Glycine betaine-assisted protein folding in a lysA mutant of Escherichia coli.

Osmoprotectants exogenously supplied to a hyperosmotic culture medium are efficiently imported and amassed by stressed cells of Escherichia coli. In addition to their evident role in the recovery and maintenance of osmotic balance, these solutes should play an important role on the behavior of cellular macromolecules, for example in the process of protein folding. Using a random chemical mutagenesis approach, a conditional lysine auxotrophic mutant was obtained. The growth of this mutant was restored by addition of either lysine or osmoprotectants including glycine betaine (GB) in the minimal medium. The growth rate increased proportionally with the augmentation of the intracellular GB concentration. The mutation was located in the lysA gene and resulted in the substitution of the Ser at position 384 by Phe of the diaminopimelate decarboxylase (DAPDC), which catalyzes the conversion of meso-diaminopimelate to L-lysine. We purified both the wild type DAPDC and the mutated DAPDC-sf and demonstrated that GB was capable of activating DAPDC-sf in vitro, thus confirming the in vivo results. Most importantly, we showed that the activation was correlated with a conformational change of DAPDC-sf. Taken together, these results show, for the first time, that GB may actively assist in vivo protein folding in a chaperone-like manner.

Analysis of the integration functions of phi304L: an integrase module among corynephages.

Plasmid p12929 was shown to integrate into the chromosome of Corynebacterium glutamicum RM3 and BL15. The minimal integrating fragment was subsequently defined. The arms flanking the integrated plasmid (attL and attR) were identified, allowing for the determination of the attP and the attB attachment sites. The attB site is located at the 3' end of an ORF presenting 62-78% identity with L19 ribosomal proteins. Integration in the attB site does not result in the inactivation of this gene because its end is also present on the attR arm of the integrated plasmid and is reconstituted. The minimal integrating fragment is 1663 bp long and contains two ORFs. The int ORF was identified as phi304L integrase on the basis of the amino acid homologies it shared with the tyrosine recombinases of the lambda integrase family. Moreover this integrase is highly homologous throughout its sequence with the integrase of phi16 corynephage, the percentage of identity reaching 89% at the NH2 end. The identity also extends upstream of the initiation codon, while both phages are elsewhere nonhomologous. An integrase module was proposed to explain this extensive homology.

Cloning and sequence determination of a phi AAU2 gene whose product aborts the phage lytic cycle.

This communication describes the cloning of a 1.8-kb fragment from the genome of the corynephage phi AAU2, which aborts the phage lytic cycle when cloned on a high-copy shuttle vector. The associated phenotype, called Apld (aborting phage lytic development), was revealed by noting the reduced plaque size and lower efficiencies of plaquing of phi AAU2 cp, a virulent derivative of phi AAU2, on "Arthrobacter aureus"-C70 Apld+ cells. Adsorption and phage DNA transfection experiments showed evidence that Apld acted once the phage DNA had entered into the cell; apld was confined to a single open reading frame (ORF), encoding a putative 63-aa polypeptide which did not show any homology to proteins contained in the databanks; apld is followed by an ORF the product of which shows homology with a protein expressed by the early region of the Streptomyces phage phi C31.

Genetic characterization of site-specific integration functions of phi AAU2 infecting "Arthrobacter aureus" C70.

All the essential genetic determinants for site-specific integration of corynephage phi AAU2 are contained within a 1,756-bp DNA fragment, carried on the integrative plasmid p5510, and are shown to be functional in Escherichia coli. One open reading frame, ORF4, encoding a protein of 266 amino acids was shown to represent the phi AAU2 integrase. The nucleotide sequence of the phi AAU2 attachment site, attP, and the attB, attL, and attR sequences in the host "Arthrobacter aureus" C70 were determined. Identical nucleotide sequences were shown to be responsible for the integration of p5510 in the chromosomes of Corynebacterium glutamicum, Brevibacterium divaricatum, and B. lactofermentum, and a sequence almost identical to attB was found to be present in these three strains. In contrast to other phage site-specific recombination systems, a plasmid encompassing only int-attP failed to integrate into the host chromosome. This led to the identification of an 800-bp noncoding region, immediately upstream of int, absolutely required for site-specific integration of p5510.

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.

Structural organization of the Corynebacterium glutamicum plasmid pCG100.

pCG100, a 3 kb cryptic plasmid of Corynebacterium glutamicum ATCC 13058, probably identical with pSR1 from C. glutamicum ATCC 19223, was characterized. The minimum region for autonomous replication was shown to be contained on a 1.9 kb BglII-NcoI fragment; a 380 bp HindIII-SphI fragment can replicate in the presence of the parental plasmid, which presumably provides a trans-acting replication factor. Derivatives of pCG100 are able to replicate in several Corynebacterium, Brevibacterium and Arthrobacter strains. pCG100 is compatible with pBL1, a cryptic plasmid of Brevibacterium lactofermentum. Shuttle plasmid vectors, containing the kanamycin-resistance gene from Tn903 or from Streptococcus faecalis as selectable markers and the AmpR, TetR or lacZ alpha genes for insertional inactivation, were constructed using the minimum replication fragment of pCG100.

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.

Isolation and preliminary characterization of twenty bacteriophages infecting either brevibacterium or arthrobacter strains.

Thirty-seven bacteriophages plaquing on Corynebacterium, Brevibacterium, or Arthrobacter strains were isolated from soil or vegetation samples. Restriction analysis of phage DNA indicated that 20 phages were unique; one of them produced entirely turbid plaques on Brevibacterium ketoglutamicum and was characterized as temperate. All these phages were assigned to group B of the classification of Bradley (Bacteriol. Rev. 31:230-314, 1967) and had relatively narrow host ranges.

Plasmids with the uidA reporter gene for the detection of promoters and transcription signals.

We have constructed promoter-probe plasmids, pNB4 and pNB5, based on the promoterless gene for beta-glucuronidase (uidA) of Escherichia coli. Unique restriction sites for EcoRI, SacI, KpnI, SmaI, XmaI, XbaI, SalI, SphI and HindIII in pNB4 and for HindIII, PstI and BglII in pNB5 were included upstream of the uidA structural gene. The usefulness of these plasmids was demonstrated by cloning the promoter-operator region of the E. coli uxaB gene. We observed that expression of the uxaB-uidA operon fusion followed the transcription-regulating properties of the uxaB promoter. Another construct, pNB2, can be used to detect operator and terminator signals. Recipient cells transformed with such recombinant plasmids can be revealed by growth on medium containing a chromogenic beta-glucuronidase substrate.

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.

Structural characteristics of the Corynebacterium lilium bacteriophage CL31.

Bacteriophage CL31 was isolated on a Corynebacterium lilium strain. Out of 30 strains tested, only CL31 was able to form plaques on Corynebacterium glutamicum ATCC 13287, Brevibacterium lactofermentum ATCC 21086, and Arthrobacter sp. strain SI55, but at a very low frequency. This phage belongs to group B of Bradley's classification (D. E. Bradley, Bacteriol. Rev. 31:230-314; 1967). Its head is 53 nm in diameter, and its tail is 396 nm in length. The phage capsid contains three major proteins, of 12.5, 29.0, and 37.0 kilodaltons, and five minor ones (23.9, 26.0, 27.0, 40.0, and 55.4 kilodaltons). CL31 DNA is a linear molecule of 48 kilobases with cohesive ends. Restriction mapping was performed for endonucleases BglII, EcoRI, SalI, and KpnI. The expression of CL31 genes in Escherichia coli was studied by the maxicell technique; 12 different proteins were detected.

Thirteen Virulent and Temperate Bacteriophages of Lactobacillus bulgaricus and Lactobacillus lactis Belong to a Single DNA Homology Group.

Thirteen virulent phages and two temperate phages of two closely related bacterial species (Lactobacillus lactis and L. bulgaricus) were compared for their protein composition, their antigenic properties, their restriction endonuclease patterns, and their DNA homology. The immunoblotting studies and the DNA-DNA hybridizations showed that the phages could be differentiated into two groups. One group contained 2 temperate phages of L. bulgaricus and 11 virulent phages of L. lactis. Inside each group, at least two common proteins of identical sizes could be detected for each phage. These proteins were able to cross-react in immunoblotting experiments with an antiserum raised against one phage of the same group. Temperate phage DNAs showed partial homology with DNAs from some virulent phages. These homologies seem to be located on the region coding for the structural proteins since recombinant plasmids coding for one of the major phage proteins of one phage were able to hybridize with the DNAs from phages of the same group. These results suggest that temperate and virulent phages may be related to one another.

Physical and genetic characterization of the genome of Lactobacillus lactis bacteriophage LL-H.

Bacteriophage LL-H is a virulent phage of Lactobacillus lactis LL23. A restriction map of the phage genome was constructed with various restriction endonucleases. This chromosome has a 34-kilobase size and seems to be circularly permuted. We used a bank of LL-H restriction fragments to study the expression of five of the seven main phage particle proteins. Immunoblotting experiments permitted the mapping on the chromosome of several genes coding for phage particle proteins. We also show that the gene of the main capsid protein is expressed from its own promoter in an Escherichia coli strain.