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.     

Intraspecific gene expression variability in the yeast Kluyveromyces lactis revealed by micro-array analysis

Using the Genolevures sequencing data, we developed an expression micro-array for the yeast Kluyveromyces lactis consisting of 482 genes, mainly involved in central metabolism, compound transport facilitators and stress response. The array was validated using the LAC/GAL system. By comparing gene expression in the laboratory reference strain CBS2359 and in an industrial strain B1, we demonstrated the influence of two carbon sources, glucose and lactose, on the expression of genes involved in the respiratory and in the fermentative metabolic pathways. We also showed that the two strains, although both originating from dairies, display unexpected differences in gene expression on each type of carbon source.     

Discrepancies between the phenotypic and genotypic characterization of Lactococcus lactis cheese isolates

AIMS: The use of randomly amplified polymorphic DNA (RAPD)-PCR fingerprinting and plasmid profiles to determine at the strain level, the similarity of Lactococcus lactis isolates obtained during sampling of traditional cheeses and to verify its correspondence to the selected phenotypic characteristics. METHODS AND RESULTS: A total of 45 L. lactis isolates were genotypically analysed by RAPD-PCR fingerprinting and plasmid patterns. Phenotypic traits used to compare strains were proteolytic, acidifying, aminotransferase (aromatic and branched chain aminotransferase) and alpha-ketoisovalerate decarboxylase (Kivd) activities. The results show that 23 isolates could be grouped in clusters that exhibited 100% identity in both their RAPD and plasmid patterns, indicating the probable isolation of dominant strains during the cheese sampling process. However, there were phenotypic differences between isolates within the same cluster that included the loss of relevant technological properties such as proteinase activity and acidifying capacity or high variation in their amino acid converting enzyme activities. Likewise, the analysis of a specific attribute, Kivd activity, indicated that 7 of 15 isolates showed no detectable activity despite the presence of the encoding (kivd) gene. CONCLUSION: Phenotypic differences found between genotypically similar strains of L. lactis strains could be linked to differences in enzymatic expression. SIGNIFICANCE AND IMPACT OF THE STUDY: Phenotypic analysis of L. lactis isolates should be considered when selecting strains with new cheese flavour forming capabilities.     

Inhibition of tyrosine-sensitive 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase by glyphosate in Candida maltosa

Abstract A gene block controlling sucrose-fermenting ability, nisin resistance and nisin production was found to be transmissible by a conjugation-like process. The 'pSN' (sucrose nisin) plasmid was transferred from 8 different nisin-producing donor strains into MG1614, a plasmid-free derivative of Streptococcus lactis 712. In the new host low yields of a plasmid of approx. 30 MDa were isolated but its authenticity as a pSN plasmid has not yet been established. Possibilities for increased nisin yield by genetic manipulation in S. lactis 712 must exist.     

Cloning and sequence analysis of the gene encoding Lactococcus lactis malolactic enzyme: Relationships with malic enzymes

Abstract Malolactic enzyme is the key enzyme in the degradation of L-malic acid by lactic acid bacteria. Using degenerated primers designed from the first 20 N-terminal amino acid sequence of lactococcal malolactic enzyme, a 60-bp DNA fragment containing part of the mleS gene was amplified from Lactococcus lactis in a polymerase chain reaction. This specific probe was used to isolate two contiguous fragments covering the gene as a whole. The 1.9-kb region sequenced contains an open reading frame of 1623 bp, coding a putative protein of 540 amino acids. The deduced amino acid sequence reveals that lactococcal putative protein (Mlep) is highly homologous to the malic enzyme of other organisms. Expression of the mleS gene in Escherichia coli results in malolactic activity.     

Structure and properties of novel inclusions in Shewanella putrefaciens

Cytoplasmic inclusions surrounded by a bilayer membrane were seen in thin sections. negatively stained and freeze-fractured preparations of Shewanella putrefaciens. Cells harvested from the late exponential and early stationary phase showed a higher number of these vesicles than bacteria isolated from early exponential or late stationary phase. Chemical dyes for polyphosphate or poly-beta-hydroxybutyrate did not stain the material enclosed within these vesicles. Elemental analysis of the material indicated that the content was organic in nature and might be a protein. HPLC analysis of the material showed that it was probably not a carbon source, nor an electron acceptor used by S. putrefaciens.