Six putative two-component regulatory systems isolated from Lactococcus lactis subsp. cremoris MG1363.

The genetic elements specifying six putative two-component regulatory systems (2CSs) were identified on the chromosome of Lactococcus lactis MG1363. These 2CSs appear to represent distinct loci, each containing a histidine kinase and response-regulator-encoding gene pair. Transcriptional analysis of the six 2CSs was performed either by generating transcriptional fusions to a reporter gene or by primer extension.

Disruption of the sole ldhL gene in Lactobacillus sakei prevents the production of both L- and D-lactate.

A 7 kb DNA fragment was cloned from Lactobacillus sakei which contains the IdhL gene encoding the L(+)-lactate dehydrogenase (L-LDH). Analysis of the DNA sequence, Northern experiments and primer extension experiments showed that IdhL is transcribed from a single promoter, leading to a monocistronic 1.15 kb mRNA which yields the L-LDH.

Identification by PCR of genes encoding multiple response regulators.

Environmental sensing in bacteria often involves the concerted action of sensor kinases and response regulators. Degenerate oligonucleotide primers were designed on the basis of amino acid similarity in the response regulators of these two-component systems. The primers were used in PCR to specifically amplify an internal DNA segment corresponding to the receiver module domain from genes encoding response regulators.

Efficient transformation of Lactobacillus sake by electroporation.

A procedure to transform intact cells by electroporation was developed through a systematic examination of the effect of changes in various parameters on the transformation efficiency of strain 64F. The most critical factors were found to be the electrical parameters, the composition of washing and electroporation/storage solutions, and the presence of MgCI in the expression medium. Under optimal conditions transformation efficiencies up to 10 transformants (μg supercoiled DNA) were obtained.