Frank Michiels

Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii

Two plasmids, pLAB1000 and pLAB2000 (3.3 and 9.1 kb, respectively), have been isolated from a grass silage strain of Lactobacillus hilgardii. Both plasmids were cloned in Escherichia coli and characterized through restriction mapping. A 1.6-kb XbaI-SacI fragment of pLAB1000 appeared to be sufficient for autonomous replication in Lactobacillus plantarum and in Bacillus subtilis. Different shuttle vectors for E. coli and gram-positive bacteria were developed using the pLAB1000 plasmid. These could stably be maintained in Lactobacillus, Enterococcus, and Bacillus under selective conditions. Plasmids sharing DNA homologies with pLAB1000 have been observed in different strains of the related species L. plantarum.

Cloning and expression of cellulase and xylanase genes in Lactobacillus plantarum

SummaryEleven cellulase genes from Gram-positive bacteria were cloned in a Lactobacillus plantarum silage inoculum. Eight of these genes were expressed as active enzymes from their original promotors and translation signals. Where tested, the enzymes produced by transformed L.plantarum had the same temperature and pH optimum as enzymes produced in the original host, or in transformed Escherichia coli. Using chloramphenicol acetyltransferase as a cell-internal marker enzyme, it could be demonstrated that at least endoglucanase D from Clostridium thermocellum was actively secreted by transformed L. plantarum. In growing L. plantarum cultures, most of the enzymes were irreversibly inactivated when the pH decreased below 4.5. If the transformed strains were to be applied as an inoculum in silage, this pH inactivation might be useful in preventing overdigestion of the crop fibre.

Genetic maps of mouse Chromosome 17 including 12 new anonymous DNA loci and 25 anchor loci

An interspecific backcross between lab mice and Mus spretus was used to construct a multilocus map of Chromosome 17 consisting of 12 new anonymous loci and 9 anchor loci. In addition, 7 anonymous DNA loci were added to the Chr 17 map for the BXD strains. Although we were able to identify readily the most likely gene order in the interspecific backcross, we found no evidence for an unambiguous gene order using the BXD recombinant inbred strains. Comparison of the interspecific backcross map and the BXD RI strain map revealed evidence in the interspecific backcross for a longer total genetic length, enhanced recombination distal to H-2, a segment showing suppressed recombination, and strong interference.

Characterization and heterologous expression of the tetL gene and identification of iso-ISS1 elements from Enterococcus faecalis plasmid pJH1.

The tetracycline-resistance (TcR) determinant of the Enterococcus faecalis plasmid pJH1 has been identified and located on a 2.2-kb RsaI-EcoRI fragment. The fragment was cloned in Escherichia coli, and specified TcR in this host. The nucleotide (nt) sequence of the cloned fragment showed the presence of an open reading frame (ORF) of 1374 bp, designated tetL. The nt sequence of tetL from pJH1 was identical to that of the tetL present on pLS1 from Streptococcus agalactiae. Upstream of the pJH1 tetL, part of another ORF was found that, except for two single-nt substitutions, was identical to an iso-ISS1 element from Lactococcus lactis. Hybridization studies indicated the presence of several ISS1-like elements in plasmid pJH1, but not on the En. faecalis chromosome. To study its usefulness as a marker in Gram+ organisms, the pJH1 tetL was cloned on the broad-host-range plasmid pNZ124, resulting in pNZ280, that was found to give resistance to 40 micrograms Tc/ml in Lc. lactis and Bacillus subtilis.