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PubMed1995

Novel insertion sequence-like element IS982 in lactococci.

A novel insertion sequence-like (IS) element, designated IS982, was found on the lactose plasmid, pSK11L, from Lactococcus lactis subsp. cremoris SK11 and was located between the origin of replication and the oligopeptide transport gene cluster. The 1003-base pair (bp) IS982 was flanked by 18-bp perfect inverted repeats. IS982 contained an open reading frame encoding a putative transposase of 296 amino acids. An almost identical IS-like element (99% DNA sequence identity) was cloned and partially sequenced from the chromosome of Lactococcus lactis subsp. cremoris Wg2 with 17-bp perfect inverted repeats. Southern analysis indicated that in 12 lactococcal strains examined, IS982 was present with copy numbers ranging from 1 to at least 20. IS982 displayed sequence homology to the putative IS element RSBst-alpha from Bacillus stearothermophilus CU21, IS982 and RSBst-alpha were not related to other known insertion sequences and may represent a new family of IS elements.

Amino Acid Sequence↗
PubMed1996

Loss of plasmid-mediated oligopeptide transport system in lactococci: another reason for slow milk coagulation.

Fast milk-coagulating (Fmc+) strains of lactococci are known to segregate slow milk-coagulating (Fmc-) variants, which has been attributed to loss of proteinase (Prt) activity encoded by plasmid DNA. It was found that the Fmc- phenotype could also be due to loss of a plasmid encoding an oligopeptide permease (Opp) system. In Lactococcus lactis subsp. lactis (L. lactis) C2O, lactose metabolism (Lac) and Prt were linked to pJK550 and the Opp system to pJK430. In Lactococcus lactis subsp. cremoris SK11, known to possess Prt on a 78-kb plasmid, DNA sequence analysis of a 7.4-kb region from the Lac plasmid, pSK11L, revealed that it possessed the Opp system. The Lac plasmid in L. lactis C2 encoded both the Prt and Opp systems. Fmc- derivatives of L. lactis C2 were missing the prt genes and had Opp integrated into the chromosome, possibly due to transposition events. Growth studies showed the Opp systems were functional and, in combination with Prt, produced the Fmc+ phenotype.

Animals↗
PubMed2001

Analysis of the complete DNA sequence of the temperate bacteriophage TP901-1: evolution, structure, and genome organization of lactococcal bacteriophages.

A complete analysis of the entire genome of the temperate lactococcal bacteriophage TP901-1 has been performed and the function of 21 of 56 TP901-1-encoded ORFs has been assigned. This knowledge has been used to propose 10 functional modules each responsible for specific functions during bacteriophage TP901-1 proliferation. Short regions of microhomology in intergenic regions present in several lactococcal bacteriophages and chromosomal fragments of Lactococcus lactis are suggested to be points of exchange of genetic material through homologous recombination. Our results indicate that TP901-1 may have evolved by homologous recombination between the host chromosome and a mother phage and support the observation that phage remnants as well as prophages located in the Lactococcus chromosome contribute significantly to bacteriophage evolution. Some proteins encoded in the early transcribed region of the TP901-1 genome were more homologous to proteins encoded by phages infecting gram-positive hosts other than L. lactis. This protein homology argues for the occurrence of horizontal genetic exchange among these bacteriophages and indicates that they have access to a common gene pool.

Bacteriophages↗
PubMed1995

16S-23S and 23S-5S intergenic spacer regions of Streptococcus thermophilus and Streptococcus salivarius, primary and secondary structure.

The 16S-23S intergenic spacer region (spacer region 1) of Streptococcus salivarius, S. thermophilus, and Lactococcus lactis subsp. cremoris and the 23S-5S intergenic spacer region (spacer region 2) of S. salivarius and L. lactis subsp. cremoris were sequenced and compared with the spacer regions 1 and 2 of other streptococci. A high degree of intraspecific conservation was observed for S. thermophilus and L. lactis, and very similar sequences were found for S. salivarius and S. thermophilus. Whereas spacer region 1 is highly conserved in the genus Streptococcus sensu-stricto, only the tRNA gene and the rRNA processing stems are highly conserved in the three genera: Streptococcus sensu-stricto, Lactococcus, and Enterococcus. The presence of a unique tRNA(Ala) gene without the 3' terminal CCA sequence seems to be a general feature of the streptococci spacer region 1. A secondary structure model was built to show the interaction between the spacer regions 1 and 2 of S. thermophilus and S. salivarius. The rapid evolution of spacer region 1 in streptococci is in part due to insertions and deletions of small RNA stem/loop structures.

Base Sequence↗
PubMed1996

Attachment of lactic acid bacteria to beef-muscle surfaces.

The effect of immersion time and cell concentration in the attachment of several lactic acid bacteria with antibacterial activity to beef-muscle surface was studied. The number of firmly attached bacteria increased with immersion time in the case of Pediococcus acidilacti, Lactobacillus sake, Lactococcus cremoris (two strains) and Pediococcus acidilacti. Pediococcus pentosaceus, Lactococcus lactis and Lactobacillus curvatus reached maximum adhesion after 15-30 min. The highest strength of attachment (Sr values) were observed after 15-30 min of contact time except for P. pentosaceus. For all strains, the number of bacteria adhering to meat increased with increasing cell concentration in the adhesion medium. The highest strength of attachment was observed at a cell concentration of 10(5)/mL mainly for L. sake, L. lactis and L. cremoris. Due to their attachment characteristics, L. sake, L. lactis and L. cremoris are proposed as potential biocontrol agents because they could grow on meat surface and limit the potential attachment of pathogenic microorganisms.

Animals↗
PubMed2003

Transcriptional analysis of the genetic elements involved in the lysogeny/lysis switch in the temperate lactococcal bacteriophage phiLC3, and identification of the Cro-like protein ORF76.

A transcriptional analysis of the lysogeny-related genes of the temperate bacteriophage Lactococcus lactis phiLC3 was performed using Northern blot hybridization during lysogeny and lytic infection by the phage. The lysogeny-related gene cluster was found to contain four promoters (P(1), P(2), Pint and P(173)), while the P(87) promoter directed transcription of orf80 and the putative gene orf87, which are located between the integrase gene and the cell lysis genes. The start sites of the transcripts were determined by primer extension. The divergently oriented lysogenic P(1) and lytic P(2) promoters located in the genetic switch region are responsible for transcription of orf286 which encodes the phage repressor, and the genes orf63 - orf76 - orf236 - orf110 - orf82 - orf57, respectively, while orf173 is transcribed from P(173). orf76 was identified as the gene encoding the Cro-like protein of phiLC3, and it was shown that ORF76 is able to bind specifically to the genetic switch region, albeit with lower affinity than does the phage repressor ORF286. ORF76 also competed with ORF286 for binding to this region. The functionality of P(1) and P(2), and their regulation by ORF286 and ORF76, was investigated using a reporter gene. In general, P(2) was a stronger promoter than P(1), but expression from both promoters, especially P(2), was regulated and modulated by flanking sequences and the presence of orf286 and orf76. ORF286 and ORF76 were both able to repress transcription from P(1) and P(2), while ORF286 was able to stimulate its own synthesis by tenfold. This work reveals the complex interplay between the regulatory elements that control the genetic switch between lysis and lysogeny in phiLC3 and other temperate phages of Lactococcus.

Bacteriophages↗
PubMed2006

The effect of decreasing alkalinity on microbial community dynamics in a sulfate-reducing bioreactor as analyzed by PCR-SSCP.

PCR-single-strand conformation polymorphism (SSCP) and Southern blotting techniques were adopted to investigate microbial community dynamics in a sulfate-reducing bioreactor caused by decreasing influent alkalinity. Experimental results indicated that the sulfate-removal rate approached 87% in 25 d under the conditions of influent alkalinity of 4000 mg/L (as CaCO3) and sulfate-loading rate of 4.8 g/(L*d), which indicated that the bioreactor started up successfully. The analysis of microbial community structure in this stage showed that Lactococcus sp., Anaerofilum sp. and Kluyvera sp. were dominant populations. It was found that when influent alkalinity reduced to 1000 mg/L, sulfate-removal rate decreased rapidly to 35% in 3 d. Then influent alkalinity was increased to 3000 mg/L, the sulfate-removal rate rose to 55%. Under these conditions, the populations of Dysgonomonas sp., Sporobacte sp., Obesumbacterium sp. and Clostridium sp. got to rich, which predominated in the community together with Lactococcus sp., Anaerofilum sp. and Kluyvera sp. However, when the alkalinity was decreased to 1500 mg/L, the sulfate-removal rate rose to and kept stable at 70% and populations of Dysgonomonas sp., Sporobacter sp. and Obesumbacterium sp. died out, while some strains of Desulfovibrio sp. and Clostridium sp. increased in concentration. In order to determine the minimum alkalinity value that the system could tolerate, the influent alkalinity was decreased from 1500 to 400 mg/L secondly. This resulted in the sulfate-removal rate, pH value and effluent alkalinity dropping quickly. The amount of Petrotoga sp., Prevotella sp., Kluyvera sp. and Neisseria sp. reduced obviously. The result data from Southern blotting indicated that the amount of sulfate-reducing bacteria (SRBs) decreased with influent alkalinity dropping. Analysis of the microbial community structure and diversity showed that the SRBs populations were very abundant in the inoculated activated sludge and the alkalinity decrease caused the reduction of the populations noted. Most of resident populations in the bioreactor were fermentative acidogenic bacteria (FABs), among which the phylum Firmicute was in the majority, but SRBs were very few. This community structure demonstrates the cooperation between SRBs and FABs, which sustains the system's high sulfate-removal and operation stability.

Bioreactors↗
PubMed1991

Effect of lactic cultures on Escherichia coli in ewes' milk stored at low temperatures.

The behaviour of Escherichia coli in pasteurized ewes' milk inoculated with different lactic starter cultures and incubated at temperatures in the range 4-16 degrees C for 96 h was investigated. Growth temperature of lactic starter cultures before inoculation had a significant effect on inhibition of E. coli. The growth temperature of lactic starter inoculum which resulted in the highest inhibitory activity was 24 degrees C. Size of lactic starter inoculum also significantly influenced growth of E. coli, with a higher inhibition for 1% inoculum than for 0.1% or 0.3% inocula. Single cultures of Lactococcus lactis showed a stronger inhibitory activity than single cultures of Leuconostoc cremoris or Leuconostoc dextranicum. A lactic starter culture comprising Lactococcus lactis, Leuconostoc cremoris and Leuconostoc dextranicum resulted in the strongest inhibition. Stimulation of E. coli by the lactic starter cultures was frequently recorded at 4 degrees C and 8 degrees C. However, none or a very limited growth of E. coli was seen at these temperatures.

Animals↗
PubMed1994

Utilization of cheddar cheese containing nisin as an antimicrobial agent in other foods.

Cheddar cheese made with nisin-producing lactococci contained between 400 and 1200 IU of nisin per gram of cheese. Cultures used were Lactococcus lactis ssp. cremoris JS102, a nisin-producing transconjugant developed in the laboratories of Dr. L.L. McKay and Lactococcus lactis ssp. lactis NCDO 1404 obtained from the National Collection of Food Bacteria, Reading, England. Pasteurized process cheese spreads with 53% and 60% moisture and 0, 301 and 387 IU nisin/g were manufactured and inoculated with 2000 spores of Clostridium sporogenes PA 3679 during manufacture. The heat process did not reduce nisin activity in the cheese spreads. The spreads were incubated at 22 degrees and 37 degrees C for 90 days. Spoilage was detected by the presence of gas and/or odor in the packages. The shelf-life of the nisin-containing cheese spreads was significantly greater than that of the control cheese spreads at the lower temperature at both moisture levels, whereas the keeping quality of the higher moisture cheeses at the higher temperature was not significantly different. Club cheese or cold pack cheese spreads with moisture levels of 44% and 60% and 0, 100 and 300 IU nisin/g were made. These cold processed cheese spreads were inoculated with 1000 cfu per g of Listeria monocytogenes V7, Staphylococcus aureus 196E and spores of C. sporogenes PA 3679. Heat shocked spores of PA 3769 at the same number were added to separate lots of the cheese spread. The cold pack cheese spreads were incubated at 23 degrees and 37 degrees C for up to 8 weeks. Samples were taken weekly and analyzed for surviving organisms. Significant reductions in numbers of the non-sporeforming test microbes were noted at both temperatures, at both moisture levels and both levels of nisin. Heat shocking the spores was needed to show reduction in numbers during the storage of the cold pack cheese spreads. The data obtained in this study suggest that the use of nisin-containing cheese as an ingredient in pasteurized process cheese or cold pack cheese spreads could be an effective method of controlling the growth of undesirable microorganisms in these processed foods.

Cheese↗
PubMed1996

Identification and sequence analysis of IS1297, an ISS1-like insertion sequence in a Leuconostoc strain.

The insertion sequence (IS) ISS1 from Lactococcus lactis was amplified from lactococcal genomic DNA using a primer to the 18-bp inverted repeat sequence. The amplified product hybridized to a single EcoRI fragment in a total genomic DNA digest of Leuconostoc mesenteroides ssp. dextranicum NZDRI 2218. The DNA sequence of this ISS1-like element (IS1297) and the Le. mesenteroides sequences flanking the IS were determined and compared with other iso-ISS1 elements. No direct repeats were found immediately flanking IS1297; however, direct repeats were present approximately 60 bp on either side of the insertion site. IS1297 contained a major open reading frame (ORF) of 681 bp, encoding a putative 226-amino-acid protein with 96.5% homology to the presumed transposase of ISS1. An overlapping ORF of 174 bp in the same orientation was also present. A putative ORF in the opposite orientation to the transposase ORF, which has been shown in some iso-ISS1 elements, was not present in IS1297. IS1297 was shown to hybridize with other dairy Leuconostoc strains. This is the first sequence of an ISS1-like element from a genus other than Lactococcus; however, IS1297 has close similarity to the lactococcal iso-ISS1 elements, especially the iso-ISS1 element from the lactose plasmid, pTD1.

Base Sequence↗
PubMed2006

Artisanal and experimental Pecorino Siciliano cheese: microbial dynamics during manufacture assessed by culturing and PCR-DGGE analyses.

Traditional artisanal Pecorino Siciliano (PS) cheeses, and two experimental PS cheeses were manufactured using either raw or pasteurised ewes' milk with the addition of starter cultures. The bacterial diversity and dynamics of the different cheese types were evaluated both by culturing and characterisation of isolates, and a culture-independent approach based on the 16S ribosomal RNA (rRNA) gene. Following cultivation, artisanal and experimental cheese types showed similar microbial counts, and isolates belonging to Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecalis and Leuconostoc mesenteroides were identified by phenotypic characterisation and comparison of the restriction fragment length polymorphism (RFLP) of the 16S rRNA gene to that of reference species. The culture-independent fingerprinting technique PCR and denaturing gradient gel electrophoresis (DGGE) of V6 to V8 regions of the 16S rRNA gene of samples taken during artisanal PS cheese manufacture, from raw milk to the ripened cheese, indicated relevant shifts in the microbial community structure. The dominance of Streptococcus bovis and Lactococcus lactis species in the traditional artisanal PS was revealed by 16S rRNA gene sequencing. Comparison of DGGE profiles of samples from milk to ripened cheese, derived from artisanal procedure and the two experimental PS cheeses during production showed similar trends with the presence of intense bands in common. Nevertheless, the profiles of several artisanal cheeses from different farms appeared more diverse, and these additional species are probably responsible for the generally superior flavour and aroma development of traditional PS cheese.

Animals↗
PubMed2006

Characterization of lactic acid bacteria isolated from the one humped camel milk produced in Morocco.

One hundred and twenty (120) strains of lactic acid bacteria (LAB) were enumerated and isolated from raw dromedary milk in Morocco using various cultured media. Strains isolated were characterized by phenotypic, physiological and biochemical properties. Results showed that high counts of LAB were found. Presumptive lactobacilli counts ranged from 2.5x10(2) to 6x10(7)cfu/ml, presumptive lactococci levels varied from 5x10(2) to 6x10(7)cfu/ml, presumptive streptococci counts varied from 4.2x10(2) to 8x10(7)cfu/ml, presumptive leuconostoc levels ranged from 5.4x10(2) to 5.4x10(7)cfu/ml. Results showed also that Lactobacillus and Lactococcus were the predominant genera with 37.5% and 25.8%, respectively. The dominated species found were Lactococcus lactis subsp. lactis (17.5%), Lactobacillus helveticus (10%), Streptococcus salivarius subsp. thermophilus (9.20%), Lactobacillus casei subsp. casei (5.80%) and Lactobacillus plantarum (5%). This is the first report on the characterization of LAB strains isolated from the one humped camel milk produced in Morocco.

Animals↗
PubMed2003

Molecular organization of exopolysaccharide (EPS) encoding genes on the lactococcal bacteriophage adsorption blocking plasmid, pCI658.

The lactococcal plasmid pCI658 (58 kb) isolated from Lactococcus lactis ssp. cremoris HO2 encodes the production of a hydrophilic exopolysaccharide (EPS) which consists primarily of galactose and glucuronic acid and which interferes with adsorption of phages ø712 and øc2 to cell surface receptors. Examination of the nucleotide sequence of a 21.8-kb region of the plasmid revealed a large genetic cluster consisting of at least 23 putative EPS biosynthetic determinants in addition to the presence of insertion sequences at the 5(') and 3(') ends. According to homology searches, the genes were organized in specific regions involved in regulation, synthesis and export of the EPS. The predicted products of individual genes exhibited significant homology to exopolysaccharide, capsular polysaccharide (CPS), and lipopolysaccharide (LPS) gene products from a variety of Gram positive and Gram negative bacteria. Evidence of a gene encoding UDP-glucose dehydrogenase is also presented and this is the first description of such a gene in Lactococcus.

Adsorption↗
PubMed2001

Nisin-producing organisms during traditional 'Fior di latte' cheese-making monitored by multiplex-PCR and PFGE analyses.

In this work we studied using different molecular methods the population dynamics of nisin-producing organisms and the persistence of such organisms within a complex ecosystem, 'Fior di latte' cheese, a traditional high-moisture pasta filata cheese. Using the primers targeting the eubacterial 16S-23S rRNA spacer region, together with those amplifying the nisA or nisZ gene, we were able to provide a rapid species identification of the isolates. Inhibitors of Lactococcus lactis subsp. lactis DSM 20481T used as indicator occurred during the whole process of cheese manufacture as a significant part of lactic microflora; however, only 12 among 109 isolates of bacteriocin producers were nisin producers. Amplification of the nisA or nisZ gene, using DNA extracted directly from dairy samples as templates, showed that the nisin structural gene was detected during cheese-making from milk samples up to the end of curd ripening but not in the final cheese. In order to monitor nisin-producing strains during cheese manufacturing, the 12 Lactococcus lactis nis+ strains were analysed by low frequency restriction fragment and PFGE. Nine isolates among the 12 nisin-producers exhibited an unique and distinct DNA banding pattern and are considered to be genetically diverse. The other three isolates from curd after ripening showed the same restriction pattern and could be the same strain. In fact, it was also isolated 2 months after the first analysis of cheese-making of 'Fior di latte'.

Bacteriocins↗
PubMed2001

The microbiology of South African traditional fermented milks.

A total of 15 samples of traditional fermented milk were collected from individual households in South Africa and Namibia. Lactic acid bacteria dominated the microflora of these samples, especially the genera Leuconostoc, Lactococcus and Lactobacillus. Other groups identified included pyogenic streptococci and enterococci. The dominant lactococci species was Lactococcus lactis subsp. lactis. Eighty-three percent of the leuconostoc isolates were identified as Leuconostoc mesenteroides subsp. dextranicum. Other species identified included Leuconostoc citreum, Leuconostoc lactis, Lactobacillus delbrueckii subsp. lactis and Lactobacillus plantarum.

Animals↗
PubMed1997

Lactic acid bacteria of foods and their current taxonomy.

Application of molecular genetic techniques to determine the relatedness of food-associated lactic acid bacteria has resulted in significant changes in their taxonomic classification. During the 1980s the genus Streptococcus was separated into the three genera Enterococcus, Lactococcus and Streptococcus. The lactic acid bacteria associated with foods now include species of the genera Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella. The genus Lactobacillus remains heterogeneous with over 60 species (ymol% G+C content ranging from 33 to 55), of which about one-third are strictly heterofermentative. However, many changes have been made and reorganization of the genus along lines that do not follow previous morphological or phenotypic differentiation from Leuconostoc and Pediococcus is being studied. Phylogenetically belonging to the Actinomyces branch of the bacteria, Lactobacillus bifidus has been moved to the genus Bifidobacterium also on account of its greater than 50 mol% G+C content. It is therefore no longer considered one of the lactic acid bacteria senso strictu, which form part of the Clostridium branch of the bacteria. The new genus Weissella has been established to include one member of the genus Leuconostoc (Leuc, paramesenteroides) and heterofermentative lactobacilli with unusual interpeptide bridges in the peptidoglycan. Contrary to the clear-cut division of the streptococci, morphological and physiological features of Weissella do not directly support this grouping which now incorporates species that produce D(-)- as well as DL-lactate. The new genus Carnobacterium is morphologically similar to the lactobacilli, but it shares some physiological similarities (e.g. growth at pH 9.5) and a common phylogenetic branch with the genus Enterococcus. The review includes information on the taxonomic changes and the relationship of the bacteria of food fermentation and spoilage.

Enterococcus↗
PubMed2003

Evidence of a relationship between autolysis of starter bacteria and lipolysis in cheddar cheese during ripening.

Cell viability, autolysis and lipolysis were studied in Cheddar cheese made using Lactococcus lactis subsp. cremoris AM2 or Lactococcus lactis subsp. cremoris HP. Cheddar cheese was made in triplicate over a 3 month period and ripened for 238 days at 8 degrees C. Cell viability in cheese was lower for AM2 (a non-bitter strain) than for strain HP (a bitter strain). Autolysis, monitored by the level of the intracellular marker enzyme, lactate dehydrogenase (EC 1.1.1.27) in cheese 'juice' extracted by hydraulic pressure, was much greater in the cheese made using AM2 than that made with HP. Lipolysis was determined by the increase during ripening of individual free fatty acids (FFA) from butyric (C4:0) to linolenic acid (C18:3) measured using a high performance liquid chromatographic technique. Levels of individual FFA from butyric (C4:0) to linolenic (C18:3) acids increased significantly (P<0.05) during ripening in cheeses made with either starter culture. Palmitic (C16:0) and oleic (C18:1) acids were the most abundant FFA throughout ripening in all cheeses. Levels of caprylic (C8:0), myristic (C14:0), palmitic (C16:0) and stearic (C18:0) acids were significantly higher (P<0.05) in cheeses manufactured with Lc. lactis subsp. cremoris AM2 than in cheeses manufactured with Lc. lactis subsp. cremoris HP. Differences in levels of lipolysis between strains was not due to differences in the specific lipolytic or esterolytic activities in cell free extracts of the strains as measured by activity on triolein (lipase) and p-nitrophenylbutyrate (esterase) substrates. Therefore, evidence is provided for a relationship between the extent of starter cell autolysis and the level of lipolysis during Cheddar cheese ripening.

Bacteriolysis↗
PubMed2004

Diversity of lactic acid bacteria isolated from AOC Salers cheese.

The objective of this work was to describe the diversity of lactic acid bacteria in traditional raw milk Salers cheeses at the species and strain levels. The characterization of 381 strains isolated during ripening and various strain collections was investigated using physiological analysis and molecular techniques: Rep-PCR, species and genus specific amplifications and the sequence analysis of 16S rDNA for strain typing and taxonomic identification. The strains belonged to Lactobacillus plantarum, Lactobacillus paracasei, Lactococcus lactis, Lactococcus garviae, Enterococcus faecalis, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Streptococcus salivarius, Streptococcus millieri, Streptococcus macedonicus and Pediococcus pentosaceus. A wide phenotypic and genomic heterogeneity was observed within the different species (Lactobacillus plantarum, Lactobacillus paracasei and Leuconostoc mesenteroides) according to the origin and the time of ripening. The natural microflora was different from strain collection and each method must be combined to identify and characterize natural microflora. This study revealed the low selectivity of selective media used for the isolation of different groups of lactic acid bacteria except the Facultatively Heterofermentative lactobacilli medium selecting mesophile lactobacilli and SB medium selective for Enterococcus. The study reveals, for the first time, the microbial lactic acid bacteria community of Salers cheese and its diversity. A better knowledge of microbial flora will be useful to improve understanding of sensory quality of cheeses.

Cheese↗