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.

Isolation and characterization of IS1165, an insertion sequence of Leuconostoc mesenteroides subsp. cremoris and other lactic acid bacteria.

We have cloned and characterized an insertion sequence from Leuconostoc mesenteroides subsp. cremoris strain DB1165. This element, designated IS1165, is 1553 bp, has imperfect inverted repeat ends, contains an open reading frame of 1236 bp, and is not related to any previously described insertion sequence. The copy number of IS1165 varies from 4 to 13 in L. mesenteroides subsp. cremoris strains allowing genetic fingerprinting of strains based on location and number of bands on hybridization. IS1165 or closely related elements have been detected by hybridization in L. lactis, L. oenos, Pediococcus sp., Lactobacillus helveticus, and Lb. casei but not in Lactococcus.

Molecular size of [3H]WB-4101-binding sites in rat cortex as determined by radiation inactivation.

The molecular weight of the [3H]WB-4101-binding sites in rat cerebral cortex was estimated by the irradiation-inactivation technique. The molecular weight was found to be dependent on the assay concentrations of the radioligand in the binding assay. Assays with a [3H]WB-4101 concentration of 0.25 nM showed a molecular weight of 62,100 daltons and 5.1 nM showed 50,800 daltons. Scatchard transformation of the [3H]WB-4101-binding data shows two binding sites (high-affinity: KD = 0.09 nM, Bmax = 9.1 pmoles/g; low-affinity: KD = 20 nM, Bmax = 80 pmoles/g). It is suggested that the two binding exist at two distinct molecules and in that case the observed molecular weights of 62,100 and 50,800 daltons are not true values because the determinations are carried out on a mixture of the two molecule populations. The distribution of the two binding sites was calculated for the two radioligand concentrations, 0.25 nM and 5.1 nM; and on this background the "true" molecular weights of the two [3H]WB-4101-binding sites were estimated to be 68,300 daltons for the high-affinity molecule and 41,400 daltons for the low-affinity molecule. Competition studies with a variety of adrenergic agonists and antagonists against [3H]WB-4101 supported the hypothesis that only the high-affinity binding site is an alpha-1-adrenoceptor.