Functional analysis of promoters in the nisin gene cluster of Lactococcus lactis

Abstract

The promoters in the nisin gene cluster nisABTCIPRKFEG of Lactococcus lactis were characterized by primer extension and transcriptional fusions to the Escherichia coli promoterless ␤-glucuronidase gene (gusA). Three promoters preceding the nisA, nisR, and nisF genes, which all give rise to gusA expression in the nisin-producing strain L. lactis NZ9700, were identified. The transcriptional autoregulation of nisA by signal transduction involving the sensor histidine kinase NisK and the response regulator NisR has been demonstrated previously (O. P. Kuipers, M. M. Beerthuyzen, P. G. G. A. de Ruyter, E. J. Luesink, and W. M. de Vos, J. Biol. Chem. 270: 27299-27304, 1995), and therefore the possible nisin-dependent expression of gusA under control of the nisR and nisF promoters was also investigated. The nisR promoter was shown to direct nisin-independent gusA expression in L. lactis MG1363, which is a nisin-transposon-and plasmid-free strain. L. lactis NZ9800, which does not produce nisin because of a deletion in the nisA gene, containing the nisF-gusA fusion plasmid, gave rise to ␤-glucuronidase production only after induction by nisin. A similar regulation was found in L. lactis NZ3900, which contains a single copy of the nisR and nisK genes but no other genes of the nisin gene cluster. In contrast, when the nisK gene was disrupted, no ␤-glucuronidase activity directed by the nisF promoter could be detected even after induction with nisin. These results show that, like the nisA promoter, the nisF promoter is nisin inducible. The nisF and nisA promoter sequences have significant similarities and contain a conserved region that could be important for transcriptional control.

Monitoring the function of membrane transport proteins in detergent-solubilized form

“…(9). L. lactis NZ9000 (15) and expression plasmid pNZ8048 (23) were obtained from W. Konings (University of Gronigen, Haren, The Netherlands) through H. R. Kaback (Universtiy of California, Los Angeles, CA). Recombinant tyt1 [containing a His 10 coding region before the tyt1 start codon (9)] was subcloned into pNZ8048 by using the BstBI/HindIII restriction enzyme combination, followed by transformation of electrocompetent L. lactis NZ9000 with the ligation mix.…”

Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria

“…This vector contains the following features ( Fig. 2): the lactococcal pNZ212 origin of replication; a chloramphenicol acetyltransferase gene for selection; the P nisA promoter to direct inducible expression of PA fusions [23]; the lactococcal USP45 secretion signal to drive secretion [24]; the gene fragment encoding the PA (the C-terminal 218 amino acid cell-wall binding domain of AcmA of L. lactis [25] including its stop codon); and a transcriptional terminator is present 445 bp downstream of the PA gene fragment. Genes can be inserted in the unique NcoI site, resulting in a transcriptional fusion with the Met residue located at position +5 of the mature secreted protein.…”

Function of Lactococcus lactis Nisin Immunity Genes nisI and nisFEG after Coordinated Expression in the Surrogate Host Bacillus subtilis

“…Whereas nisI was found on a transcriptional unit nisABTCIP together with the structural gene (nisA) and genes involved in posttranslational modifications (nisBC), the processing (nisP) and transport (nisT) of nisin expression of nisFEG is controlled by its own promoter (47). C, nisI and nisFEG were fused into a single transcriptional unit under the control of the P spac promoter (pHZ51) and integrated into the chromosome of B. subtilis MO1099.…”