The cold shock response inLactococcus lactis subsp.lactis

The lactic acid bacterium,Lactococcus lactis subsp.lactis IL1403 was subjected to defferent cold temperatures for various times. Physiological experiments showed that this strain had an improved survival capacity in stationary phase as the temperature decreased. Two-dimensional electrophoresis of proteins extracted from cold-temperature exposed cultures showed that a dozen proteins are overexpressed up to threefold compared with exposure at 30°C. Most of these proteins are overexpressed first, temporarily and second, in the first 10 h after the transfer to 8°C. These observations indicate that response to cold stress inL. lactis subsp.lactis is an active phenomenon.     

Genetic evidence for plasmid-encoded lactococcin production inLactococcus lactis subsp.lactis 484

Lactococcus lactis subsp.lactis 484 produced a proteinaceous antibacterial substance designated as lactococcin capable of inhibiting members of theLactococcus group,Bacillus cereus, Staphylococcus aureus, andSalmonella typhi. Growth of this culture in the presence of 2–30 g/ml of ethidium bromide or acriflavin or novobiocin, and at elevated temperature (39° and 41°C), could not produce any lactococcin-negative (Lap^–) variants. However, protoplast-induced curing with lysozyme was successful in developing Lap^– derivatives. Two types of cured derivatives, namely Lac^– Lap⁺ and Lac^– Lap^–, were obtained. Lap^– variants were also lacking sucrose-fermenting ability (Suc⁺) and lactococcin resistance (Lap^r). The lactose-negative (Lac^–) variants and Lap⁺ were clearly lacking the largest (65 Md) plasmid. However, Lap^– Suc^– Lap^s variants lost a 2 Md plasmid.L. lactis subsp.lactis 484 transferred lactose-fermenting ability as well as Lap⁺ Suc⁺ Lap^r phenotypes simultaneously toL. lactis subsp.lactis LM 2306 and LM 0230 by surface mating at a frequency of 10^–4 and 10^–1 per donor respectively. However, cured Lac^– Lap^– transconjugants could not transfer Lac⁺ Lap⁺ Suc⁺ Lap^r phenotypes to any of these recipient strains. Our results indicate that Lac⁺ and Lap⁺ Suc⁺ Lap^r phenotypes are associated with 65 Md and 2 Md plasmids respectively. Conjugal transfer of 2 Md plasmid is possible only in the presence of a conjugative 65 Md plasmid.     

Inducible gene expression systems inLactococcus lactis

Lactococcus lactis is industrially important microorganism used in many dairy fermentations. Numerous genes and gene expression signals from this organism have now been identified and characterized. Recently, several naturally occurring, inducible gene-expression systems have also been described inL. lactis. The main features of these systems can be exploited to design genetically engineered expression cassettes for controlled production of various proteins and enzymes. Novel gene-expression systems inLactococcus have great potential for development of industrial cultures with desirable metabolic traits for a variety of bioprocessing applications.     

Cold shock response inLactococcus lactis ssp.diacetylactis

The acquired freeze-thaw tolerance was investigated forLactococcus lactis ssp.diacetylactis. Pretreatment of microorganisms at less severe temperatures to initiate cold tolerance gaveL. lactis ssp.diacetylactis improved cell viability after successive freezings and thawings. The ability of cells to survive freeze-thaw was dependent on factors experienced prior to freezing. Factors affecting lactic acid bacteria survival during freeze-thaw cycles were found to be different diluents, growth phase, and different cold temperatures. Viability experiments showed that this strain displaying cold shock cryotolerance had an improved survival capacity in stationary phase. The plasmid contents of lactic acid bacteria isolated from different types, DRC-2 and DRC-2C, were examined and compared with the plasmid contents of culture collection strains both before and after cold shock treatment. Using agarose gel electrophoresis, no obvious correlation between the cold shock response and the number of plasmids in the cell could be observed.     

Characterization of the Heat Shock Response in Lactococcus lactis subsp. lactis

The heat shock response in Lactococcus lactis subsp. lactis was characterized with respect to synthesis of a unique set of proteins induced by thermal stress. A shift in temperature from 30 to 42°C was sufficient to arrest the growth of L. lactis subsp. lactis, but growth resumed after a shift back to 30°C. Heat shock at 50°C reduced the viable cell population by 10³; however, pretreatment of the cells at 42°C made them more thermoresistant to exposure at 50°C. The enhanced synthesis of approximately 13 proteins was observed in cells labeled with ³⁵S upon heat shock at 42°C. Of these heat shock-induced proteins, two appeared to be homologs of GroEL and DnaK, based on their molecular weights and reactivity with antiserum against the corresponding Escherichia coli proteins. Therefore, we conclude that L. lactis subsp. lactis displays a heat shock response similar to that observed in other mesophilic bacteria.     

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.     

PCR Amplification of the Gene acmA Differentiates Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris

The occurrence of the acmA gene, encoding the lactococcal N-acetylmuramidase in new lactococcal isolates from raw milk cheeses, has been determined. Isolates were genotypically identified to the subspecies level with a PCR technique. On the basis of PCR amplification of the acmA gene, the presence or absence of an additional amplicon of approximately 700 bp correlated with Lactococcus lactis subspecies. L. lactis subsp. lactis exhibits both the expected 1,131-bp product and the additional amplicon, whereas L. lactis subsp. cremoris exhibits a single 1,131-bp fragment.     

PCR amplification of the gene acmA differentiates Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris.

The occurrence of the acmA gene, encoding the lactococcal N-acetylmuramidase in new lactococcal isolates from raw milk cheeses, has been determined. Isolates were genotypically identified to the subspecies level with a PCR technique. On the basis of PCR amplification of the acmA gene, the presence or absence of an additional amplicon of approximately 700 bp correlated with Lactococcus lactis subspecies. L. lactis subsp. lactis exhibits both the expected 1,131-bp product and the additional amplicon, whereas L. lactis subsp. cremoris exhibits a single 1,131-bp fragment.     

Histidine biosynthesis genes in Lactococcus lactis subsp. lactis.

The genes of Lactococcus lactis subsp. lactis involved in histidine biosynthesis were cloned and characterized by complementation of Escherichia coli and Bacillus subtilis mutants and DNA sequencing. Complementation of E. coli hisA, hisB, hisC, hisD, hisF, hisG, and hisIE genes and the B. subtilis hisH gene (the E. coli hisC equivalent) allowed localization of the corresponding lactococcal genes. Nucleotide sequence analysis of the 11.5-kb lactococcal region revealed 14 open reading frames (ORFs), 12 of which might form an operon. The putative operon includes eight ORFs which encode proteins homologous to enzymes involved in histidine biosynthesis. The operon also contains (i) an ORF encoding a protein homologous to the histidyl-tRNA synthetases but lacking a motif implicated in synthetase activity, which suggests that it has a role different from tRNA aminoacylation, and (ii) an ORF encoding a protein that is homologous to the 3'-aminoglycoside phosphotransferases but does not confer antibiotic resistance. The remaining ORFs specify products which have no homology with proteins in the EMBL and GenBank data bases.     

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.     

Heat shock-induced redistribution of a 160-kDa nuclear matrix protein.

In this paper we describe a 160-kDa protein (p160) which is present in the nuclear matrix of rat, mouse, and human cells. Biochemical and ultrastructural analysis shows that p160 is associated with the internal matrix and is not present in the lamina-pore complex. Immunoelectron microscopy shows that the protein is part of the extranucleolar, fibrogranular network of the nuclear matrix. During an in vivo 42 degrees C heat treatment of HeLa cells, A431 human epidermoid cells, and T24 human bladder carcinoma cells, p160 transiently formed large clusters inside the nucleus. These p160 clusters are associated with the nuclear matrix network, as judged by immunolabeling on isolated nuclear matrices. The percentage of cells showing p160 clusters increased proportionally with longer heat treatments, reaching a maximum after a period of 3 h. At this time 70 +/- 5% of the cells displayed these clusters. Clustering decreased after longer heat treatments and the anti-p160 staining pattern became diffuse granular again. Other nuclear components, such as the A1 antigen of hnRNP (ribonucleoprotein), the Sm antigen of snRNPs, and lamins A and C, did not cluster during the 42 degrees C treatment, indicating that this reallocation is characteristic for the p160 matrix protein. These results demonstrate that p160 is an internal nuclear matrix element with a dynamic spatial distribution.     

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.     

Antisense mRNA-Mediated Bacteriophage Resistance in Lactococcus lactis subsp. lactis

Resistance to a broad class of isometric bacteriophages that infect strains of Lactococcus lactis has been engineered into a dairy starter by expression of antisense mRNA targeted against a conserved bacteriophage gene. Maximum protection is obtained only when the entire 1,654-bp coding sequence for a 51-kDa protein is positioned in the antisense orientation with respect to a promoter sequence that functions in L. lactis subsp. lactis. Expression of the antisense mRNA results in more than 99% reduction of the total number of PFU. Plaques that do form are characterized by their relatively small size and irregular shape. A variety of truncated genes, including the open reading frame expressed in the sense orientation, fail to provide any significant measure of resistance as compared with that of the intact open reading frame. Southern hybridization with probes specific for the conserved region reveal that the [ill] plasmid constructs are maintained despite the presence of a large complement of other indigenous plasmids. Strains harboring the antisense mRNA plasmid construct grow and produce acid at a rate equivalent to that of the host strain alone, suggesting that antisense expression is not deleterious to normal cellular metabolism.     

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.     

Glutamate Biosynthesis in Lactococcus lactis subsp. lactis NCDO 2118

Unlike other lactic acid bacteria, Lactococcus lactis subsp. lactis NCDO 2118 was able to grow in a medium lacking glutamate and the amino acids of the glutamate family. Growth in such a medium proceeded after a lag phase of about 2 days and with a reduced growth rate (0.11 h⁻¹) compared to that in the reference medium containing glutamate (0.16 h⁻¹). The enzymatic studies showed that a phosphoenolpyruvate carboxylase activity was present, while the malic enzyme and the enzymes of the glyoxylic shunt were not detected. As in most anaerobic bacteria, no α-ketoglutarate dehydrogenase activity could be detected, and the citric acid cycle was restricted to a reductive pathway leading to succinate formation and an oxidative branch enabling the synthesis of α-ketoglutarate. The metabolic bottleneck responsible for the limited growth rate was located in this latter pathway. As regards the synthesis of glutamate from α-ketoglutarate, no glutamate dehydrogenase was detected. While the glutamate synthase-glutamine synthetase system was detected at a low level, high transaminase activity was measured. The conversion of α-ketoglutarate to glutamate by the transaminase, the reverse of the normal physiological direction, operated with different amino acids as nitrogen donor. All of the enzymes assayed were shown to be constitutive.     

Nucleolytic activities in Lactococcus lactis subsp. lactis NCDO 497

A plasmid known to be associated with mupirocin resistance of Staphylococcus aureus has been isolated and a restriction enzyme map constructed. An EcoRI fragment of 4.05 kb from this plasmid has been cloned into an Escherichia coli-Staphylococcus aureus shuttle vector and shown to carry the gene for resistance to mupirocin. The DNA sequence of a small section of the gene has been determined and the derived amino acid sequence compared with a data bank. The amino acid sequence is identical for eight amino acids with the sequence of isoleucyl tRNA synthetase of E. coli. This finding adds to the evidence that mupirocin resistance is the result of a modified isoleucyl tRNA synthetase.     

Components of fermentation medium regulate bacteriocin synthesis by the recombinant strain Lactococcus lactis subsp. lactis F-116

The regulation of the synthesis of bacteriocin produced by the recombinant strain Lactococcus lactis subsp. lactis F-116 has been studied. The synthesis is regulated by the components of the fermentation medium, the content of inorganic phosphate (KH2PO4), yeast autolysate (source of amine nitrogen), and changes in carbohydrates and amino acids. The strain was obtained by fusion of protoplasts derived from two related L. lactis subsp. lactis strains, both exhibiting a weak ability to synthesize the bacteriocin nisin. Decreasing the content of KH2PO4 from 2.0 to 1.0 or 0.5% caused bacteriocin production to go down from 4100 to 2800 or 1150 IU/ml, respectively; the base fermentation medium contained 1.0% glucose, 0.2% NaCl, 0.02% MgSO4, and yeast autolysate (an amount corresponding to 35 mg % ammonium nitrogen). The substitution of sucrose for glucose (as the source of carbon) increased the antibiotic activity by 26%, and the addition of isoleucine, by 28.5%. Elevation of the concentration of yeast autolysate in the low-phosphate fermentation medium stimulated both the growth of the lactococci and the synthesis of bacteriocin. Introduction of 1% KH2PO4, yeast autolysate (in an amount corresponding to 70 mg % ammonium nitrogen), 2.0% sucrose, and 0.1% isoleucine increased the bacteriocin-producing activity of the strain by 2.4 times.