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Identification and characterisation of a gene encoding aminoacylase activity from Lactococcus lactis MG1363.

Analysis of the sequence of a randomly cloned chromosomal DNA fragment (3.2 kb) from Lactococcus lactis revealed the presence of part of an open reading frame, designated amd1, which specifies a protein displaying significant similarity to aminoacylases from various bacteria. The presence of an immobilised copy of an IS982 element immediately upstream of the coding region of amd1 has probably resulted in the displacement of amd1's native promoter. This genetic organisation was shown to be retained in seven other dairy strains, one of which was only slightly different. The amd1 gene was overexpressed in L. lactis NZ9800 under the control of the inducible nisA promoter and the deacetylating capacity of its gene product was measured on a number of substrates.

Amidohydrolases↗

Expression of green fluorescent protein in Lactococcus lactis.

The gfp gene from Aequorea victoria, encoding the green fluorescent protein (GFP) has been expressed in Lactococcus lactis subsp. lactis biovar cremoris MG1363, upon construction and introduction of plasmid pLS1GFP into this host. GFP was monitored in living cells during growth to evaluate its use in molecular and physiological studies. Quantification of the levels of GFP expressed by cultures was feasible by fluorescence spectroscopy. Phase-contrast and fluorescence microscopy allowed us to distinguish, in mixed cultures, lactococcal cells expressing GFP. Our results indicate that GFP can be used as a reporter in L. lactis.

Genes, Reporter↗

Novel sucrose transposons from plant strains of Lactococcus lactis.

Lactococcus lactis strains isolated from vegetable products transferred the ability to ferment sucrose in conjugation experiments with the recipient strain L. lactis MG1614. Nisin production and sucrose fermentation were transferred together from two strains, but transfer also occurred from several other strains which did not produce nisin. Pulsed-field gel electrophoresis analysis showed that all transconjugants had acquired large chromosomal insertions at two main sites. Nisin sucrose transconjugants had gained inserts of 70 kb, while those that fermented sucrose without nisin production contained inserts of between 50 and 110 kb. Transconjugants from one donor had acquired a separate insertion of 55 kb which correlated with enhanced bacteriophage resistance, but contained neither nisin nor sucrose fermentation genes.

Bacteriophages↗

Zinc uptake, oxidative stress and the FNR-like proteins of Lactococcus lactis.

Lactococcus lactis ssp. cremoris MG1363 contains two FNR homologues, FlpA and FlpB, encoded by the distal genes of two paralogous operons (orfX(A/B)-orfY(A/B)-flpA/B). An flpA flpB double mutant strain is hypersensitive to hydrogen peroxide and has a depleted intracellular Zn(II) pool. The phenotypes of the flp mutant strains suggest that FlpA and FlpB control the expression of high and low affinity ATP-dependent Zn(II) uptake systems, respectively. Plate tests revealed that expression from a orfX(B)::lac reporter was activated by Cd(II), consistent with other Zn(II)-regulated systems. The link between a failure to acquire Zn(II) and hypersensitivity to oxidative stress suggests that Zn(II) may be required to protect vulnerable protein thiols from oxidation.

Adenosine Triphosphate↗

The LlaGI restriction and modification system of Lactococcus lactis W10 consists of only one single polypeptide.

The naturally occurring 12.1-kb plasmid, pEW104, in Lactococcus lactis ssp. cremoris W10 was found to confer decreased bacteriophage sensitivity to its host. Plasmid pEW104 encodes a non-classic restriction and modification (R/M) system, named LlaGI, consisting of only one single polypeptide. Analysis of the amino acid sequence revealed the presence of a catalytic motif and seven helicase-like motifs (DEAD-box motifs) characteristic of type I and III endonucleases, followed by four conserved methylase motifs characteristic of adenine-methylases. A comparison between LlaGI and the very similar R/M system, LlaBIII, suggests that the C-terminal region of LlaGI, apparently containing no known motifs, could possibly specify target DNA recognition. Conceivably, the LlaGI gene is included in the operon of the plasmid replication machinery. Finally, it is proposed that LlaGI represents a variant of the type I R/M systems.

Amino Acid Sequence↗

Conversion of methionine to methional by Lactococcus lactis.

Lactic acid bacteria were screened for methional production from 4-methylthio-2-ketobutanoate. Only Lactococcus lactis IFPL730 produced high amounts of methional. It was demonstrated that production of this compound was an exclusively enzymatic reaction. The present work describes for the first time that L. lactis can convert enzymatically methionine to methional in a process mediated by aminotransferase and alpha-ketoacid decarboxylase activities. The activity seems to be strain dependent.

Aldehydes↗

Lactococcin MMFII, a novel class IIa bacteriocin produced by Lactococcus lactis MMFII, isolated from a Tunisian dairy product.

A novel bacteriocin, lactococcin MMFII, produced by Lactococcus lactis MMFII isolated from a Tunisian dairy product had been identified. The bacteriocin was purified to homogeneity from fresh overnight M17 broth culture by sulfate ammonium precipitation, cation-exchange chromatography, sep-pack chromatography and two steps of reverse-phase chromatography. The purified bacteriocin was heat stable, pH resistant and protease sensitive. Its amino acid sequence, obtained by Edman degradation, revealed a 37-amino acid peptide with two cysteine residues in positions 9 and 14 and a calculated mass of 4144.6 Da. Laser desorption mass spectrometry analysis gave a molecular mass of 4142.6, suggesting the presence of a disulfide bond within the purified bacteriocin. Lactococcin MMFII contains the N-terminal YGNGV consensus motif and is active against Listeria. Thus, it belongs to the class IIa bacteriocins figuring the first example of such a bacteriocin produced by a lactococcal strain.

Amino Acid Sequence↗

Characterization of genes homologous to the general stress-inducible gene gls24 in Enterococcus faecalis and Lactococcus lactis.

Three genes (Ef0604, ymgG, and ytgH) were identified as homologous to the gls24 gene (encoding the general stress protein Gls24) in Enterococcus faecalis V583 and Lactococcus lactis IL1403 genomes sequences, and therefore are part of the 'gls24 family'. The other proteins encoded by the different surrounding genes in each of their respective operons are also highly similar. Our transcriptional analysis showed that Ef0604 and ymgG are not induced under the stress conditions tested. On the other hand, ytgH is induced at the onset of glucose starvation and by heat, ethanolic, osmotic, and CdCl(2) stresses. The transcriptional start site of this operon was identified and the promoter region appears to contain an 'extended -10 Box'. Moreover, the over-expression under several stress conditions of the YtgH protein observed on 2D gel electrophoresis confirms that it corresponds to a general stress protein in L. lactis.

Bacterial Proteins↗

A distinct physiological state of Lactococcus lactis cells that confers survival against a direct and prolonged exposure to severe stresses.

When exponential phase cultures of Lactococcus lactis were directly exposed to severe stresses (acid, bile salt, heat, and hydrogen peroxide) for a prolonged period, most of the cells were quickly killed, however, a small number of the cells, approximately 0.01% of the population, was found to survive. How these 'survivor' cells might have survived the stresses, when other supposedly-the-same cells could not, was investigated. The cultures were not exposed to any mild stresses prior to the exposure to the severe stresses, and therefore adaptation can be ruled out as the cause of survival. When the survivor cells were re-cultured and re-exposed to the same severe stresses a similar pattern of survival was displayed, indicating that the survivor cells were not stress-resistant mutants. Furthermore, the survivor cells displayed typical growth kinetics once they were freed of the stresses. The survivor cells appear to be in a distinct physiological state, because when they were tested against a second stress they exhibited significantly greater survival against that stress than the normal cells exposed to the same stress. Also, cells at different time points of synchronously growing culture displayed different levels of survival against stress. It is proposed that the difference in survival of exponential phase cells is due to the difference in the protein makeup of cells at different stages of the cell cycle.

Acids↗

Lactococcus lactis AbiD1 abortive infection efficiency is drastically increased by a phage protein.

Sensitivity of phage bIL66 to the AbiD1 Lactococcus lactis abortive infection mechanism was previously shown to be determined by the phage middle-time-expressed operon composed of four orfs. Using spontaneous bIL66 mutants resistant to AbiD1, we established that this sensitivity is determined by the orf1 encoded protein. Overproduction of Orf1 in trans in AbiD1(+) cells was shown to increase AbiD1 efficiency on both wild-type phage bIL66 and mutants resistant to AbiD1. Such an increase was not observed following overproduction of mutant Orf1. We propose that wild-type, but not a mutant Orf1, activates AbiD1 expression or activity.

Amino Acid Sequence↗

Transduction of concatemeric plasmids containing the cos site of Lactococcus lactis bacteriophage sk1.

Lactococcus lactis bacteriophage sk1 can transduce plasmids containing the phage cos site and surrounding DNA sequences at frequencies as high as 2x10(-3) transductants per PFU. Deletion analysis demonstrated that the presence of phage DNA spanning cos and putative R sites were the most important for efficient plasmid transduction. Inserts of 440 bp containing cos and the R sites were sufficient to induce transduction frequencies of 10(-4) transductants per PFU. The role of the R1 site was investigated by altering 14 of the 19 bases in the site. This resulted in a two-fold decrease in transduction frequency compared to a 26-fold decrease in transduction following deletion of the entire site. It was demonstrated that transducing plasmids were packaged as linear trimeric concatemers commencing at the cos site.

Bacteriophages↗

Effect of bacteriocin-induced cell damage on the branched-chain amino acid transamination by Lactococcus lactis.

The effect of the bacteriocin lacticin 3147 on the branched-chain amino acid transamination by Lactococcus lactis IFPL359 was investigated. The bacteriocin provokes membrane permeabilisation of the cells, rendering them non-viable but metabolically active. Free diffusion of amino acids into the cell was facilitated. In addition, membrane permeabilisation promotes further cell lysis. Both facts render the enzymes more accessible to their substrates and hence increase branched-chain amino acid transamination. This research broadens the spectrum of technological applications of lacticin 3147 in the development of cheese flavour.

Amination↗

Conservation of key elements of natural competence in Lactococcus lactis ssp.

Natural competence is active in very diverse species of the bacterial kingdom and probably participates in horizontal gene transfer. Recently, the genome sequence of various species, including Lactococcus lactis, revealed the presence of homologues of competence genes in bacteria, which were not previously identified as naturally transformable. We investigated the conservation among lactococcal strains of key components of the natural competence process in streptococci: (i) comX which encodes a sigma factor, allowing the expression of the late competence genes involved in DNA uptake, (ii) its recognition site, the cin-box and (iii) dprA which encodes a protein shown to determine the fate of incoming DNA. The comX and dprA genes and the cin-box appeared conserved among strains, although some L. lactis ssp. lactis strains presented an inactivated dprA gene. We established that ComX controls the expression of the late competence genes in L. lactis. In conclusion, our work strongly suggests that ComX has the same role in streptococci and L. lactis, i.e. the regulation of late competence genes. It also allowed the identification of a set of L. lactis strains and the construction of a comX overexpression system, which should facilitate the investigation of the natural competence activity in lactococci.

Amino Acid Sequence↗

Spontaneous resistance in Lactococcus lactis IL1403 to the lantibiotic lacticin 3147.

The ability and frequency at which target organisms can develop resistance to bacteriocins is a crucial consideration in designing and implementing bacteriocin-based biocontrol strategies. Lactococcus lactis ssp. lactis IL1403 was used as a target strain in an attempt to determine the frequency at which spontaneously resistant mutants are likely to emerge to the lantibiotic lacticin 3147. Following a single exposure to lacticin 3147, resistant mutants only emerged at a low frequency (10(-8)-10(-9)) and were only able to withstand low levels of the bacteriocin (100 AU mL(-1)). However, exposure to increasing concentrations, in a stepwise manner, resulted in the isolation of eight mutants that were resistant to moderately higher levels of lacticin 3147 (up to 600 AU mL(-1)). Interestingly, in a number of cases cross-resistance to other lantibiotics such as nisin and lacticin 481 was observed, as was cross-resistance to environmental stresses such as salt. Finally, reduced adsorption of the bacteriocin in to the cell was documented for all resistant mutants.

Adsorption↗

Lactococcus garvieae infections in humans: possible association with aquaculture outbreaks.

Lactococcus garvieae is an important pathogen in aquaculture, outbreaks of which significantly affect production. It is a rare pathogen with a low virulence in human infection. The relation between the aquaculture outbreak and the human infection has not been clarified. Prospective and retrospective epidemiologic surveillance of the four patients with L. garvieae infection between 2000 and 2003 and their relations to the aquaculture outbreaks of L. garvieae were conducted. All the four patients with L. garvieae infection were associated with gastrointestinal disorders. Three of the four patients gave a history of consuming raw fish and in three of the four patients, the infection occurred in summer between June and August while there is a decrease of fisheries production and an increase in L. garvieae infection in aquaculture farms. There was a 100% identity of 16S rDNA sequence of L. garvieae isolates from patient 1 and from the squid muscle obtained from the restaurant where patient 1 consumed the raw fish. Sporadic occurrence of L. garvieae infection in human appears to correlate with the seasonal aquaculture outbreaks of L. garvieae infection. The presence of gastro-intestinal disorder may facilitate L. garvieae infection.

Aged↗

Control analysis as a tool to understand the formation of the las operon in Lactococcus lactis.

In Lactococcus lactis the enzymes phosphofructokinase (PFK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) are uniquely encoded in the las operon. We used metabolic control analysis to study the role of this organization. Earlier studies have shown that, at wild-type levels, LDH has no control over glycolysis and growth rate, but high negative control over formate production (C(Jformate)LDH=-1.3). We found that PFK and PK exert no control over glycolysis and growth rate at wild-type enzyme levels but both enzymes exert strong positive control on the glycolytic flux at reduced activities. PK exerts high positive control over formate (C(Jformate)PK=0.9-1.1) and acetate production (C(Jacetate)PK=0.8-1.0), whereas PFK exerts no control over these fluxes at increased expression. Decreased expression of the entire las operon resulted in a strong decrease in the growth rate and glycolytic flux; at 53% expression of the las operon glycolytic flux was reduced to 44% and the flux control coefficient increased towards 3. Increased las expression resulted in a slight decrease in the glycolytic flux. At wild-type levels, control was close to zero on both glycolysis and the pyruvate branches. The sum of control coefficients for the three enzymes individually was comparable with the control coefficient found for the entire operon; the strong positive control exerted by PK almost cancels out the negative control exerted by LDH on formate production. Our analysis suggests that coregulation of PFK and PK provides a very efficient way to regulate glycolysis, and coregulating PK and LDH allows cells to maintain homolactic fermentation during glycolysis regulation.

Bacterial Proteins↗

AcmA of Lactococcus lactis is an N-acetylglucosaminidase with an optimal number of LysM domains for proper functioning.

AcmA, the major autolysin of Lactococcus lactis MG1363 is a modular protein consisting of an N-terminal active site domain and a C-terminal peptidoglycan-binding domain. The active site domain is homologous to that of muramidase-2 of Enterococcus hirae, however, RP-HPLC analysis of muropeptides released from Bacillus subtilis peptidoglycan, after digestion with AcmA, shows that AcmA is an N-acetylglucosaminidase. In the C-terminus of AcmA three highly similar repeated regions of 45 amino acid residues are present, which are separated by short nonhomologous sequences. The repeats of AcmA, which belong to the lysine motif (LysM) domain family, were consecutively deleted, removed, or, alternatively, one additional repeat was added, without destroying the cell wall-hydrolyzing activity of the enzyme in vitro, although AcmA activity was reduced in all cases. In vivo, proteins containing no or only one repeat did not give rise to autolysis of lactococcal cells, whereas separation of the producer cells from the chains was incomplete. Exogenously added AcmA deletion derivatives carrying two repeats or four repeats bound to lactococcal cells, whereas the derivative with no or one repeat did not. In conclusion, these results show that AcmA needs three LysM domains for optimal peptidoglycan binding and biological functioning.

Acetylglucosaminidase↗

Treatment of murine colitis by Lactococcus lactis secreting interleukin-10.

The cytokine interleukin-10 (IL-10) has shown promise in clinical trials for treatment of inflammatory bowel disease (IBD). Using two mouse models, we show that the therapeutic dose of IL-10 can be reduced by localized delivery of a bacterium genetically engineered to secrete the cytokine. Intragastric administration of IL-10-secreting Lactococcus lactis caused a 50% reduction in colitis in mice treated with dextran sulfate sodium and prevented the onset of colitis in IL-10(-/-) mice. This approach may lead to better methods for cost-effective and long-term management of IBD in humans.

Animals↗