Lactococcus lactis SpOx spontaneous mutants: a family of oxidative-stress-resistant dairy strains

Numerous industrial bacteria generate hydrogen peroxide (H(2)O(2)), which may inhibit the growth of other bacteria in mixed ecosystems. We isolated spontaneous oxidative-stress-resistant (SpOx) Lactococcus lactis mutants by using a natural selection method with milk-adapted strains on dairy culture medium containing H(2)O(2). Three SpOx mutants displayed greater H(2)O(2) resistance. One of them, SpOx3, demonstrated better behavior in different oxidative-stress situations: (i) higher long-term survival upon aeration in LM17 and milk and (ii) the ability to grow with H(2)O(2)-producing Lactobacillus delbrueckii subsp. delbrueckii strains. Furthermore, the transit kinetics of the SpOx3 mutant in the digestive tract of a human flora-associated mouse model was not affected.     

The MG1363 and IL1403 Laboratory Strains of Lactococcus lactis and Several Dairy Strains Are Diploid

Bacteria are normally haploid, maintaining one copy of their genome in one circular chromosome. We have examined the cell cycle of laboratory strains of Lactococcus lactis, and, to our surprise, we found that some of these strains were born with two complete nonreplicating chromosomes. We determined the cellular content of DNA by flow cytometry and by radioactive labeling of the DNA. These strains thus fulfill the criterion of being diploid. Several dairy strains were also found to be diploid while a nondairy strain and several other dairy strains were haploid in slow-growing culture. The diploid and haploid strains differed in their sensitivity toward UV light, in their cell size, and in their D period, the period between termination of DNA replication and cell division.In contrast to higher eukaryotes, bacteria are haploid (6, 19); i.e., they store their genetic information in a single chromosome, which is then duplicated during the cell cycle. If the growth rate is sufficiently low, bacteria are born with a single copy of the chromosome, which will then be duplicated before the bacterium divides.There are a few reports about bacteria that have more than one genome per cell, i.e., that are polyploid. Deinococcus radiodurans has been shown to have 4 to 10 copies of its genome (13, 14). The diplococcal bacterium Neisseria gonorrhoeae was found to be diploid per coccal unit (31). Azotobacter vinelandii bacteria amplify the genome during growth in rich medium more than 40 times (20, 24, 27). The giant bacterium Epulopiscium fishelsoni has been shown to amplify its genome into a polytene chromosome of 3,000-fold ploidy (2). In addition, noncomplementing diploid bacteria have been isolated from protoplast fusions in Bacillus subtilis (11) and, as a result of zygogenesis, in Escherichia coli (10). A few other bacteria with two to six different chromosomes have been reported (15, 30).The normal cell cycle is divided into three periods: (i) the B period from cell division until initiation of replication, (ii) the C period in which the cell replicates its DNA, and (iii) the D period from termination of productive replication until cell division. The D period thus includes processes such as proofreading and deconcatenation. The B period is found only in cells whose generation times exceed the length of the combined C and D periods. If the generation times become shorter than the combined lengths of the C and D periods, then the initiations of replication move into previous cell cycles (16). Fast-growing bacteria will therefore have more than one ongoing round of DNA replication at the same time; they might have 4, 8, or even 16 origins of replication (4). Normal haploid cells are born with one chromosome, either replicating or nonreplicating, and always with one terminus of replication. Not until the replication has ended do the cells have two termini. If the D period becomes longer than the generation time, which happens at high growth rates, the cells will be born with two termini as a result of the overlapping cell cycles. Long D periods are discussed further in the Discussion.We have examined the cell cycle of Lactococcus lactis subsp. cremoris MG1363 in order to determine the cell cycle periods. To our surprise, we found that slow-growing cultures of these bacteria were born with two complete chromosomes, which were replicated into four chromosomes during the C period. This strain thus fulfills the criterion of being diploid without overlapping chromosomal replication cycles. Comparison with other L. lactis strains showed that both of the subspecies, L. lactis subsp. cremoris and L. lactis subsp. lactis, had members that were either diploid, like MG1363, or haploid, like most bacteria.     

Nisin-Producing Lactococcus lactis Strains Isolated from Human Milk

Characterization by partial 16S rRNA gene sequencing, ribotyping, and green fluorescent protein-based nisin bioassay revealed that 6 of 20 human milk samples contained nisin-producing Lactococcus lactis bacteria. This suggests that the history of humans consuming nisin is older than the tradition of consuming fermented milk products.     

A Food-Grade Cloning System for Industrial Strains of Lactococcus lactis

We have previously reported the construction of a food-grade cloning vector for Lactococcus using the ochre suppressor, supB, as the selective marker. This vector, pFG1, causes only a slight growth inhibition in the laboratory strain MG1363 but is unstable in the industrial strains tested. As supB suppresses both amber and ochre stop codons, which are present in 82% of all known lactococcal genes, this undesirable finding may result from the accumulation of elongated mistranslated polypeptides. Here, we report the development of a new food-grade cloning vector, pFG200, which is suitable for overexpressing a variety of genes in industrial strains of Lactococcus lactis. The vector uses an amber suppressor, supD, as selectable marker and consists entirely of Lactococcus DNA, with the exception of a small polylinker region. Using suppressible pyrimidine auxotrophs, selection and maintenance are efficient in any pyrimidine-free medium including milk. Importantly, the presence of this vector in a variety of industrial strains has no significant effect on the growth rate or the rate of acidification in milk, making this an ideal system for food-grade modification of industrially relevant L. lactis strains. The usefulness of this system is demonstrated by overexpressing the pepN gene in a number of industrial backgrounds.     

Molecular Differentiation of Lactococcus lactis Subspecies lactis and cremoris Strains by Ribotyping and Site Specific-PCR

Twenty-five strains of Lactococcus lactis subspecies lactis and subspecies cremoris obtained from dairy industry and environmental collections were examined by 16S RNA automated ribotyping profiles and site-specific PCR (S-PCR). By automated ribotyping, the majority of strains were classified in accordance with phenotypic characterization, with the exception of one lactis (220) and two cremoris (BO32 and 140) strains. A complete differentiation of subspecies lactis and cremoris in agreement with conventional phenotypic methods was achieved by S-PCR with a set of site-specific primer pairs (PR1, RM4, and F3) designed particularly from a deletion region found in subspecies cremoris, but not in lactis. Therefore, S-PCR with primers (PR1, RM4, and F3) is a rapid and very sensitive method for the distinction of lactis and cremoris subspecies in dairy production. Received: 19 June 2000 / Accepted: 17 July 2000     

Comparative Analyses of Prophage-Like Elements Present in Two Lactococcus lactis Strains

In this study, we describe the genetic organizations of six and five apparent prophage-like elements present in the genomes of the Lactococcus lactis subsp. cremoris strains MG1363 and SK11, respectively. Phylogenetic investigation as well bioinformatic analyses indicates that all 11 prophages belong to subdivisions of the lactococcal P335 group of temperate bacteriophages.     

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.     

Increasing Acidification of Nonreplicating Lactococcus lactis ΔthyA Mutants by Incorporating ATPase Activity

Lactococcus lactis MBP71 ΔthyA (thymidylate synthase) cannot synthesize dTTP de novo, and DNA replication is dependent on thymidine in the growth medium. In the nonreplicating state acidification by MBP71 was completely insensitive to bacteriophages (M. B. Pedersen, P. R. Jensen, T. Janzen, and D. Nilsson, Appl. Environ. Microbiol. 68:3010-3023, 2002). For nonreplicating MBP71 the biomass increased 3.3-fold over the first 3.5 h, and then the increase stopped. The rate of acidification increased 2.3-fold and then started to decrease. Shortly after inoculation the lactic acid flux was 60% of that of exponentially growing MBP71. However, when nonspecific ATPase activity was incorporated into MBP71, the lactic acid flux was restored to 100% but not above that point, indicating that control over the flux switched from ATP demand to ATP supply (i.e., to sugar transport and glycolysis). As determined by growing nonreplicating cells with high ATPase activity on various sugar sources, it appeared that glycolysis exerted the majority of the control. ATPase activity also stimulated the rate of acidification by nonreplicating MBP71 growing in milk, and pH 5.2 was reached 40% faster than it was without ATPase activity. We concluded that ATPase activity is a functional means of increasing acidification by nonreplicating L. lactis.     

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.     

Characterization of the Highly Autolytic Lactococcus lactis subsp. cremoris Strains CO and 2250

Two highly autolytic Lactococcus lactis subsp. cremoris strains (CO and 2250) were selected and analyzed for their autolytic properties. Both strains showed maximum lysis when grown in M17 broth containing a limiting concentration of glucose (0.4 to 0.5%) as the carbohydrate source. Lysis did not vary greatly with pH or temperature but was reduced when strains were grown on lactose or galactose. Growth in M17 containing excess glucose (1%) prevented autolysis, although rapid lysis of L. lactis subsp. cremoris CO did occur in the presence of 1% glucose if sodium fluoride (an inhibitor of glycolysis) was added to the medium. Maximum cell lysis in a buffer system was observed early in the stationary phase, and for CO, two pH optima were observed for log-phase and stationary-phase cells (6.5 and 8.5, respectively). Autolysins were extracted from the cell wall fraction of each strain by using either 4% sodium dodecyl sulfate (SDS), 6 M guanidine hydrochloride, or 4 M lithium chloride, and their activities were analyzed by renaturing SDS-polyacrylamide gel electrophoresis on gels containing Micrococcus luteus or L. lactis subsp. cremoris CO cells as the substrate. More than one lytic band was observed on each substrate, with the major band having an apparent molecular mass of 48 kDa for CO. Each lytic band was present throughout growth and lysis. These results suggest that at least two different autolytic enzymes are present in the autolytic L. lactis subsp. cremoris strains. The presence of the lactococcal cell wall hydrolase gene, acmA (G. Buist, J. Kok, K. J. Leenhouts, M. Dabrowska, G. Venema, and A. J. Haandrikman, J. Bacteriol. 177:1554-1563, 1995), in strains 2250 and CO was confirmed by Southern hybridization. Analysis of an acmA deletion mutant of 2250 confirmed that the gene was involved in cell separation and had a role in cell lysis.     

Zirex: a Novel Zinc-Regulated Expression System for Lactococcus lactis

Here, we report a new zinc-inducible expression system for Lactococcus lactis, called Zirex, consisting of the pneumococcal repressor SczA and P_czcD. P_czcD tightly regulates the expression of green fluorescent protein in L. lactis. We show the applicability of Zirex together with the nisin-controlled expression system, enabling simultaneous but independent regulation of different genes.     

Development and Use of a Screening Procedure for Production of (alpha)-Acetolactate by Lactococcus lactis subsp. lactis biovar diacetylactis Strains

A method was developed to screen and isolate mutagenized Lactococcus lactis subsp. lactis biovar diacetylactis strains accumulating (alpha)-acetolactate. This compound is accumulated by (alpha)-acetolactate decarboxylase-deficient strains and undergoes spontaneous degradation into diacetyl on agar plates. The diacetyl produced is detected by a colorimetric reaction yielding a red halo around the colonies.     

Identification of a RecA-like protein in Lactococcus lactis

We have identified in Lactococcus lactis, an analogue of Escherichia coli RecA protein. Physiological responses such as ultraviolet (UV) and chemical mutagenesis and induction of prophage have been characterized and suggest the existence of RecA-like functions in this commercially important species. The putative RecA protein was detected at the position of an apparent molecular weight of 39 kDa by Western blot analysis by using antiserum against E coli RecA protein. In addition, the protein level is significantly increased after UV irradiation in a wild-type strain compared to the recombination deficient mutant strain.     

Restoration of a defective Lactococcus lactis xylose isomerase

The genes (xylA) encoding xylose isomerase (XI) from two Lactococcus lactis subsp. lactis strains, 210 (Xyl(-)) and IO-1 (Xyl(+)), were cloned, and the activities of their expressed proteins in recombinant strains of Escherichia coli were investigated. The nucleotide and amino acid sequence homologies between the xylA genes were 98.4 and 98.6%, respectively, and only six amino acid residues differed between the two XIs. The purified IO-1 XI was soluble with K(m) and k(cat) being 2.25 mM and 184/s, respectively, while the 210 XI was insoluble and inactive. Site-directed mutagenesis on 210 xylA showed that a triple mutant possessing R202M/Y218D/V275A mutations regained XI activity and was soluble. The K(m) and k(cat) of this mutant were 4.15 mM and 141/s, respectively. One of the IO-1 XI mutants, S388T, was insoluble and showed negligible activity similar to that of 210 XI. The introduction of a K407E mutation to the IO-1 S388T XI mutant restored its activity and solubility. The dissolution of XI activity in L. lactis subsp. lactis involves a series of mutations that collectively eliminate enzyme activity by reducing the solubility of the enzyme.     

Restoration of a Defective Lactococcus lactis Xylose Isomerase

The genes (xylA) encoding xylose isomerase (XI) from two Lactococcus lactis subsp. lactis strains, 210 (Xyl⁻) and IO-1 (Xyl⁺), were cloned, and the activities of their expressed proteins in recombinant strains of Escherichia coli were investigated. The nucleotide and amino acid sequence homologies between the xylA genes were 98.4 and 98.6%, respectively, and only six amino acid residues differed between the two XIs. The purified IO-1 XI was soluble with K_m and k_cat being 2.25 mM and 184/s, respectively, while the 210 XI was insoluble and inactive. Site-directed mutagenesis on 210 xylA showed that a triple mutant possessing R202M/Y218D/V275A mutations regained XI activity and was soluble. The K_m and k_cat of this mutant were 4.15 mM and 141/s, respectively. One of the IO-1 XI mutants, S388T, was insoluble and showed negligible activity similar to that of 210 XI. The introduction of a K407E mutation to the IO-1 S388T XI mutant restored its activity and solubility. The dissolution of XI activity in L. lactis subsp. lactis involves a series of mutations that collectively eliminate enzyme activity by reducing the solubility of the enzyme.     

Dairy biofilm: Bacterial community diversity assessment and impact of the Lactococcus lactis bio adhesion on biofilm growth

Biofilms are the most common mode of bacterial growth in nature. Their formation occurs on organic or inorganic solid surfaces in contact with a liquid, on gas-liquid and liquid-liquid boundaries as well. The aims of this study were, by combining cell enumeration, scanning electron microscopy and denaturing gel gradient electrophoresis (DGGE), to characterize the structural dynamics of dairy biofilm growth in the environments with a nutrient flow, and to evaluate the impact of adhesion of Lactococcus lactis on the biofilm community depending on the incubation time. Significantly higher values of biofilm volume and thickness were observed under dynamic conditions after 55 h. The populations of gram-positive bacteria and fungi exhibited a significantly higher biofilm organization after 2 days of cultivation than that of gram-negative bacteria. Also, results showed that Lc. lactis was able to adhere to silicone surface and the produced biofilm in which the number of adhered gram-positive and gram-negative bacteria decreased by nine orders of magnitude after 48 h of contact. This study constitutes a step ahead in developing the strategies to prevent microbial colonization by lactococcal protective biofilm.     

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.     

Batch cultures of recombinant Lactococcus lactis subsp. lactis in a stirred fermentor

Summary The transfer of plasmids was studied in a stirred fermentor in the course of mixed batch cultures combining recombinant strains of Lactococcus lactis subsp. lactis (donor strains) with L. lactis subsp. lactis CNRZ 268M3 (recipient strain). Donor strains contained one or two of the following plasmids (coding for erythromycin or chloramphenicol resistance): pIL205 (self-transmissible), pIL252, pIL253 (non-transmissible but mobilizable by pIL205, respectively small and large copy number) and pE194 (inserted in the chromosome). Only self-transmissible plasmid pIL205 was transferred, with frequencies ranging from 10^–7 to 10^–8 after 12 h of fermentation. These frequencies were 60–400 times lower than in unstirred M17 broth and 100 000 times lower than on agar medium. In the latter case, non-transmissible plasmids pIL252 and pIL253 were mobilized by pIL205 with a frequency of about 10^–5–10^–6. Correspondence to: C.-Y. Boquien