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PubMed2003

Intracellular esterase from Lactobacillus casei LILA: nucleotide sequencing, purification, and characterization.

An esterase gene (estC) was isolated from a genomic library of Lactobacillus casei LILA. The estC gene consisted of a 777 bp open reading frame encoding a putative peptide of 28.9 kDa. A recombinant EstC fusion protein containing a C-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of EstC revealed that it was a serine-dependent dimeric enzyme. Optimum temperature, NaCl concentration, and pH for EstC were determined to be 30 degrees C, 0% NaCl, and pH 5.5, respectively. EstC had significant activity under conditions simulating those of ripening cheese (10 degrees C, 4% NaCl, and pH 5.1). Kinetic constants (KM and Vmax) were determined for EstC action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, the previously studied EstA from Lactococcus lactis MG1363 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstC and EstA.

Amino Acid Sequence↗
PubMed1999

Genetic diversity among dairy lactococcal strains investigated by polymerase chain reaction with three arbitrary primers.

Randomly amplified polymorphic DNA (RAPD) has been used for the rapid typing of Lactococcus lactis strains isolated from raw milk from the Camembert region of Normandy. It is thought that the diversity and perhaps the area strain specificity due to climatic and geographical factors of such wild-type lactococcal strains could contribute to the flavour differences and specific features detected for the same product in different areas. The patterns from 58 isolates were analysed by UPGMA dendrograms. At a similarity level of 50%, four RAPD clusters were distinguished. Clusters 1 and 2 contained strains of subspecies lactis and cluster 3 contained strains related to the C2 strain which is genetically cremoris but phenotypically lactis. The type strain of cremoris subspecies was significantly differentiated from these strains with primers P2 and P3. Thus, there was a real genetic diversity in pattern, making it possible to detect potential typical RAPD fragments.

Animals↗
PubMed2001

Characterization of lactic acid bacteria in the larval midgut of the keratinophagous lepidopteran, Hofmannophila pseudospretella.

In two of eight Hofmannophila pseudospretella specimens studied by microscopy, the larval midgut contained an unidentified micro-organism. Although not seen microscopically in midgut sections, bacteria were isolated from dissected midgut. Microscopy, carbohydrate utilization and ribosomal sequence data all separated the isolates into the same three classes. These were identified as Lactococcus lactis, Carnobacterium piscicola and, tentatively, Bacillus subtilis, the first two being facultative anaerobes and the latter, an aerobe. The bacteria were therefore not specifically adapted to the reducing conditions of the midgut.

Animals↗
PubMed2004

Insights into the DNA repair process by the formamidopyrimidine-DNA glycosylase investigated by molecular dynamics.

Formamidopyrimidine-DNA glycosylase (Fpg) identifies and removes 8-oxoguanine from DNA. All of the X-ray structures of Fpg complexed to an abasic site containing DNA exhibit a common disordered region present in the C-terminal domain of the enzyme. However, this region is believed to be involved in the damaged base binding site when the initial protein/DNA complex is formed. The dynamic behavior of the disordered polypeptide (named Loop) in relation to the supposed scenario for the DNA repair mechanism was investigated by molecular dynamics on different models, derived from the X-ray structure of Lactococcus lactis Fpg bound to an abasic site analog-containing DNA and of Bacillus stearothermophilus Fpg bound to 8-oxoG. This study shows that the presence of the damaged base influences the dynamics of the whole enzyme and that the Loop location is dependent on the presence and on the conformation of the 8-oxoG in its binding site. In addition, from our results, the conformation of the 8-oxoG seems to be favored in syn in the L. lactis models, in agreement with the available X-ray structure from B. stearothermophilus Fpg and with a possible catalytic role of the flexibility of the Loop region.

Bacterial Proteins↗
PubMed1995

Characterization of the lactococcal abiD1 gene coding for phage abortive infection.

Lactococcal phage abortive infection (AbiD1) determined by plasmid pIL105 is active on both prolate- and small-isometric-head phages of the C6A and 936 phage groups, respectively, which are considered two different species. The Abi phenotype was found to be encoded by a single gene, designated abiD1. The abiD1-encoded protein (351 amino acids) does not show homology with any known protein and has a deduced isoelectric point of 10. It also possesses two helix-turn-helix structures and an unusually high content of asparagine, isoleucine, and lysine. A consensual promoter with a TGy extension to the -10 box was mapped 76 bp upstream of the start codon. Transcription initiated at this strong promoter stops at a terminator located 48 bp downstream from the promoter. The termination process is very efficient, and transcripts corresponding to the abiD1 gene were not visible in our experimental conditions with or without phage infection. Expression of abiD1 under the control of a T7 promoter induced a lag phase in Lactococcus lactis cell growth, suggesting that overproduction of AbiD1 could be toxic for the cells. AbiD1 protein was visualized in Escherichia coli by using a tightly controlled expression system.

Amino Acid Sequence↗
PubMed2005

Llama antibodies against a lactococcal protein located at the tip of the phage tail prevent phage infection.

Bacteriophage p2 belongs to the most prevalent lactococcal phage group (936) responsible for considerable losses in industrial production of cheese. Immunization of a llama with bacteriophage p2 led to higher titers of neutralizing heavy-chain antibodies (i.e., devoid of light chains) than of the classical type of immunoglobulins. A panel of p2-specific single-domain antibody fragments was obtained using phage display technology, from which a group of potent neutralizing antibodies were identified. The antigen bound by these antibodies was identified as a protein with a molecular mass of 30 kDa, homologous to open reading frame 18 (ORF18) of phage sk1, another 936-like phage for which the complete genomic sequence is available. By the use of immunoelectron microscopy, the protein is located at the tip of the tail of the phage particle. The addition of purified ORF18 protein to a bacterial culture suppressed phage infection. This result and the inhibition of cell lysis by anti-ORF18 protein antibodies support the conclusion that the ORF18 protein plays a crucial role in the interaction of bacteriophage p2 with the surface receptors of Lactococcus lactis.

Amino Acid Sequence↗
PubMed2006

Genetic variation in Drosophila melanogaster resistance to infection: a comparison across bacteria.

Insects use a generalized immune response to combat bacterial infection. We have previously noted that natural populations of D. melanogaster harbor substantial genetic variation for antibacterial immunocompetence and that much of this variation can be mapped to genes that are known to play direct roles in immunity. It was not known, however, whether the phenotypic effects of variation in these genes are general across the range of potentially infectious bacteria. To address this question, we have reinfected the same set of D. melanogaster lines with Serratia marcescens, the bacterium used in the previous study, and with three additional bacteria that were isolated from the hemolymph of wild-caught D. melanogaster. Two of the new bacteria, Enterococcus faecalis and Lactococcus lactis, are gram positive. The third, Providencia burhodogranaria, is gram negative like S. marcescens. Drosophila genotypes vary highly significantly in bacterial load sustained after infection with each of the four bacteria, but mean loads are largely uncorrelated across bacteria. We have tested statistical associations between immunity phenotypes and nucleotide polymorphism in 21 candidate immunity genes. We find that molecular variation in some genes, such as Tehao, contributes to phenotypic variation in the suppression of only a subset of the pathogens. Variation in SR-CII and 18-wheeler, however, has effects that are more general. Although markers in SR-CII and 18-wheeler explain >20% of the phenotypic variation in resistance to L. lactis and E. faecalis, respectively, most of the molecular polymorphisms tested explain <10% of the total variance in bacterial load sustained after infection.

Animals↗
PubMed2002

[The resistance conferred by the R/M system LlaBIII against bacteriophages].

LlaBIII, isolated from the naturally occurring plasmid pJW566 from Lactococcus lactis subsp. cremoris W56, encoded a restriction and modification (R/M) system. The plasmid pJK1 carrying the R/M system LlaBIII was transformed into L. lactis IL1403 with type I R/M system located on chromosome and the strain MG1614[pAW601] with AbiS gene on plasmid pAW601, respectively. The transformants obtained showed stacking resistance against bacteriophages. The plasmid pJK1 was transformed into industrial strains L. lactis SMQ86 and CHCC2281, the transformants showed the EOP of the bacteriophages decreased by 10(-3) and 10(-5), respectively. The results indicated that the R/M system LlaBIII could protect strains from bacteriophages in dairy fermentation.

Bacterial Proteins↗
PubMed2000

Surface of lactic acid bacteria: relationships between chemical composition and physicochemical properties.

The surface chemical composition and physicochemical properties (hydrophobicity and zeta potential) of two lactic acid bacteria, Lactococcus lactis subsp. lactis bv. diacetilactis and Lactobacillus helveticus, have been investigated using cells harvested in exponential or stationary growth phase. The surface composition determined by X-ray photoelectron spectroscopy (XPS) was converted into a molecular composition in terms of proteins, polysaccharides, and hydrocarbonlike compounds. The concentration of the last was always below 15% (wt/wt), which is related to the hydrophilic character revealed by water contact angles of less than 30 degrees. The surfaces of L. lactis cells had a polysaccharide concentration about twice that of proteins. The S-layer of L. helveticus was either interrupted or crossed by polysaccharide-rich compounds; the concentration of the latter was higher in the stationary growth phase than in the exponential growth phase. Further progress was made in the interpretation of XPS data in terms of chemical functions by showing that the oxygen component at 531.2 eV contains a contribution of phosphate in addition to the main contribution of the peptide link. The isoelectric points were around 2 and 3, and the electrophoretic mobilities above pH 5 (ionic strength, 1 mM) were about -3.0 x 10(-8) and -0.6 x 10(-8) m(2) s(-1) V(-1) for L. lactis and L. helveticus, respectively. The electrokinetic properties of the latter reveal the influence of carboxyl groups, while the difference between the two strains is related to a difference between N/P surface concentration ratios, reflecting the relative exposure of proteins and phosphate groups at the surface.

Electrochemistry↗
PubMed1996

Cloning vectors for lactococci based on a plasmid encoding resistance to cadmium.

An 8.8-kb plasmid (pND302) was identified in Lactococcus lacti spp lactis M71 which encodes cadmium resistance (CdR). Most of the commercial lactococcal strains tested were sensitive to cadmium. Therefore, CdR should provide a useful selectable marker for constructing cloning vectors in lactococci. pND302 was mapped with a number of restriction enzymes and found to contain a unique EcoRI site suitable for cloning. Two E. coli/L lactis shuttle cloning vectors, pND304 and pND624, were constructed by subcloning of the E. coli plasmids pBR322 and pGEM-7Zf(+) containing a 1.6-kb gene encoding nisin resistance (NisR) of lactococcal origin into the EcoRI site of pND302, separately. The E. coli DNA component of pND624 was removed and the resulting plasmid, pND625, consisted of only lactococcal DNA, expressing NisR and CdR, with two synthetic polylinkers that contain multiple restriction sites for versatile cloning. Both pND302 and pND625 can be transformed by electroporation into L. lactis LMO230 at 10(3)/micrograms DNA and maintained stably in LMO230. The results indicated that pND302 and pND625 are potential food-grade cloning vectors for lactococci.

Base Sequence↗
PubMed1997

Combined effect of bacteriocin-producing lactic acid bacteria and lactoperoxidase system activation on Listeria monocytogenes in refrigerated raw milk.

The bactericidal activity of three bacteriocin-producing lactic acid bacteria alone and in combination with milk lactoperoxidase (LP) system activation against Listeria monocytogenes in refrigerated raw milk was studied. After 4 d at 4 degrees C, the population of L. monocytogenes in milk inoculated with bacteriocin-producing Lactococcus lactis subsp. lactis ATCC 11454, L. lactis subsp. lactis ESI 515 or Enterococcus faecalis INIA 4 was reduced by 0.21-0.24 log units. Activation of the LP system did not enhance inhibition at this temperature. After 4 d at 8 degrees C, L. monocytogenes levels in the non-activated LP system milk inoculated with L. lactis subsp. lactis ATCC 11454, L. lactis subsp. lactis ESI 515 or Ent. faecalis INIA 4 were reduced by 1.87, 1.54 and 1.11 log units compared to control milk, whereas in the activated LP system milk, this reduction was 1.99, 2.10 and 1.06, respectively. The higher nisin production by L. lactis subsp. lactis ESI 515 in milk with activated LP system than in non-activated LP system milk was responsible for the more pronounced decrease of L. monocytogenes counts in the former.

Animals↗
PubMed2001

Hop resistance in the beer spoilage bacterium Lactobacillus brevis is mediated by the ATP-binding cassette multidrug transporter HorA.

Lactobacillus brevis is a major contaminant of spoiled beer. The organism can grow in beer in spite of the presence of antibacterial hop compounds that give the beer a bitter taste. The hop resistance in L. brevis is, at least in part, dependent on the expression of the horA gene. The deduced amino acid sequence of HorA is 53% identical to that of LmrA, an ATP-binding cassette multidrug transporter in Lactococcus lactis. To study the role of HorA in hop resistance, HorA was functionally expressed in L. lactis as a hexa-histidine-tagged protein using the nisin-controlled gene expression system. HorA expression increased the resistance of L. lactis to hop compounds and cytotoxic drugs. Drug transport studies with L. lactis cells and membrane vesicles and with proteoliposomes containing purified HorA protein identified HorA as a new member of the ABC family of multidrug transporters.

ATP-Binding Cassette Transporters↗
PubMed2006

CopY-like copper inducible repressors are putative 'winged helix' proteins.

CopY of Enterococcus hirae is a well characterized copper-responsive repressor involved in copper homeostasis. In the absence of copper, it binds to the promoter. In high copper, the CopZ copper chaperone donates copper to CopY, thereby releasing it from the promoter and allowing transcription of the downstream copper homeostatic genes of the cop operon. We here show that the CopY-like repressors from E. hirae, Lactococcus lactis, and Streptococcus mutans have similar affinities not only for their native promoters, but also for heterologous cop promoters. CopZ of L. lactis accelerated the release of CopY from the promoter, suggesting that CopZ of L. lactis acts as copper chaperone, similar to CopZ in E. hirae. The consensus binding motif of the CopY-like repressors was shown to be TACAxxTGTA. The same binding motif is present in promoters controlled by BlaI of Bacillus licheniformis, MecI of Staphylococcus aureus and related repressors. BlaI and MecI have known structures and belong to the family of 'winged helix' proteins. In the N- terminal domain, they share significant sequence similarity with CopY of E. hirae. Moreover, they bind to the same TACAxxTGTA motif. NMR analysis of the N-terminal DNA binding domain of CopY of L. lactis showed that it contained the same alpha-helical content like the same regions of BlaI and MecI. These findings suggest that the DNA binding domains of CopY-like repressors are also of the 'winged helix' type.

Amino Acid Sequence↗
PubMed1996

A general system for generating unlabelled gene replacements in bacterial chromosomes.

A general system is described that facilitates gene replacements such that the recombinant strains are not labelled with antibiotic resistance genes. The method is based on the conditional replication of derivatives of the lactococcal plasmid pWV01, which lacks the repA gene encoding the replication initiation protein. Replacement vectors can be constructed in and isolated from gram-positive and gram-negative helper strains that provide RepA in trans. Cointegrate formation of the integration vectors with the chromosome of the target strain is selected by antibiotic resistance. Resolution of the cointegrate structure is identified in the second step of the procedure by the loss of the lacZ reporter gene present in the delivery vector. The second recombination event results either in gene replacement or in restoration of the original copy of the gene. As no antibiotic resistance marker is present in the genome of the mutant the system can be used to introduce multiple mutations in one strain. A feasibility study was performed using Lactococcus lactis and Bacillus subtilis as model organisms. The results indicate that the method should be applicable to any non-essential gene in numerous bacterial species.

Bacillus subtilis↗
PubMed1993

Carnocin UI49, a potential biopreservative produced by Carnobacterium piscicola: large scale purification and activity against various gram-positive bacteria including Listeria sp.

This paper describes a simple purification method for the purification of carnocin UI49, a potential biopreservative produced by Carnobacterium piscicola UI49. The protocol was also applicable for the isolation of nisin Z, which is a biopreservative produced by Lactococcus lactis SIK-83. The protocol consists of only two purification steps, XAD chromatography and cation exchange chromatography. It is quick, easy, and can be used for large scale purification of these lantibiotics. The bactericidal activity of carnocin UI49 against carnobacteria, lactococci and Listeria was compared with that of nisin Z. The carnobacteria showed similar sensitivity towards carnocin UI49 and nisin. The nisin producing L. lactis strains were very sensitive towards carnocin UI49, while the non-producing L. lactis strains were more sensitive to nisin. The Listeria strains were weakly sensitive to carnocin UI49, lower concentrations of nisin were needed to inhibit growth.

Animals↗
PubMed1991

Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1.

The nucleotide sequence of the Lactococcus lactis broad-host-range plasmid pWVO1, replicating in both gram-positive and gram-negative bacteria, was determined. This analysis revealed four open reading frames (ORFs). ORF A appeared to encode a trans-acting 26.8-kDa protein (RepA), necessary for replication. The ORF C product was assumed to play a regulatory role in replication. Both RepA and the ORF C product showed substantial sequence similarity with the Rep proteins of the streptococcal plasmid pLS1. In addition, the plus origin of replication was identified on the basis of strong similarity with the plus origin of pLS1. Derivatives of pWVO1 produced single-stranded (ss) DNA in Bacillus subtilis and L. lactis, suggesting that this plasmid uses the rolling-circle mode of replication. In B. subtilis, but not in L. lactis, the addition of rifampicin resulted in increased levels of ssDNA, indicating that in the former organism the host-encoded RNA polymerase is involved in the conversion of the ssDNA to double-stranded plasmid DNA (dsDNA). Apparently, in L. lactis the conversion of ss to ds pWVO1 DNA occurs by a mechanism which does not require the host RNA polymerase.

Amino Acid Sequence↗
PubMed2004

Conjugation mediates transfer of the Ll.LtrB group II intron between different bacterial species.

Some self-splicing group II introns (ribozymes) are mobile retroelements. These retroelements, which can insert themselves into cognate intronless alleles or ectopic sites by reverse splicing, are thought to be the evolutionary progenitors of the widely distributed eukaryotic spliceosomal introns. Lateral or horizontal transmission of introns (i.e. between species), although never experimentally demonstrated, is a well-accepted model for intron dispersal and evolution. Horizontal transfer of the ancestral bacterial group II introns may have contributed to the dispersal and wide distribution of spliceosomal introns present in modern eukaryotic genomes. Here, the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis was used as a model system to address the dissemination of introns in the bacterial kingdom. We report the first experimental demonstration of horizontal transfer of a group II intron. We show that the Ll.LtrB group II intron, originally discovered on an L. lactis conjugative plasmid (pRS01) and within a chromosomally located sex factor in L. lactis 712, invades new sites using both retrohoming and retrotransposition pathways after its transfer by conjugation. Ll.LtrB lateral transfer is shown among different L. lactis strains (intraspecies) (retrohoming and retrotransposition) and between L. lactis and Enterococcus faecalis (interspecies) (retrohoming). These results shed light on long-standing questions about intron evolution and propagation, and demonstrate that conjugation is one of the mechanisms by which group II introns are, and probably were, broadly disseminated between widely diverged organisms.

Base Sequence↗