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PubMed2003

Diversity of oligopeptide transport specificity in Lactococcus lactis species. A tool to unravel the role of OppA in uptake specificity.

The specific oligopeptide transport system Opp is essential for growth of Lactococcus lactis in milk. We examined the biodiversity of oligopeptide transport specificity in the L. lactis species. Six strains were tested for (i) consumption of peptides during growth in a chemically defined medium and (ii) their ability to transport these peptides. Each strain demonstrated some specific preferences for peptide utilization, which matched the specificity of peptide transport. Sequencing of the binding protein OppA in some strains revealed minor differences at the amino acid level. The differences in specificity were used as a tool to unravel the role of the binding protein in transport specificity. The genes encoding OppA in four strains were cloned and expressed in L. lactis MG1363 deleted for its oppA gene. The substrate specificity of these engineered strains was found to be similar to that of the L. lactis MG1363 parental strain, whichever oppA gene was expressed. In situ binding experiments demonstrated the ability of OppA to interact with non-transported peptides. Taken together, these results provide evidence for a new concept. Despite that fact that OppA is essential for peptide transport, it is not the (main) determinant of peptide transport specificity in L. lactis.

ATP-Binding Cassette Transporters↗
PubMed2003

Thr-431 and Arg-433 are part of a conserved sequence motif of the glutamine amidotransferase domain of CTP synthases and are involved in GTP activation of the Lactococcus lactis enzyme.

A conserved sequence motif within the class 1 glutamine amidotransferase (GATase) domain of CTP synthases was identified. The sequence motif in the Lactococcus lactis enzyme is (429)GGTLRLG(435). This motif was present only in CTP synthases and not in other enzymes that harbor the GATase domain. Therefore, it was speculated that this sequence was involved in GTP activation of CTP synthase. Other members of the GATase protein family are not activated allosterically by GTP. Residues Thr-431 and Arg-433 were changed by site directed mutagenesis to the sterically similar residues valine and methionine, respectively. The resulting enzymes, T431V and R433M, had both lost the ability for GTP to activate the uncoupled glutaminase activity and showed reduced GTP activation of the glutamine-dependent CTP synthesis reaction. The T431V enzyme had a similar activation constant, K(A), for GTP, but the activation was only 2-3-fold compared with 35-fold for the wild type enzyme. The R433M enzyme was found to have a 10-15-fold lower K(A) for GTP and a concomitant decrease in V(app). The activation by GTP of this enzyme was about 7-fold. The kinetic parameters for saturation with ATP, UTP, and NH(4)Cl were similar for wild type and mutant enzymes, except that the R433M enzyme only had half the V(app) of the wild type enzyme when NH(4)Cl was the amino donor. The mutant enzymes T431V and R433M apparently had not lost the ability to bind GTP, but the signal transmitted through the enzyme to the active sites upon binding of the allosteric effector was clearly disrupted in the mutant enzymes.

Adenosine Triphosphate↗
PubMed2003

Sterol transport by the human breast cancer resistance protein (ABCG2) expressed in Lactococcus lactis.

The human breast cancer resistance protein (BCRP, also know as ABCG2, MXR, or ABCP) is one of the more recently discovered ATP-binding cassette (ABC) transporters that confer resistance on cancer cells by mediating multidrug efflux. In the present study, we have obtained functional expression of human BCRP in the Gram-positive bacterium Lactococcus lactis. BCRP expression conferred multidrug resistance on the lactococcal cells, which was based on ATP-dependent drug extrusion. BCRP-mediated ATPase and drug transport activities were inhibited by the BCRP-specific modulator fumitremorgin C. To our knowledge these data represent the first example of the functional expression of a mammalian ABC half-transporter in bacteria. Although members of the ABCG subfamily (such as ABCG1 and ABCG5/8) have been implicated in the transport of sterols, such a role has not yet been established for BCRP. Interestingly, the BCRP-associated ATPase activity in L. lactis was significantly stimulated by (i) sterols including cholesterol and estradiol, (ii) natural steroids such as progesterone and testosterone, and (iii) the anti-estrogen anticancer drug tamoxifen. In addition, BCRP mediated the efflux of [3H]estradiol from lactococcal cells. Our findings suggest that BCRP may play a role in the transport of sterols in human, in addition to its ability to transport multiple drugs and toxins.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2003

Lactococcus lactis dihydroorotate dehydrogenase A mutants reveal important facets of the enzymatic function.

Dihydroorotate dehydrogenases (DHODs) are flavoenzymes catalyzing the oxidation of (S)-dihydroorotate to orotate in the biosynthesis of UMP, the precursor of all other pyrimidine nucleotides. On the basis of sequence, DHODs can be divided into two classes, class 1, further divided in subclasses 1A and 1B, and class 2. This division corresponds to differences in cellular location and the nature of the electron acceptor. Herein we report a study of Lactococcus lactis DHODA, a representative of the class 1A enzymes. Based on the DHODA structure we selected seven residues that are highly conserved between both main classes of DHODs as well as three residues representing surface charges close to the active site for site-directed mutagenesis. The availability of both kinetic and structural data on the mutant enzymes allowed us to define the roles individual structural segments play in catalysis. We have also structurally proven the presence of an open active site loop in DHODA and obtained information about the interactions that control movements of loops around the active site. Furthermore, in one mutant structure we observed differences between the two monomers of the dimer, confirming an apparent asymmetry between the two substrate binding sites that was indicated by the kinetic results.

Binding Sites↗
PubMed2004

ydaG and ydbA of Lactococcus lactis encode a heterodimeric ATP-binding cassette-type multidrug transporter.

Multidrug resistance (MDR)-type transporters mediate the active extrusion of structurally and functionally dissimilar compounds from the cells, thereby rendering cells resistant to a range of drugs. The ydaG and ydbA genes of Lactococcus lactis encode two ATP-binding cassette half-transporters, which both share homology with MDR proteins such as LmrA from L. lactis or the mammalian P-glycoprotein. The ydaG/ydbA genes were cloned and expressed separately and jointly in L. lactis using the nisin-inducible system. When both proteins are co-expressed, several structurally dissimilar drugs such as ethidium, daunomycin, and BCECF-AM are extruded from the cell. YdaG and YdbA could be co-purified as a stable heterodimer. ATPase activity was found to be associated with the YdaG/YdbA heterodimer only and not with the individual subunits. Both the ydaG and ydbA genes are up-regulated in multidrug-resistant L. lactis strains selected for growth in the presence of a variety of toxic compounds. This is the first demonstration of a functional heterodimeric ATP-binding cassette-type MDR transporter.

ATP-Binding Cassette Transporters↗
PubMed2004

Lactococcus lactis uses MscL as its principal mechanosensitive channel.

The functions of the mechanosensitive channels from Lactococcus lactis were determined by biochemical, physiological, and electrophysiological methods. Patch-clamp studies showed that the genes yncB and mscL encode MscS and MscL-like channels, respectively, when expressed in Escherichia coli or if the gene products were purified and reconstituted in proteoliposomes. However, unless yncB was expressed in trans, wild type membranes of L. lactis displayed only MscL activity. Membranes prepared from an mscL disruption mutant did not show any mechanosensitive channel activity, irrespective of whether the cells had been grown on low or high osmolarity medium. In osmotic downshift assays, wild type cells survived and retained 20% of the glycine betaine internalized under external high salt conditions. On the other hand, the mscL disruption mutant retained 40% of internalized glycine betaine and was significantly compromised in its survival upon osmotic downshifts. The data strongly suggest that L. lactis uses MscL as the main mechanosensitive solute release system to protect the cells under conditions of osmotic downshift.

Amino Acid Sequence↗
PubMed2005

Interaction between ArgR and AhrC controls regulation of arginine metabolism in Lactococcus lactis.

The expression of arginine metabolism in Lactococcus lactis is controlled by the two homologous transcriptional regulators ArgR and AhrC. Genome sequence analyses have shown that the occurrence of multiple homologues of the ArgR family of transcriptional regulators is a common feature of many low-G + C Gram-positive bacteria. Detailed studies of ArgR type regulators have previously only been carried out in bacteria containing single regulators. Here, we present a first characterization of the two L. lactis arginine regulators by means of gel retardation and DNase I footprinting. ArgR of L. lactis was shown to bind to the promoter regions of both the arginine biosynthetic argCJDBF operon and the arginine catabolic arcABD1C1C2TD2yvaD operon, but in an arginine-independent manner. Surprisingly, AhrC alone was unable to bind to DNA. Arginine-dependent DNA binding was obtained by mixing the two regulators in gel retardation assays. With both regulators present, the addition of arginine led to increased binding of ArgR-AhrC to the biosynthetic argC promoter but also to diminished binding to the catabolic arcA promoter. Footprinting showed ArgR-AhrC protection of regions containing ARG box operator sequences preceding argC. In the absence of AhrC, ArgR protected sites in the arcA promoter region with similarity to ARG box half-sites, here called ARC boxes. We propose a model for repression of arginine biosynthesis and activation of catabolism by anti-repression, involving arginine-dependent interaction between the two L. lactis regulator proteins, ArgR and AhrC.

Arginine↗
PubMed2005

The Lactococcus lactis CodY regulon: identification of a conserved cis-regulatory element.

CodY of Lactococcus lactis MG1363 is a transcriptional regulator that represses the expression of several genes encoding proteins of the proteolytic system. DNA microarray analysis, comparing the expression profiles of L. lactis MG1363 and an isogenic strain in which codY was mutated, was used to determine the CodY regulon. In peptide-rich medium and exponentially growing cells, where CodY exerts strong repressing activity, the expression of over 30 genes was significantly increased upon removal of codY. The differentially expressed genes included those predominantly involved in amino acid transport and metabolism. In addition, several genes belonging to other functional categories were derepressed, stressing the pleiotropic role of CodY. Scrutinizing the transcriptome data with bioinformatics tools revealed the presence of a novel over-represented motif in the upstream regions of several of the genes derepressed in L. lactis MG1363DeltacodY. Evidence is presented that this 15-bp cis-sequence, AATTTTCWGAAAATT, serves as a high affinity binding site for CodY, as shown by electrophoretic mobility shift assays and DNase I footprinting analyses. The presence of this CodY-box is sufficient to evoke CodY-mediated regulation in vivo. A copy of this motif is also present in the upstream region of codY itself. It is shown that CodY regulates its own synthesis and requires the CodY-box and branched-chain amino acids to interact with its promoter.

Bacterial Proteins↗
PubMed2005

Role of mRNA stability during genome-wide adaptation of Lactococcus lactis to carbon starvation.

The stability of mRNA was investigated for the first time at the genomic scale during carbon starvation adaptation of Lactococcus lactis IL1403. In exponential phase, mRNA half-lives were correlated positively to open reading frame length. A polypurine sequence, AGGAG, was identified as a putative 5'-stabilizer and inverted repeated sequences as a 3'-destabilizer. These original findings suggested that multiple pathways of mRNA degradation should coexist: internal cleavage, endonuclease cleavage initiated at the 5'-end, and exonuclease attack at the 3'-end. During carbon starvation adaptation, mRNA stability globally increased, but specific mechanisms allowing a wide range of stabilization factors between genes and differential kinetic evolution were involved. A formal method allowing the quantification of the relative influences of transcription and degradation on the mRNA pool control was developed and applied in L. lactis. Gene expression was mostly controlled by altered transcription prior to carbon source exhaustion, while the influence of mRNA stability increased during the starvation phase. This study highlighted that stability modulation in response to adverse growth conditions can govern gene regulation to the same extent as transcription in bacteria.

Carbon↗
PubMed2006

Modular structure of the receptor binding proteins of Lactococcus lactis phages. The RBP structure of the temperate phage TP901-1.

Lactococcus lactis is a gram-positive bacterium widely used by the dairy industry. Several industrial L. lactis strains are sensitive to various distinct bacteriophages. Most of them belong to the Siphoviridae family and comprise several species, among which the 936 and P335 are prominent. Members of these two phage species recognize their hosts through the interaction of their receptor-binding protein (RBP) with external cell wall saccharidices of the host, the "receptors." We report here the 1.65 A resolution crystal structure of the RBP from phage TP901-1, a member of the P335 species. This RBP of 163 amino acids is a homotrimer comprising three domains: a helical N terminus, an interlaced beta-prism, and a beta-barrel, the head domain (residues 64-163), which binds a glycerol molecule. Fluorescence quenching experiments indicated that the RBP exhibits high affinity for glycerol, muramyl-dipeptide, and other saccharides in solution. The structural comparison of this RBP with that of lactococcal phage p2 RBP, a member of the 936 species (Spinelli, S., Desmyter, A., Verrips, C. T., de Haard, J. W., Moineau, S., and Cambillau, C. (2006) Nat. Struct. Mol. Biol. 13, 85-89) suggests a large extent of modularity in RBPs of lactococcal phages.

Amino Acid Sequence↗
PubMed2006

Lys-D48 is required for charge stabilization, rapid flavin reduction, and internal electron transfer in the catalytic cycle of dihydroorotate dehydrogenase B of Lactococcus lactis.

Dihydroorotate dehydrogenase B (DHODB) catalyzes the oxidation of dihydroorotate (DHO) to orotate and is found in the pyrimidine biosynthetic pathway. The Lactococcus lactis enzyme is a dimer of heterodimers containing FMN, FAD, and a 2Fe-2S center. Lys-D48 is found in the catalytic subunit and its side-chain adopts different positions, influenced by ligand binding. Based on crystal structures of DHODB in the presence and absence of orotate, we hypothesized that Lys-D48 has a role in facilitating electron transfer in DHODB, specifically in stabilizing negative charge in the reduced FMN isoalloxazine ring. We show that mutagenesis of Lys-D48 to an alanine, arginine, glutamine, or glutamate residue (mutants K38A, K48R, K48Q, and K48E) impairs catalytic turnover substantially (approximately 50-500-fold reduction in turnover number). Stopped-flow studies demonstrate that loss of catalytic activity is attributed to poor rates of FMN reduction by substrate. Mutation also impairs electron transfer from the 2Fe-2S center to FMN. Addition of methylamine leads to partial rescue of flavin reduction activity. Nicotinamide coenzyme oxidation and reduction at the distal FAD site is unaffected by the mutations. Formation of the spin-interacting state between the FMN semiquinone-reduced 2Fe-2S centers observed in wild-type enzyme is retained in the mutant proteins, consistent with there being little perturbation of the superexchange paths that contribute to the efficiency of electron transfer between these cofactors. Our data suggest a key charge-stabilizing role for Lys-D48 during reduction of FMN by dihydroorotate, or by electron transfer from the 2Fe-2S center, and establish a common mechanism of FMN reduction in the single FMN-containing A-type and the complex multicenter B-type DHOD enzymes.

Catalysis↗
PubMed2006

Regulation of the Dha operon of Lactococcus lactis: a deviation from the rule followed by the Tetr family of transcription regulators.

Dihydroxyacetone (Dha) kinases are a novel family of kinases with signaling and metabolic functions. Here we report the x-ray structures of the transcriptional activator DhaS and the coactivator DhaQ and characterize their function. DhaQ is a paralog of the Dha binding Dha kinase subunit; DhaS belongs to the family of TetR repressors although, unlike all known members of this family, it is a transcriptional activator. DhaQ and DhaS form a stable complex that in the presence of Dha activates transcription of the Lactococcus lactis dha operon. Dha covalently binds to DhaQ through a hemiaminal bond with a histidine and thereby induces a conformational change, which is propagated to the surface via a cantilever-like structure. DhaS binding protects an inverted repeat whose sequence is GGACACATN6ATTTGTCC and renders two GC base pairs of the operator DNA hypersensitive to DNase I cleavage. The proximal half-site of the inverted repeat partially overlaps with the predicted -35 consensus sequence of the dha promoter.

Amino Acid Sequence↗
PubMed2006

The alpha-phosphoglucomutase of Lactococcus lactis is unrelated to the alpha-D-phosphohexomutase superfamily and is encoded by the essential gene pgmH.

alpha-Phosphoglucomutase (alpha-PGM) plays an important role in carbohydrate metabolism by catalyzing the reversible conversion of alpha-glucose 1-phosphate to glucose 6-phosphate. Isolation of alpha-PGM activity from cell extracts of Lactococcus lactis strain MG1363 led to the conclusion that this activity is encoded by yfgH, herein renamed pgmH. Its gene product has no sequence homology to proteins in the alpha-d-phosphohexomutase superfamily and is instead related to the eukaryotic phosphomannomutases within the haloacid dehalogenase superfamily. In contrast to known bacterial alpha-PGMs, this 28-kDa enzyme is highly specific for alpha-glucose 1-phosphate and glucose 6-phosphate and showed no activity for mannose phosphate. To elucidate the function of pgmH, the metabolism of glucose and galactose was characterized in mutants overproducing or with a deficiency of alpha-PGM activity. Overproduction of alpha-PGM led to increased glycolytic flux and growth rate on galactose. Despite several attempts, we failed to obtain a deletion mutant of pgmH. The essentiality of this gene was proven by using a conditional knock-out strain in which a native copy of the gene was provided in trans under the control of the nisin promoter. Growth of this strain was severely impaired when alpha-PGM activity was below the control level. We show that the novel L. lactis alpha-PGM is the only enzyme that mediates the interconversion of alpha-glucose 1-phosphate to glucose 6-phosphate and is essential for growth.

Amino Acid Sequence↗
PubMed2004

Phylogenetic analysis of Lactococcus lactis subspecies based on decoding the sequence of the pepT tripeptidase gene, the pepV dipeptidase gene and 16S rRNA.

Tripeptidase (PepT) and dipeptidase (PepV), the enzymes located in the final stage of the intracellular proteolytic system, were demonstrated to be distributed widely in lactic acid bacteria, especially in lactococci. Both the tripeptidase genes (pepT) and dipeptidase genes (pepV) of 15 lactococcal strains consisting of the type and domestic strains were cloned and sequenced using normal and TAIL PCR methods. Amino acid sequences of these enzymes were highly conserved among strains. Evolutionary distance trees based on the sequence of 1239 nucleotides of pepT and 1416 nucleotide of pepV showed a similar cluster as that obtained from the 1499 fragment of the 16S rRNA. Based on this profile, the species Lactococcus lactis is reasonably divided into three subspecies groups, subsp. lactis, cremoris, and hordniae, as in the current classification. Figure of trees from pepT and pepV were essentially identical to each other and slightly more intricate than that from 16S rRNA. The K nuc values obtained from pepT and pepV genes were approximately ten times as high as that from 16S rRNA. Considering these results, phylogenetic analysis based on pepT and pepV genes may aid in a more precise index of classification of L. lactis subspecies. PepT and PepV seem to have evolved in similar directions in lactococci.

Amino Acid Sequence↗
PubMed2005

Lactococcus lactis is capable of improving the riboflavin status in deficient rats.

Lactococcus lactis is a commonly used starter strain that can be converted from a vitamin B2 consumer into a vitamin B2 'factory' by over-expressing its riboflavin biosynthesis genes. The present study was conducted to assess in a rat bioassay the response of riboflavin produced by GM or native lactic acid bacteria (LAB). The riboflavin-producing strains were able to eliminate most physiological manifestations of ariboflavinosis such as stunted growth, elevated erythrocyte glutathione reductase activation coefficient values and hepatomegalia that were observed using a riboflavin depletion-repletion model. Riboflavin status and growth rates were greatly improved when the depleted rats were fed with cultures of L. lactis that overproduced this vitamin whereas the native strain did not show the same effect. The present study is the first animal trial with food containing living bacteria that were engineered to overproduce riboflavin. These results pave the way for analysing the effect of similar riboflavin-overproducing LAB in human trials.

Animals↗
PubMed2002

Endocarditis caused by Lactococcus cremoris.

We describe a case of subacute endocarditis due to Lactococcus cremoris associated with consumption of unpasteurized milk. Treatment with amoxicillin-clavulanic acid and subsequently penicillin resulted in prompt sterilization of this patient's bloodstream and full recovery.

Aged↗