Glycine betaine loses its osmoprotective activity in a bspA strain of Erwinia chrysanthemi.

Erwinia chrysanthemi insertion mutants were isolated that grew poorly specifically in the presence of glycine betaine (GB) or its analogues in high-salt media. Transposon insertions were found to affect the bspA gene, which forms an operon including the psd locus coding for phosphatidylserine decarboxylase. Initial GB uptake is not affected by the bspA mutation. However, in high-salt medium, its initial accumulation is followed by a reduced glucose uptake and a release of GB but not a loss of viability. BspA is homologous to the widespread MscS channel, YggB, but does not seem to constitute a mechanosensitive channel. We suggest that BspA is a protein sensing both intracellular GB and the extracellular salt content of the medium, the hypothesis being built on the observation that BspA is necessary to maintain the GB pool during osmoadaptation in high-salt media containing this osmoprotectant.

Carnitine acts as a compatible solute in Brevibacterium linens.

Carnitine is a trimethyl amino acid found at relatively high concentrations in materials of animal origin. Exogenously provided L-carnitine was found to stimulate growth of Brevibacterium linens ATCC 19391 in media with inhibitory osmotic strength. Its osmoprotective ability was as potent as that of glycine betaine. Electrophoretic and spectroscopic (NMR) analysis showed that this compound is only transiently accumulated, but in significant amounts, by B. linens under hyperosmotic stress and is converted into glycine betaine. The L-carnitine/glycine betaine pathway is inducible by L-carnitine in B. linens. The D-enantiomer did not improve growth of B. linens, even though this solute is accumulated by B. linens at the same level as glycine betaine. The two isomeric forms of carnitine repress the build-up of ectoine, the main endogenous osmolyte in B. linens.

Characterization of the Erwinia chrysanthemi osmoprotectant transporter gene ousA.

Growth of Erwinia chrysanthemi in media of elevated osmolarity can be achieved by the uptake and accumulation of various osmoprotectants. This study deals with the cloning and sequencing of the ousA gene-encoded osmoprotectant uptake system A from E. chrysanthemi 3937. OusA belongs to the superfamily of solute ion cotransporters. This osmotically inducible system allows the uptake of glycine betaine, proline, ectoine, and pipecolic acid and presents strong similarities in nucleotide sequence and protein function with the proline/betaine porter of Escherichia coli encoded by proP. The control of ousA expression is clearly different from that of proP. It is induced by osmotic strength and repressed by osmoprotectants. Its expression in E. coli is controlled by H-NS and is rpoS dependent in the exponential phase but unaffected by the stationary phase.

Prophage distribution in coryneform bacteria.

Four temperate bacteriophages of corynebacteria were isolated after UV induction. Phages phi 304L and phi 304S were both induced from Corynebacterium glutamicum ATCC 13058, ATCC 21488, ATCC 21649 and ATCC 21650 strains, and have no known sensitive host. Phages phi 15 and phi 16 were both induced from ATCC 14020 and ATCC 21792. Phage phi 15 formed turbid plaques on Corynebacterium sp. ATCC 21857 and on C. glutamicum ATCC 13058, ATCC 21488, ATCC 21649 and ATCC 21650. Phage phi 16 produced turbid plaques only on C. glutamicum ATCC 21792 cured of prophage phi 16. All these phages belong to the Siphoviridae family. Their genomes consist of a double-stranded DNA with cohesive ends and share no homology with each other. Prophages phi 16, phi 304L and phi 304S were integrated into their respective host chromosomes, whereas prophage phi 15 seemed to persist free in the cell. Cross-hybridizations between phage DNAs and total cellular DNA obtained from 20 strains belonging to the genera Corynebacterium and Brevibacterium did not show the presence of these prophages in strains other than their respective hosts.

Characterization of the osmotically inducible gene osmE of Escherichia coli K-12.

osmE, an osmotically inducible gene of Escherichia coli, was physically mapped on the bacterial chromosome, cloned and sequenced. osmE appeared to encode a 12,021 Da protein of unknown function, with a lipoprotein-type signal sequence at the amino-terminus. The osmE reading frame was confirmed by sequencing the junction of an osmE-phoA gene fusion. osmE was demonstrated to be transcribed as a single cistron. A phi [osmEp-lac] operon fusion was constructed, and analysis of its expression demonstrated that osmE osmotic regulation probably occurs at the transcriptional level. The osmE promoter was identified by both S1 nuclease and primer extension mapping of the 5' end of the osmE mRNA, by deletion analysis and by identification of a point mutation reducing its activity. Sequence information sufficient for expression and osmotic regulation is present on a DNA fragment extending from positions -37 to +52 with respect to the osmE transcription start. Uninduced expression of the osmE-lac fusion was increased in the presence of mutations in the hns and himA genes. The osmE promoter overlaps a promoter for a gene transcribed in the opposite direction, efg. Transcription from the efg promoter is only weakly affected by osmotic pressure and is independent of the presence of an intact OsmE protein.

Transfer of plasmid DNA to Brevibacterium lactofermentum by electrotransformation.

The Escherichia coli-Brevibacterium lactofermentum shuttle vector pBLA was introduced into intact cells of B. lactofermentum by electrotransformation. Several parameters of this procedure such as voltage and cell concentration were analysed. Optimal conditions gave an efficiency of 10(6) transformants per microgram of DNA. Two recalcitrant strains could be electrotransformed when an ampicillin pretreatment step was used. Electrotransformation experiments using DNAase or different structural forms of plasmid DNA showed that the electrotransformation process is quite different from natural transformation involving competence development. Restriction-modification-proficient B. lactofermentum could be efficiently electrotransformed with pBLA DNA isolated from E. coli. This restriction-modification system therefore seems to be overcome by electrotransformation. Thus electrotransformation may efficiently replace the protoplast bacterial transformation method.

Isolation and characterization of a restriction and modification deficient mutant of Brevibacterium lactofermentum.

In order to facilitate genetic engineering in amino-acid producing bacteria we have isolated two restriction-deficient Brevibacterium lactofermentum strains. They have been selected for their ability to obtain a high yield of plaques from CL31 phage which was grown on Corynebacterium lilium. These mutant strains do not restrict either phage DNA by transfection or DNA from the shuttle vector pBLA extracted from Escherichia coli by protoplast transformation. These mutants have also lost modification activity. We also report the presence of a restriction modification system in C. lilium ATCC 15990.

Characterization of the corynebacteriophage CG33.

Bacteriophage CG33 was isolated from a strain of Corynebacterium glutamicum that had become contaminated during an industrial fermentation. CG33 was assigned to Bradley's group B since it had a polyhedral head 40 nm wide and a short non-contractile and striated tail 78 nm long. Adsorption to its host, C. glutamicum ATCC 13287, was enhanced in the presence of Ca2+. The latent period was 18 min at 34 degrees C; the burst size was 16 p.f.u. ml-1. CG33 also formed plaques on C. lilium ATCC 15990 but at a low frequency. Its genome consisted of a linear double stranded DNA molecule of 13.4 kb with cohesive ends. A restriction map of the genome was obtained by using various endonucleases.