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PubMed2000

Homologous recombination between a lactococcal bacteriophage and the chromosome of its host strain.

Genetic exchanges constitute a significant means by which bacteriophages acquire novel characteristics. Phages of Lactococcus lactis occupy a particular niche, the dairy factory environment, where their populations are subjected to constant changes. Little is known about the mechanisms of evolution that lead to the genetic diversity of lactococcal phages. In this study, we described two DNA exchanges involving the lytic phage ul36, a member of the P335 species, and its L. lactis host. They occurred by homologous recombination with phage-related sequences present in the host chromosome. Both mutants generated by these recombination events are insensitive to the phage resistance mechanism AbiK and one has a reduced burst size as well as a new origin of replication. We propose that this type of DNA exchange with prophages or remnants of prophages occurs frequently within the P335 species as supported by DNA-DNA comparisons between P335-like phages.

Bacterial Proteins↗
PubMed2003

PH-sensing 96-well microtitre plates for the characterization of acid production by dairy starter cultures.

A new method for characterization of acid production by dairy starter cultures is presented. Microplates with integrated optical pH sensors are developed. Two fluorophores, a pH-sensitive and a pH-insensitive one are immobilised at the bottom of a polystyrene 96-well microtitre plate. The pH-insensitive fluorophore serves as an internal reference and makes calibration unnecessary. The sensor measures pH accurately in optically well-defined media. Particles and fluorophores contained in the bulk medium disturbed the measurements. Despite these disturbances it was possible to clearly sense differences in inoculum type and in inoculum sizes of cultures of Lactococcus lactis and of Streptococcus thermophilus at 30 and 37 degrees C. Besides a pH-related signal there is information about other changes during milk fermentation. The cultivation results were compared with those from the established CINAC-method. From this comparison it can be concluded that the new method can be used reliably to characterize particularly a large number of strains for screening purposes but also for quality control.

Animals↗
PubMed1996

Dramatic decay of phage transcripts in lactococcal cells carrying the abortive infection determinant AbiB.

The abortive infection determinant AbiB prevents growth of the sensitive phage bIL170, but not of the resistant phage bIL41, on Lactococcus lactis strain IL1403. Here we show that AbiB promotes a dramatic degradation of sensitive phage transcripts, starting 10-15 min after infection. The decay of the transcripts is the probable cause of the arrest of the sensitive phage development. Mapping of the 5' end of degradation products established that they result from endonucleolytic cleavage preferentially at U/U, A/U and U/A sites. We propose that an early product of the sensitive phage either induces the synthesis or stimulates the activity of an RNase in an AbiB+ cell.

Bacterial Proteins↗
PubMed2006

Proteolytic enzyme activities in Cheddar cheese juice made using lactococcal starters of differing autolytic properties.

AIMS: To determine proteolytic enzyme activities released in Cheddar cheese juice manufactured using lactococcal starter strains of differing autolytic properties. METHODS AND RESULTS: The activities of residual chymosin, cell envelope proteinase and a range of intracellular proteolytic enzymes were determined during the first 70 days of ripening when starter lactococci predominate the microbial flora. In general, in cell free extracts (CFE) of the strains, the majority of proteolytic activities was highest for Lactococcus lactis HP, intermediate for L. lactis AM2 and lowest for L. lactis 303. However, in cheese juice, as ripening progressed, released proteolytic activities were highest for the highly autolytic strain L. lactis AM2, intermediate for L. lactis 303 and lowest for L. lactis HP. CONCLUSIONS: These results indicate that strain related differences in autolysis influence proteolytic enzyme activities released into Cheddar cheese during ripening. No correlation was found between proteolytic potential of the starter strains measured in CFE prior to cheese manufacture and levels of activities released in cheese juice. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings further support the importance of autolysis of lactococcal starters in determining the levels of proteolytic activities present in cheese during initial stages of ripening.

Cheese↗
PubMed2005

Lantibiotic structures as guidelines for the design of peptides that can be modified by lantibiotic enzymes.

Lantibiotics are (methyl)lanthionine-containing bacterial peptides. (Methyl)lanthionines are posttranslationally introduced into the prepropeptides by biosynthetic enzymes that dehydrate serines and threonines and couple these dehydrated residues to cysteine residues. Thirty seven lantibiotic primary structures have been proposed to date, but little is known about the substrate specificity of the lantibiotic modifying enzymes. To define rules for the rational design of modified peptides, we compared all known lantibiotic structures by in silico analysis. Although no strict sequence motifs can be defined that govern the modification, statistical analysis demonstrates that dehydratable serines and threonines are more often flanked by hydrophobic than by hydrophilic amino acids. Serine residues escape dehydration more often than threonines. With these rules, novel hexapeptides were designed that either were predicted to become modified or will escape modification. The hexapeptides were fused to the nisin leader and expressed in a Lactococcus lactis strain containing the nisin modifying and export enzymes. The excreted peptides were analyzed by mass spectrometry. All designed fusion peptides were produced, and the presence or absence of modifications was found to be in full agreement with the predictions based on the statistical analysis. These findings demonstrate the feasibility of the rational design of a wide range of novel peptides with dehydrated amino acid residues.

Alanine↗
PubMed2004

Bacteriophage Tuc2009 encodes a tail-associated cell wall-degrading activity.

Tuc2009 is a P335-type member of the tailed-phage supergroup Siphoviridae and was originally identified as a resident prophage of the gram-positive bacterium Lactococcus lactis UC509. A Tuc2009 gene designated tal2009 which is located within the morphogenic module was shown to specify a lytic activity within the 3' portion of its coding region. Comparative sequence analysis indicated that the cell wall-degrading part of Tal2009 is a member of the M37 protein family and that Tal2009 lacks a cell-binding domain, a finding supported by binding studies. Tal2009 appears to undergo self-mediated posttranslational processing in both L. lactis and Escherichia coli. Antibodies directed against a purified C-terminal portion of Tal2009 were used for immunoelectron microscopy, which showed that Tal2009 is located at the tail tip of Tuc2009. Antibody neutralization studies demonstrated that Tal2009-directed antibodies inhibited the ability of phage to mediate host lysis by more than 100-fold. These data indicate that tal2009 encodes a tail-associated lysin involved in localized cell wall degradation, thus allowing the Tuc2009 DNA injection machinery access to the membrane of its bacterial host.

Catalysis↗
PubMed2005

Characterization and kinetic analysis of enzyme-substrate recognition by three recombinant lactococcal tripeptidases.

Tripeptidases from Lactococcus lactis subsp. lactis (L9PepTR), L. lactis subsp. cremoris (L6PepTR), and L. lactis subsp. hordniae (hTPepTR) were cloned, overexpressed, purified, and characterized. Although these enzymes contained three to seven naturally occurring amino acid differences, both metal-binding and catalytic sites were highly conserved. The k(cat) values of hTPepTR were approximately 1.5- to 2-fold higher than those of L9PepTR, while, for L6PepTR, they were approximately 0.8- to 1.4-times the L9PepTR values. The K(m) of tripeptidase from subsp. lactis (L9PepTR) was considerably larger when glycine was the amino acid located at both the N- and C-terminus of the peptide substrate. In addition, the K(m) values of L9PepTR increased in the following order for YGG, LGG, FGG, SGG, and alpha-aminoisobutyrylglycylglycine, while the k(cat)/K(m) decreased in the same order. These results suggest that the dipole moment and steric hindrance of the N-terminal amino acid side chain may be the most important factors controlling substrate specificity.

Amino Acid Sequence↗
PubMed2000

Identification of a DNA region from lactococcal phage sk1 protecting phage 712 from the abortive infection mechanism AbiF.

Bacteriophage 712 is a small isometric-headed phage which is sensitive to the lactococcal abortive infection mechanism AbiF. Its 29.6-kb DNA genome was characterized by restriction mapping and transcriptional analysis. Construction of a gene bank of lactococcal phage sk1, which is insensitive to the action of AbiF, in Lactococcus lactis containing AbiF resulted in the identification of a 324-bp DNA fragment which reduced the effect of the abortive infection mechanism on phage 712. Analysis of this region provided evidence that the action of AbiF is related to the cos ends of small isometric-headed phages. Sequence analysis of a 3.2-kb segment containing the middle operon and the cos ends of phage 712 genome allowed comparison of this part of the phage 712 genome with the equivalent sequences of four other small isometric-headed phages.

Cloning, Molecular↗
PubMed2001

Studies on the genomic organization of recombinant Streptococcus gordonii and the development of a novel intergenic integration site for foreign gene expression.

The methods currently employed to produce recombinant Streptococcus gordonii strains for use as vaccines and/or protein expression vectors result in the insertion of foreign genes into an unknown integration site with no information on the transcriptional context or potential phenotypic consequences. Therefore, the genomic organization surrounding the insertion site of a recombinant strain of S. gordonii (GP1223) containing a portion of the emm6 gene of Streptococcus pyogenes was determined. The nucleotide sequence of chromosomal walks in both directions from the insertion site revealed that the insert was flanked by a duplicated 3061-bp ClaI fragment. A consensus gram-positive promoter and a factor-independent RNA polymerase terminator sequence could be deduced in the fragment immediately upstream of the insertion site. The ClaI fragment also encoded open reading frames (ORFs) with high homology and parallel structural organization to the leucine biosynthesis operon of Lactococcus lactis subsp. lactis. Chromosomal walks downstream of the identified promoter region on the non-recombinant parental strain, GP204, yielded the sequence of two ORFs which would be normal targets of the transcription derived from this promoter. Northern analyses detected a highly expressed M6-specific transcript in recombinant strain GP1223 consistent in size with the proposed transcription unit. Transcripts analogous in length to those observed for the leucine biosynthesis operon of L. lactis subsp. lactis were also detected encompassing the homologous ORFs of S. gordonii. This information has enabled the construction of a recombinant S. gordonii strain in which the emm6 gene from S. pyogenes was targeted to a distinct intergenic locus within the S. gordonii genome. This new recombination site allows for expression of foreign gene products with minimal perturbation of the genomic organization of the wild-type S. gordonii strain and has provided information essential for further optimization of foreign gene expression levels.

Base Sequence↗
PubMed1989

Cloning of chromosomal genes of Lactococcus by heterologous complementation: partial characterisation of a putative lactose transport gene.

A cosmid gene library of the genome of Lactococcus lactis subsp. lactis 712 has been constructed in the broad host range plasmid pLAFR1 in Escherichia coli LE392. Three lactococcal genes from the bank were identified by heterologous complementation of specific mutations in strains of E. coli. A cosmid clone encoding a putative lactose transport gene was identified by complementing an E. coli lacY mutant. The complemented clone supported the uptake of 14C lactose in transport assays. The DNA fragment responsible was subcloned and localised to a 1.28 kb fragment of the lactococcal chromosome.

Biological Transport↗
PubMed1991

Comparison of lactococcal bacteriophage isolated in the United States and Argentina.

Bacteriophage of Lactococcus lactis ssp. lactis and ssp. cremoris, isolated in the United States and Argentina, were compared with respect to host range, adsorption, latent period, burst size and immunological cross-reactivity. Only 1 out of 13 U.S. culture isolates was sensitive to Argentinian phage. Argentinian L. lactis ssp. lactis C2 mutants were resistant to 13 U.S. phage isolates (4 prolate and 9 isometric). While Argentinian phage Stl-3 multiplied on U.S. culture isolate 59-1, low adsorption (38%) and insignificant burst size and latent period data were evident. Antisera prepared against U.S. phage D59-1 (prolate) and F4-1 (isometric) neutralized the lytic activities of all Argentinian prolate phage although the F4-1 antiserum was less effective. The data suggest homology especially between U.S. phage D59-1 and the Argentinian phage.

Absorption↗
PubMed2004

A new vector, pGID052, for genetic transfer in Oenococcus oeni.

Despite the large number of techniques available for the transformation of bacteria, several species are still resistant to the introduction of foreign DNA. Oenococcus oeni are among the organisms that are particularly refractory to transformation. However, conjugal experiments from Lactococcus lactis to O. oeni with a new plasmid, pGID052, were performed via mobilization with success. This plasmid, a derivative of pORI19, encompasses: (i) the oriT of pIP501, which permitted the transfer to O. oeni, (ii) the replication genes of a native Leuconostoc citreum plasmid. Frequencies of 10(-7) conjugants per recipient were found. The transfer did not affect the structure of this low-copy-number plasmid. Moreover, pGID052 seems segregationally stable and could be used in the future as an expression vector.

Alcoholic Beverages↗
PubMed2001

Examination of lactococcal bacteriophage c2 DNA replication using two-dimensional agarose gel electrophoresis.

The ori locus of the prolate-headed lactococcal bacteriophage c2 supports plasmid replication in Lactococcus lactis in the absence of phage infection. To determine whether phage c2 DNA replication is initiated at the ori locus in vivo and to investigate the mechanism of phage DNA replication, replicating intermediates of phage c2 were analyzed using neutral/neutral two-dimensional agarose gel electrophoresis (2D). The 2D data revealed that c2 replicates via a theta mechanism and localized the initiation of theta replication to the ori region of the c2 genome.

Bacteriophages↗
PubMed2001

Analysis of free fatty acids in whey products by solid-phase microextraction.

To evaluate the impact of Cheddar cheese starter cultures on the level of free fatty acids in liquid whey, a solid-phase microextraction (SPME) technique was utilized. The determination of response factors relative to an internal standard and the verification of linearity over a wide concentration range allowed for the quantitation of free fatty acids in experimentally produced liquid whey and in a wide range of dry whey ingredients. Results indicated that whey produced with a Lactococcus lactis subsp. lactis starter culture contained the highest level of total free fatty acids with significantly higher levels of lauric, myristic, and palmitic acids. Significant declines in oleic, linoleic, and palmitic acid occurred during storage. Commercial whey ingredients demonstrated a linear increase in free fatty acids with increasing percent protein, except for whey protein isolate, which had the lowest concentration. The use of SPME for the rapid analysis of free fatty acids in whey products should allow for further research to determine the importance of these compounds on the quality and stability of whey products.

Cheese↗
PubMed2002

Complete genomic sequence of bacteriophage ul36: demonstration of phage heterogeneity within the P335 quasi-species of lactococcal phages.

The complete genomic sequence of the Lactococcus lactis virulent phage ul36 belonging to P335 lactococcal phage species was determined and analyzed. The genomic sequence of this lactococcal phage contained 36,798 bp with an overall G+C content of 35.8 mol %. Fifty-nine open reading frames (ORFs) of more than 40 codons were found. N-terminal sequencing of phage structural proteins as well as bioinformatic analysis led to the attribution of a function to 24 ORFs (41%). A lysogeny module was found within the genome of this virulent phage. The putative integrase gene seems to be the product of a horizontal transfer because it is more closely related to Streptococcus pyogenes phages than it is to L. lactis phages. Comparative genome analysis with six complete genomes of temperate P335-like phages confirmed the heterogeneity among phages of P335 species. A dUTPase gene is the only conserved gene among all P335 phages analyzed as well as the phage BK5-T. A genetic relationship between P335 phages and the phage-type of the BK5-T species was established. Thus, we proposed that phage BK5-T be included within the P335 species and thereby reducing the number of lactococcal phage species to 11.

Bacteriophages↗
PubMed1990

Simultaneous conjugal transfer in Lactococcus to genes involved in bacteriocin production and reduced susceptibility to bacteriophages.

Conjugal matings were performed between Lactococcus lactis DRC1 (a lactose-fermenting (Lac+), bacteriocin-producing (Bac+) strain) and L. lactis HID113 (Lac- and Bac-). Transconjugant derivatives of HID113 were identified on the basis of lactose fermentation, resistance to the DRC1 bacteriocin (dricin) or reduced sensitivity to phage sk1. Regardless of how they were identified, all transconjugants gave fewer and smaller plaques with phages c2 and sk1 than did HID113. All but one of 275 transconjugants tested also produced dricin, suggesting some functional relationship or close genetic linkage between the reduced phage sensitivity and dricin production and resistance. Some transconjugants were also Lac+, but this property was unstable.

Bacteriocins↗
PubMed2006

Determinants of substrate specificity in KdcA, a thiamin diphosphate-dependent decarboxylase.

Thiamin diphosphate-dependent decarboxylases catalyze the non-oxidative decarboxylation of 2-keto carboxylic acids. Although they display relatively low sequence similarity, and broadly different range of substrates, these enzymes show a common homotetrameric structure. Here we describe a kinetic characterization of the substrate spectrum of a recently identified member of this class, the branched chain 2-keto acid decarboxylase (KdcA) from Lactococcus lactis. In order to understand the structural basis for KdcA substrate recognition we developed a homology model of its structure. Ser286, Phe381, Val461 and Met358 were identified as residues that appeared to shape the substrate binding pocket. Subsequently, site-directed mutagenesis was carried out on these residues with a view to converting KdcA into a pyruvate decarboxylase. The results show that the mutations all lowered the Km value for pyruvate and both the S286Y and F381W variants also had greatly increased values of k(cat) with pyruvate as a substrate.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1998

Use of 13C nuclear magnetic resonance and gas chromatography to examine methionine catabolism by lactococci.

Formation of methanethiol from methionine is widely believed to play a significant role in development of cheddar cheese flavor. However, the catabolism of methionine by cheese-related microorganisms has not been well characterized. Two independent methionine catabolic pathways are believed to be present in lactococci, one initiated by a lyase and the other initiated by an aminotransferase. To differentiate between these two pathways and to determine the possible distribution between the pathways, 13C nuclear magnetic resonance (NMR) performed with uniformly enriched [13C]methionine was utilized. The catabolism of methionine by whole cells and cell extracts of five strains of Lactococcus lactis was examined. Only the aminotransferase-initiated pathway was observed. The intermediate and major end products were determined to be 4-methylthio-2-oxobutyric acid and 2-hydroxyl-4-methylthiobutyric acid, respectively. Production of methanethiol was not observed in any of the 13C NMR studies. Gas chromatography was utilized to determine if the products of methionine catabolism in the aminotransferase pathway were precursors of methanethiol. The results suggest that the direct precursor of methanethiol is 4-methylthiol-2-oxobutyric acid. These results support the conclusion that an aminotransferase initiates the catabolism of methionine to methanethiol in lactococci.

Carbon Isotopes↗