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At least 1,423 records · Page 79Linked to original sources
PubMed2003

The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities.

LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis that is a structural and functional homologue of the human multidrug resistance P-glycoprotein MDR1 (ABCB1). LmrA is also homologous to MsbA, an essential ABC transporter in Escherichia coli involved in the trafficking of lipids, including Lipid A. We have compared the substrate specificities of LmrA and MsbA in detail. Surprisingly, LmrA was able to functionally substitute for a temperature-sensitive mutant MsbA in E. coli WD2 at non-permissive temperatures, suggesting that LmrA could transport Lipid A. LmrA also exhibited a Lipid A-stimulated, vanadate-sensitive ATPase activity. Reciprocally, the expression of MsbA conferred multidrug resistance on E. coli. Similar to LmrA, MsbA interacted with photoactivatable substrate [3H]azidopine, displayed a daunomycin, vinblastine, and Hoechst 33342-stimulated vanadate-sensitive ATPase activity, and mediated the transport of ethidium from cells and Hoechst 33342 in proteoliposomes containing purified and functionally reconstituted protein. Taken together, these data demonstrate that MsbA and LmrA have overlapping substrate specificities. Our observations imply the presence of structural elements in the recently published crystal structures of MsbA in E. coli and Vibrio cholera (Chang, G., and Roth, C. B. (2001) Science 293, 1793-1800; Chang, G. (2003) J. Mol. Biol. 330, 419-430) that support drug-protein interactions and suggest a possible role for LmrA in lipid trafficking in L. lactis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Cloning and sequencing of the gene for a lactococcal endopeptidase, an enzyme with sequence similarity to mammalian enkephalinase.

The gene specifying an endopeptidase of Lactococcus lactis, named pepO, was cloned from a genomic library of L. lactis subsp. cremoris P8-2-47 in lambda EMBL3 and was subsequently sequenced. pepO is probably the last gene of an operon encoding the binding-protein-dependent oligopeptide transport system of L. lactis. The inferred amino acid sequence of PepO showed that the lactococcal endopeptidase has a marked similarity to the mammalian neutral endopeptidase EC 3.4.24.11 (enkephalinase), whereas no obvious sequence similarity with any bacterial enzyme was found. By means of gene disruption, a pepO-negative mutant was constructed. Growth and acid production of the mutant strain in milk were not affected, indicating that the endopeptidase is not essential for growth of L. lactis in milk.

Acids↗
PubMed1993

Sequence analysis of the lysin gene region of the prolate lactococcal bacteriophage c2.

Approximately 80% of the genome of the prolate-headed lactococcal bacteriophage c2 was cloned into shuttle vectors pSA3 and pFX3 in Escherichia coli and transferred to Lactococcus lactis. A 1.67-kilobase EcoRV fragment containing the gene for the phage lysin was identified and the position and orientation of the phage lysin gene in the physical map of the phage were determined. The phage lysin was expressed in E. coli and its sequence was determined and compared with the sequences of other bacteriophage lytic genes. The sequence was similar, but not identical, to that of the related lactococcal phage m13, having a number of silent substitutions and an apparent deletion that altered the carboxy terminus of the protein. Possible alternative translation initiation codons for the lysin gene and two possible alternative mechanisms for access of the lysin enzyme to the cell wall are discussed. An open reading frame upstream of the putative lysin gene was found to be 177 base pairs longer than that reported for phage m13. A codon usage table for the lysin genes of several phages as well as for reported gene sequences from L. lactis and lactococcal bacteriophages is presented.

Amino Acid Sequence↗
PubMed2005

pIH01, a small cryptic plasmid from Leuconostoc citreum IH3.

A small cryptic plasmid pIH01 from Leuconostoc citreum IH3 was characterized. This 1.8-kb sized plasmid contains single open reading frame that encodes a RepC class protein (342 amino acids) and a conserved pT181-type double strand origin, suggesting a rolling circle replication mode. This putative replicase protein shows the highest similarity to a replicase from pFR18 plasmid of Leuconostoc mesenteroides FR52 (64% identity), one of the pT181-type rolling circle plasmid family and contains a strictly conserved RepC-type active site sequence of pT181 family. A shuttle vector that was developed on the basis of this cryptic plasmid by insertion of both erythromycin resistance gene (ermC) from pE194 and Escherichia coli ColE1 origin was able to transform Leuconostoc strains, Lactobacillus plantarum, and Lactococcus lactis. Therefore, pIH01 derivative plasmids might be useful for the manipulation of Leuconostoc strains.

Amino Acid Sequence↗
PubMed2004

Identification, characterisation and specificity of a cell wall lytic enzyme from Lactobacillus fermentum BR11.

Screening of a genomic library with an antiserum raised against whole Lactobacillus fermentum BR11 cells identified a clone expressing an immunoreactive 37-kDa protein. Analysis of the 3010-bp DNA insert contained within the clone revealed four open reading frames (ORFs). One ORF encodes LysA, a 303 amino acid protein which has up to 35% identity with putative endolysins from prophages Lj928 and Lj965 from Lactobacillus johnsonii and Lp1 and Lp2 from Lactobacillus plantarum as well as with the endolysin of Lactobacillus gasseri bacteriophage Phiadh. The immunoreactive protein was shown to be encoded by a truncated ORF downstream of lysA which has similarity to glutamyl-tRNA synthetases. The N-terminus of LysA has sequence similarity with N-acetylmuramidase catalytic domains while the C-terminus has sequence similarity with putative cell envelope binding bacterial SH3b domains. C-terminal bacterial SH3b domains were identified in the majority of Lactobacillus bacteriophage endolysins. LysA was expressed in Escherichia coli and unusually was found to have a broad bacteriolytic activity range with activity against a number of different Lactobacillus species and against Lactococcus lactis, streptococci and Staphylococcus aureus. It was found that LysA is 2 and 8000 times more active against L. fermentum than L. lactis and Streptococcus pyogenes, respectively.

Amino Acid Sequence↗
PubMed2000

Flavor enhancement of reduced fat cheddar cheese using an integrated culturing system.

Mild cheese flavor in reduced fat Cheddar cheese was enhanced by using an integrated starter culture system. Three cultures, Lactococcus lactis subsp. cremoris SK11, L. lactis subsp. lactis biovar. diacetylactis JVI, and Lactobacillus casei 7A, were carefully selected to obtain a nonbitter, mildly acid, buttery flavored cheese. Cheeses were produced from all possible combinations of these cultures with the constraint that L. lactis subsp. cremoris SK11 was used as the primary acid-producing culture. Cheeses made with SK11 were compared to cheeses produced using an L. lactis subsp. cremoris commercial starter culture. Cheeses were ripened for 150 days and periodically sampled for chemical, microbiological, and sensory analysis. Cheeses produced with L. lactis subsp. cremorisSK11 had substantially lower bitterness intensity than the cheeses produced with commercial starter culture. L. lactis subsp. lactis biovar. diacetylactis JVI significantly increased diacetylacetoin and acetate concentrations. Sensory results indicate that these cheeses had increased buttery (diacetyl) flavor.

Cheese↗
PubMed1999

Novel polymer-polymer conjugates for recovery of lactic acid by aqueous two-phase extraction.

A new family of polymer conjugates is proposed to overcome constraints in the applicability of aqueous two-phase systems for the recovery of lactic acid. Polyethylene glycol-polyethylenimine (PEI) conjugates and ethylene oxide propylene oxide-PEI (EOPO-PEI) conjugates were synthesized. Aqueous two-phase systems were generated when the conjugates were mixed with fractionated dextran or crude hydrolyzed starch. With 2% phosphate buffer in the systems, phase diagrams with critical points of 3.9% EOPO-PEI-3.8% dextran (DEX) and 3.5% EOPO-PEI-7.9% crude starch were obtained. The phase separation temperature of 10% EOPO-PEI solutions titrated with lactic acid to pH 6 was 35 degrees C at 5% phosphate, and increased linearly to 63 degrees C at 2% phosphate. Lactic acid partitioned to the top conjugate-rich phase of the new aqueous two-phase systems. In particular, the lactic acid partition coefficient was 2.1 in 10% EOPO-PEI-8% DEX systems containing 2% phosphate. In the same systems, the partitioning of the lactic acid bacterium, Lactococcus lactis subsp. lactis, was 0.45. The partitioning of propionic, succinic, and citric acids was also determined in the new aqueous two-phase systems.

Biotechnology↗
PubMed1995

Lactococcal bacteriocins: mode of action and immunity.

Bacteriocins are antimicrobial peptides produced by bacteria. Some of those synthesized by Lactococcus lactis have been characterized in great detail recently. The lactococcal bacteriocins are hydrophobic cationic peptides, which form pores in the cytoplasmic membrane of sensitive cells.

Amino Acid Sequence↗
PubMed2003

Complete DNA sequence and analysis of two cryptic plasmids isolated from Lactobacillus plantarum.

The complete nucleotide sequence of two cryptic plasmids isolated from Lactobacillus plantarum strain AS1.2986 has been determined. The smaller plasmid, designated pLP2000, encodes a 37.0kDa Rep protein and has a 17bp sequence repeated 10 times. Sequence analysis of the larger plasmid, designated pLP9000, revealed nine putative open reading frames (ORFs). Based on sequence similarity, ORF1 codes for a putative magnesium transporter protein that shows similarities to CorA from plasmid pCIS3 (Lactococcus lactis). None of the nine ORFs shows similarity to any known Rep protein. Southern blot analysis indicates these two plasmids both replicate via a rolling circle (RC) mechanism.

Genetic Variation↗
PubMed1993

Theta replication of the lactococcal plasmid pWVO2.

pWVO2 is a 3.8 kb narrow-host-range plasmid from Lactococcus lactis ssp. cremoris Wg2, which does not replicate in Bacillus subtilis or Escherichia coli. Single-stranded pWVO2 DNA was not observed in lactococcal cells, indicating that this plasmid does not replicate via a rolling-circle mechanism. The sequence of pWVO2 neither showed the structural organization typical for rolling-circle plasmids, nor were sequence similarities with known rolling-circle plasmids present. By 2-D agarose gel electrophoresis of replication intermediates, it was shown that pWVO2 replicates via a theta mechanism. This is the first proof for the existence of theta-replicating plasmids in lactococci. The pWVO2 minimal replicon is strongly related to that of several other lactococcal plasmid replicons. It contains one open reading frame encoding the replication protein, which is preceded by a 22 bp sequence tandemly repeated three and a half times. Further upstream is another 10 bp direct repeat present in an A/T-rich sequence. This structural organization resembles that of several iteron-containing theta-type plasmids from E. coli. Derivatives of pWVO2 were stably maintained in L. lactis and are good candidates for the development of stable food-grade cloning vectors for this organism.

Amino Acid Sequence↗
PubMed1998

The site-specific recombination system of the Lactobacillus species bacteriophage A2 integrates in gram-positive and gram-negative bacteria.

The region of the bacteriophage A2 genome involved in site-specific recombination with the DNA of Lactobacillus spp. has been identified. Two orfs, transcribed from the same strand, have been found immediately upstream of the phage attachment site (attP). The orf adjacent to attP predicts a 385-amino-acid protein that presents significant similarity with site-specific recombinases of the integrase family. The other orf encodes a basic polypeptide of 76 amino acid residues. The junctions of the prophage with the genomes of its hosts have been determined, allowing the identification of the host attachment site (attB), which has a common 19-nucleotide core region with attP. The attB site is located at the 3' end of the transfer RNALeu gene (anticodon CAA). Nonreplicative plasmids containing the A2-specific recombination cassette integrate into different lactobacilli but also into unrelated Gram-positive bacteria such as Lactococcus lactis and even into Escherichia coli. In Lc. lactis, integration occurs in a previously unknown intergenic region, whereas in E. coli, it maps within the rrnD operon, 5' of rrsD gene. Comparison of the integration sites in the different hosts indicates that some flexibility is permitted in the attB sequence, since Lc. lactis and E. coli only share 13 and 11 nucleotides, respectively, with the 19-nucleotide core sequence of the lactobacilli.

Amino Acid Sequence↗
PubMed2002

Analysis and practical implementation of a model for combined growth and metabolite production of lactic acid bacteria.

Next to the traditional application of lactic acid bacteria (LAB) as starter cultures for food fermentations, the use of LAB as protective cultures against microbial pathogens and spoilage organisms in other food production processes gains more and more interest. The inhibitory effect of LAB is mainly accomplished through formation of antimicrobial metabolites. In this paper, the model of Nicolaï et al. [Food Microbiol. 10 (1993) 229.], describing cell growth and production of lactic acid, which is the major end-product of LAB metabolism, is investigated. In contrast to classical predictive models, the transition of the exponential growth phase to the stationary phase is obtained through the increasing concentrations of undissociated lactic acid [LaH] and decreasing pH in the environment. To describe the variation in time of [LaH] and pH, a novel, robust calculation method is introduced. The model of Nicolaï et al. in combination with the novel method of [LaH] and pH computation is then further applied to an experimental data set of Lactococcus lactis SL05 grown in a rich medium. An accurate description of the measured values of cell concentration, total lactic acid concentration and pH is obtained.

Cell Division↗
PubMed2003

Metabolic pathway engineering in lactic acid bacteria.

Lactic acid bacteria (LAB) display a relatively simple carbon and energy metabolism where the sugar source is converted mainly to lactic acid. In Lactococcus lactis metabolic engineering has been very successful in the re-routing of lactococcal pyruvate metabolism to products other than lactic acid. Current metabolic engineering approaches tend to focus on more complex, biosynthetic pathways leading to end-products that generate a health benefit for the consumer (nutraceuticals). Several examples of research on these minor pathways in L. lactis have illustrated the potential of LAB as producers of these metabolites. Whole genome sequencing efforts and corresponding global technologies will have an impact on metabolic engineering in the future.

Bacterial Proteins↗
PubMed2003

Construction of the mobilizable plasmid pMV158GFP, a derivative of pMV158 that carries the gene encoding the green fluorescent protein.

Plasmid pMV158 has been employed to construct cloning non-mobilizable vectors for various Gram-positive organisms. Here we report the construction of a mobilizable pMV158-based plasmid that harbors the gene encoding the green fluorescent protein under the control of a promoter inducible by maltose. The plasmid was mobilized between strains of Streptococcus pneumoniae as well as from S. pneumoniae to Lactococcus lactis or Enterococcus faecalis at the same frequency as its parental. Transconjugant that received the GFP-tagged plasmid could be detected by their fluorescence, which was especially high in E. faecalis cells.

Conjugation, Genetic↗
PubMed1994

Use of the Escherichia coli beta-glucuronidase (gusA) gene as a reporter gene for analyzing promoters in lactic acid bacteria.

A transcriptional fusion vector, designated pNZ272, based on the promoterless beta-glucuronidase gene (gusA) of Escherichia coli as a reporter gene, has been constructed for lactic acid bacteria. The replicon of pNZ272 was derived from the Lactococcus lactis plasmid pSH71, allowing replication in a wide range of gram-positive bacteria and E. coli. The applicability of pNZ272 and the expression of the gusA gene in L. lactis was demonstrated in shotgun cloning experiments with lactococcal chromosomal and bacteriophage DNA. In addition, three defined lactococcal promoters were inserted in pNZ272: the plasmid-derived lacA promoter, the chromosomal usp45 promoter, and a promoter from bacteriophage phi SK11G. The three resulting plasmids showed beta-glucuronidase activity in a gusA-deficient E. coli strain and in four species of lactic acid bacteria belonging to the genera Lactobacillus, Lactococcus, and Leuconostoc. The copy numbers of the gusA-expressing plasmids were similar within a single species of lactic acid bacteria. However, the specific beta-glucuronidase activity and the gusA mRNA levels varied considerably both within a single species and among different species of lactic acid bacteria. The transcriptional start site of all three promoters was determined and found to be identical in the different species. The results of this comparative promoter analysis indicate that the requirements for efficient transcription initiation differ among the lactic acid bacteria studied.

Amino Acid Sequence↗
PubMed1995

Characterization and distribution of two insertion sequences, IS1191 and iso-IS981, in Streptococcus thermophilus: does intergeneric transfer of insertion sequences occur in lactic acid bacteria co-cultures?

A chromosomal repeated sequence from Streptococcus thermophilus was identified as a new insertion sequence (IS), IS1191. This is the first IS element characterized in this species. This 1313 bp element has 28 bp imperfect terminal inverted repeats and is flanked by short direct repeats of 8 bp. The single large open reading frame of IS1191 encodes a 391-amino-acid protein which displays homologies with transposases encodes by IS1201 from Lactobacillus helveticus (44.5% amino-acid sequence identity) and by the other ISs of the IS256 family. One of the copies of IS1191 is inserted into a truncated iso-IS981 element. The nucleotide sequences of two truncated iso-IS981s from S. thermophilus and the sequence of IS981 element from Lactococcus lactis share more than 99% identity. The distribution of these insertion sequences in L. lactis and S. thermophilus strains suggests that intergeneric transfers occur during cocultures used in the manufacture of cheese.

Bacteria↗
PubMed1999

Histidinol phosphate phosphatase, catalyzing the penultimate step of the histidine biosynthesis pathway, is encoded by ytvP (hisJ) in Bacillus subtilis.

The deduced product of the Bacillus subtilis ytvP gene is similar to that of ORF13, a gene of unknown function in the Lactococcus lactis histidine biosynthesis operon. A B. subtilis ytvP mutant was auxotrophic for histidine. The only enzyme of the histidine biosynthesis pathway that remained uncharacterized in B. subtilis was histidinol phosphate phosphatase (HolPase), catalyzing the penultimate step of this pathway. HolPase activity could not be detected in crude extracts of the ytvP mutant, while purified glutathione S-transferase-YtvP fusion protein exhibited strong HolPase activity. These observations demonstrated that HolPase is encoded by ytvP in B. subtilis and led us to rename this gene hisJ. Together with the HolPase of Saccharomyces cerevisiae and the presumed HolPases of L. lactis and Schizosaccharomyces pombe, HisJ constitutes a family of related enzymes that are not homologous to the HolPases of Escherichia coli, Salmonella typhimurium, and Haemophilus influenzae.

Amino Acid Sequence↗
PubMed2005

Comparative and functional genomics of lactococci.

Whole-genome nucleotide sequencing has revolutionized the genetic, biochemical and molecular biology research on bacteria and indeed, many higher organisms. The genome sequences of the strains of two subspecies of Lactococcus lactis, L. lactis subsp. lactis and L. lactis subsp. cremoris, have been determined. These genomic sequences have permitted two important new approaches to be applied in the research of L. lactis. The analysis of the regulation of expression of all genes under specific circumstances at a given point in time is now possible by DNA microarray technology. The elucidation of the full protein complement of the organism as a function of intrinsic or external factors has been made possible by high-throughput protein identification and analysis techniques combined with the gene-derived know-how of the total protein encoding capacity of the genome. These techniques from the genomics arena, transcriptomics and proteomics, have been recently implemented in the study of various aspects of growth and functioning of L. lactis. In this paper we discuss a number of similarities and differences between the two lactococcal genome sequences and review the current status of genomics research in L. lactis. We also propose future directions with respect to both answering fundamental questions more quickly and more completely, as well as opening new avenues for biotechnological applications.

DNA Transposable Elements↗