramolecular modes by extrapolating to zero wave vector a hydrodynamic radius of 86 +/- 4 nm was calculated. Theoretical models for random coil polymers show that this z-averaged hydrodynamic radius is consistent with the z-averaged radius of gyration, 97 +/- 3 nm, as found with GPC.

Carbohydrate Sequence↗