Lactococcus lactis and stress

It is now generally recognized that cell growth conditions in nature are often suboptimal compared to controlled conditions provided in the laboratory. Natural stresses like starvation and acidity are generated by cell growth itself. Other stresses like temperature or osmotic shock, or oxygen, are imposed by the environment. It is now clear that defense mechanisms to withstand different stresses must be present in all organisms. The exploration of stress responses in lactic acid bacteria has just begun. Several stress response genes have been revealed through homologies with known genes in other organisms. While stress response genes appear to be highly conserved, however, their regulation may not be. Thus, search of the regulation of stress response in lactic acid bacteria may reveal new regulatory circuits. The first part of this report addresses the available information on stress response in Lactococcus lactis.Acid stress response may be particularly important in lactic acid bacteria, whose growth and transition to stationary phase is accompanied by the production of lactic acid, which results in acidification of the media, arrest of cell multiplication, and possible cell death. The second part of this report will focus on progress made in acid stress response, particularly in L. lactis and on factors which may affect its regulation. Acid tolerance is presently under study in L. lactis. Our results with strain MG1363 show that it survives a lethal challenge at pH 4.0 if adapted briefly (5 to 15 minutes) at a pH between 4.5 and 6.5. Adaptation requires protein synthesis, indicating that acid conditions induce expression of newly synthesized genes. These results show that L. lactis possesses an inducible response to acid stress in exponential phase.To identify possible regulatory genes involved in acid stress response, we determined low pH conditions in which MG1363 is unable to grow, and selected at 37°C for transposition insertional mutants which were able to survive. About thirty mutants resistant to low pH conditions were characterized. The interrupted genes were identified by sequence homology with known genes. One insertion interrupts ahrC, the putative regulator of arginine metabolism; possibly, increased arginine catabolism in the mutant produces metabolites which increase the pH. Several other mutations putatively map at some step in the pathway of (p)ppGpp synthesis. Our results suggest that the stringent response pathway, which is involved in starvation and stationary phase survival, may also be implicated in acid pH tolerance.     

Divergence of Genomic Sequences between Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris

Relatedness between Lactococcus lactis subsp. cremoris and L. lactis subsp. lactis was assessed by Southern hybridization analysis, with cloned chromosomal genes as probes. The results indicate that strains of the two subspecies form two distinct groups and that the DNA sequence divergence between L. lactis subsp. lactis and L. lactis subsp. cremoris is estimated to be between 20 and 30%. The previously used phenotypic criteria do not fully discriminate between the groups; therefore, we propose a new classification which is based on DNA homology. In agreement with this revised classification, the L. lactis subsp. lactis and L. lactis subsp. cremoris strains from our collection have distinct phage sensitivities.     

Nasal administration of Lactococcus lactis improves local and systemic immune responses against Streptococcus pneumoniae

Lactococcus lactis NZ9000 is a non-pathogenic non-invasive bacterium extensively used for the delivery of antigens and cytokines at the mucosal level. However, there are no reports concerning the per se immunomodulatory capacity of this strain. The aim of the present study was to investigate the intrinsic immunostimulating properties of the nasal administration of L. lactis NZ9000 in a pneumococcal infection model. Mice were preventively treated with L. lactis (2, 5 or 7 days with 10(8) cells/day per mouse) and then challenged with Streptococcus pneumoniae. The local and the systemic immune responses were evaluated. Our results showed that nasal administration of L. lactis for 5 days (LLN5d) increased the clearance rate of S. pneumoniae from lung and prevented the dissemination of pneumococci into blood. This effect coincided with an upregulation of the innate and specific immune responses in both local and systemic compartments. LLN5d increased phagocyte activation in lung, blood and bone marrow, determined by NBT and peroxidase tests. Anti-pneumococcal immunoglobulin (Ig)A in bronchoalveolar lavages (BAL) and IgG in BAL and serum were increased in the LLN5d group. Lung tissue injury was reduced by LLN5d treatment as revealed by histopathological examination and albumin concentration and lactate dehydrogenase activity in BAL. The adjuvant effect of L. lactis in our infection model would be an important advantage for its use as a delivery vehicle of pneumococcal proteins and nasal immunization with recombinant L. lactis emerges as an effective route of vaccination for both systemic and mucosal immunity against pneumococcal infection.     

Physiological responses of Lactococcus lactis ML3 to alternating conditions of growth and starvation

Lactococcus lactis species can survive periods of carbohydrate starvation for relatively long periods of time. In the first hours of starvation, however, the maximal glycolytic and arginine deiminase (ADI) pathway activities decline rapidly. The rate of decrease of the pathway activities diminishes as soon as the cells become depleted of energy-rich intermediates. Loss of glycolytic activity is associated with loss of glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate mutase and pyruvate kinase activities. Upon addition of sugar to starved cultures these enzymatic, and thus the glycolytic, activities can be restored to 100% values. The recovery of enzymatic activities is inhibited by chloramphenicol, indicating that protein synthesis is involved. In contrast, restoration of ADI pathway activity does not require de novo synthesis of proteins. General protein degradation and synthesis have been studied in growing and starving cells using [³⁵S]methionine-labeling of proteins and two-dimensional gel analysis. The breakdown of bulk proteins in exponentially growing cells shows first-order rate kinetics (t1/2 of approximately 5 h). Following an initial breakdown of proteins with a t1/2 of 5 h during the first hour(s) of starvation, bulk proteins are degraded very slowly in starving energy-depleted cells. The breakdown of proteins during starvation appears to be (largely) nonspecific. The rate of synthesis of proteins decreases rapidly in the first hour(s) of starvation. From the onset of starvation on at least 45 proteins are no longer synthesized. During starvation relative induction of fourteen to fifteen proteins can be observed.Abbreviations ADI Arginine deiminase - ATP adenosine triphosphate - PEP phosphoenolpyruvate - membrane potential - pH pH gradient - PTS sugar phosphotransferase system - CDM chemically defined medium - TCA trichloro-acetic acid     

Genome plasticity in Lactococcus lactis

Comparative genome analyses contribute significantly to our understanding of bacterial evolution and indicate that bacterial genomes are constantly evolving structures. The gene content and organisation of chromosomes of lactic acid bacteria probably result from a strong evolutionary pressure toward optimal growth of these microorganisms in milk. The genome plasticity of Lactococcus lactis was evaluated at inter- and intrasubspecies levels by different experimental approaches. Comparative genomics showed that the lactococcal genomes are not highly plastic although large rearrangements (a.o. deletions, inversions) can occur. Experimental genome shuffling using a new genetic strategy based on the Cre-loxP recombination system revealed that two domains are under strong constraints acting to maintain the original chromosome organisation: a large region around the replication origin, and a smaller one around the putative terminus of replication. Future knowledge of the rules leading to an optimal genome organisation could facilitate the definition of new strategies for industrial strain improvement.     

Lysozyme expression in Lactococcus lactis

Summary Three lysozyme-encoding genes, one of eukaryotic and two of prokaryotic origin, were expressed in Lactococcus lactis subsp. lactis. Hen egg white lysozyme (HEL) could be detected in L. lactis lysates by Western blotting. No lysozyme activity was observed, however, presumably because of the absence of correctly formed disulphide bonds in the L. lactis product. The functionally related lysozymes of the E. coli bacteriophages T4 and were produced as biologically active proteins in L. lactis. In both cases, the highest expression levels were obtained using configurations in which the bacteriophage lysozyme genes had been translationally coupled to a short open reading frame of lactococcal origin. Both enzymes, like HEL, may prevent the growth of food-spoilage bacteria.     

Gene expression in Lactococcus lactis

Abstract Lactic acid bacteria are of major economic importance, as they occupy a key position in the manufacture of fermented foods. A considerable body of research is currently being devoted to the development of lactic acid bacterial strains with improved characteristics, that may be used to make fermentations pass of more efficiently, or to make new applications possible. Therefore, and because the lactococci are designated 'GRAS' organisms ('generally recognized as safe') which may be used for safe production of foreign proteins, detailed knowledge of homologous and heterologous gene expression in these organisms is desired. An overview is given of our current knowledge concerning gene expression in Lactococcus lactis . A general picture of gene expression signals in L. lactis emerges that shows considerable similarity to those observed in Escherichia coli and Bacillus subtilis . This feature allowed the expression of a number of L. lactis -derived genes in the latter bacterial species. Several studies have indicated, however, that in spite of the similarities, the expression signals from E. coli, B. subtilis and L. lactis are not equally efficient in these three organisms.     

Characterization of N-deoxyribosyltransferase from Lactococcus lactis subsp. lactis

A nucleoside N-deoxyribosyltransferase-homologous gene was detected by homological search in the genomic DNA of Lactococcus lactis subsp. lactis. The gene yejD is composed of 477 nucleotides encoding 159 amino acids with only 25% identity, which is low in comparison to the amino acid sequences of the N-deoxyribosyltransferases from other lactic acid bacteria, i.e. Lactobacillus leichmannii and Lactobacillus helveticus. The residues responsible for catalytic and substrate-binding sites in known enzymes are conserved at Gln49, Asp73, Asp93 (or Asp95), and Glu101, respectively. The recombinant YejD expressed in Escherichia coli shows a 2-deoxyribosyl transfer activity to and from both bases of purine and pyrimidine, showing that YejD should be categorized as a class II N-deoxyribosyltransferase. Interestingly, the base-exchange activity as well as the heat stability of YejD was enhanced by the presence of monovalent cations such as K(+), NH(4)(+), and Rb(+), indicating that the Lactococcus enzyme is a K(+)-activated Type II enzyme. However, divalent cations including Mg(2+) and Ca(2+) significantly inhibit the activity. Whether or not the yejD gene product actually participates in the nucleoside salvage pathway of Lc. lactis remains unclear, but the lactic acid bacterium possesses the gene coding for the nucleoside N-deoxyribosyltransferase activated by K(+) on its genome.     

Comparison of cell wall proteinases from Lactococcus lactis subsp. cremoris AC1 and Lactococcus lactis subsp. lactis NCDO 763

Summary Cell wall-associated proteinases were isolated from Lactococcus lactis subsp. cremoris AC1 and subsp. lactis NCDO 763 in order to compare their specificities towards different caseins. Two purification strategies were applied. Cells grown in casein-free M17 medium were a suitable starting material for purification, since electrophoretic purity could be achieved after one chromatographic step. Both enzymes has an apparent molecular mass of about 145000 daltons as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Electrophoresis and reversed phase HPLC patterns of hydrolysates of _s1-, _s2-, -, and K-caseins indicated that both proteinases had a similar specificity. The enzyme of L. lactis subsp. lactis split _s1- and _s2-caseins more extensively than that of L. lactis subsp. cremoris.     

Comparison of cell wall proteinases from Lactococcus lactis subsp. cremoris AC1 and Lactococcus lactis subsp. lactis NCDO 763

Summary The cell wall proteinases of Lactococcus lactis subsp. lactis NCDO 763 and L. lactis subsp. cremoris AC1 hydrolyse -casein with a similar specificity even though some quantitative differences can be observed for a few degradation products analysed by reverse phase HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The main peptides soluble in 1.1% trifluoroacetic acid and liberated by the two proteinases were identified and have been found to be the same for the two enzymes. They are located in two areas of the -casein sequence (53–93 and the C-terminal part: 129–209) and they include bitter tasting or physiologically active fragments. No narrow specificity was observed for these proteinases. However, glutamine and serine residues are more frequently encountered in position P1 and P1 of the sensitive peptide bond and the close environment (position P2 to P4 and P2 to P4) of the cleaved bond is mainly hydrophobic.     

Oxidative stress in Lactococcus lactis

Lactococcus lactis, the most extensively characterized lactic acid bacterium, is a mesophilic- and microaerophilic-fermenting microorganism widely used for the production of fermented food products. During industrial processes, L. lactis is often exposed to multiple environmental stresses (low and high temperature, low pH, high osmotic pressure, nutrient starvation and oxidation) that can cause loss or reduction of bacterial viability, reproducibility, as well as organoleptic and/or fermentative qualities. Among these stress factors, oxidation can be considered one of the most deleterious to the cell, causing cellular damage at both molecular and metabolic levels. During the last two decades, considerable efforts have been made to improve our knowledge of oxidative stress in L. lactis. Many genes involved with both oxidative stress resistance and control mechanisms have been identified; functionally they seem to overlap. The finding of new genes, and a better understanding of the molecular mechanisms of stress resistance in L. lactis and other lactic acid bacterium, will lead to the construction and isolation of stress-resistant strains. Such strains could be exploited for both traditional and probiotic uses.     

Time‐resolved genetic responses of Lactococcus lactis to a dairy environment

Lactococcus lactis is one of main bacterial species found in mixed dairy starter cultures for the production of semi‐hard cheese. Despite the appreciation that mixed cultures are essential for the eventual properties of the manufactured cheese the vast majority of studies on L. lactis were carried out in laboratory media with a pure culture. In this study we applied an advanced recombinant in vivo expression technology (R‐IVET) assay in combination with a high‐throughput cheese‐manufacturing protocol for the identification and subsequent validation of promoter sequences specifically induced during the manufacturing and ripening of cheese. The system allowed gene expression measurements in an undisturbed product environment without the use of antibiotics and in combination with a mixed strain starter culture. The utilization of bacterial luciferase as reporter enabled the real‐time monitoring of gene expression in cheese for up to 200 h after the cheese‐manufacturing process was initiated. The results revealed a number of genes that were clearly induced in cheese such as cysD, bcaP, dppA, hisC, gltA, rpsE, purL, amtB as well as a number of hypothetical genes, pseudogenes and notably genetic elements located on the non‐coding strand of annotated open reading frames. Furthermore genes that are likely to be involved in interactions with bacteria used in the mixed strain starter culture were identified.     

Isolation and characterization of Lactococcus lactis subsp. lactis promoters.

DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained.     

Isolation and characterization of Lactococcus lactis subsp. lactis promoters.

DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained.     

Ubiquity and diversity of multidrug resistance genes in Lactococcus lactis strains isolated between 1936 and 1995

The presence and the nucleotide sequence of four multidrug resistance genes, lmrA, lmrP, lmrC, and lmrD, were investigated in 13 strains of Lactococcus lactis ssp. lactis, four strains of Lactococcus lactis ssp. cremoris, two strains of Lactococcus plantarum, and two strains of Lactococcus raffinolactis. Multidrug resistance genes were present in all L. lactis isolates tested. However, none of them could be detected in the strains belonging to the species L. raffinolactis and L. plantarum, suggesting a different set of multidrug resistance genes in these species. The analysis of the four deduced amino acid sequences established two different variants depending on the subspecies of L. lactis. Either lmrA, or lmrP, or both were found naturally disrupted in five strains, while full-length lmrD was present in all strains.     

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Bacterial isolates from bean-sprouts were screened for anti- Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L.monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L.monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNAsequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis . Polymerase chain reaction and nucleotide sequencing revealed that thegenomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth,bean-sprout isolate HPB 1688 survived at 3–4·5°C for at least 20 d, grew at 4°Cand produced anti-listerial compoundsat 5°C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited thegrowth of L. monocytogenes at 4°C after 14d and at 10°C after 2 d. When co-inoculatedwith 10²cells g⁻¹ of L.monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10⁸cells g⁻¹) was able to reduce the number of L. monocytogenes by 1–1·4 logs after storage for 10 d at 7° and 10°C. A bacteriocin-producing Enterococcusfaecium was also able to reduce the numbers of L. monocytogenes onCaesar salad, butdid not act synergistically when co-inoculated with L. lactis subsp. lactis .     

Respiration capacity and consequences in Lactococcus lactis

We recently reported that the well-studied fermenting bacterium Lactococcus lactis could grow via a respirative metabolism in the presence of oxygen when a heme source is present. Respiration induces profound changes in L. lactis metabolism, and improvement of oxygen tolerance and long-term survival. Compared to usual fermentation conditions, biomass is approximately doubled by the end of growth, acid production is reduced, and large amounts of normally minor end products accumulate. Lactococci grown via respiration survive markedly better after long-term storage than fermenting cells. We suggest that growth and survival of lactococci are optimal under respiration-permissive conditions, and not under fermentation conditions as previously supposed.Our results reveal the uniqueness of the L. lactis respiration model. The well-studied aerobic bacteria express multiple terminal cytochrome oxidases, which assure respiration all throughout growth; they also synthesize their own heme. In contrast, the L. lactis cydABgenes encode a single cytochrome oxidase (bd), and heme must be provided. Furthermore, cydAB genes mediate respiration only late in growth. Thus, lactococci exit the lag phase via fermentation even if heme is present, and start respiration in late exponential phase. Our results suggest that the spectacularly improved survival is in part due to reduced intracellular oxidation during respiration. We predict that lactococcal relatives like the Enterococci, and some Lactobacilli, which have reported respiration potential, will display improved survival under respiration-permissive conditions.     

Integration and excision of plasmid DNA in Lactococcus lactis subsp. lactis

The capacity of the 75-kb lactose-proteinase plasmid pCI301 from Lactococcus lactis subsp. lactis UC317 to recombine with the lactococcal chromosome was examined. Low-frequency integration of pCI301 sequences was detected following protoplast transformation of strain MG136Sm with total plasmid DNA from strain UC317. Excision of integrated sequences was subsequently observed at a low level. Excised sequences were rescued through recombination with and mobilization by the conjugative enterococcal plasmid pAMB1. Transconjugants harboring novel recombinant pCI301::pAMB1 plasmids, both pAMB1 and a pCI301 derivative, and pAMB1 only were isolated. The latter represents a class of transconjugant in which an elevated level of reintegration of pCI301 DNA in the recipient chromosome has occurred.