Brigitte Cami

Sequence analysis of the cellulase-encoding celY gene of Erwinia chrysanthemi: a possible case of interspecies gene transfer

The Erwinia chrysanthemi (strain 3937) celY gene encoding the minor endoglucanase (EGY) was sequenced. The analysis of the upstream region allowed us to identify an in vivo active promoter recognized by the NtrA (sigma 54) holoenzyme. No similarity was found between the predicted amino acid (aa) sequences of EGY and either the Er. chrysanthemi major endoglucanase, EGZ, or the Er. carotovora CelS endoglucanase. In contrast, a very high level of identity, both at the nucleotide and the predicted aa levels, was found between celY and an EG-encoding gene from Cellulomonas uda, a Gram + bacterium taxonomically distant from Er. chrysanthemi. By comparing the molar G + C% of the cellulase-encoding genes and that of Er. chrysanthemi and C. uda chromosomal DNAs, we speculate that celY was transferred from Er. chrysanthemi to C. uda.

Homology between endoglucanase Z of Erwinia chrysanthemi and endoglucanases of Bacillus subtilis and alkalophilic Bacillus

Nucleotide sequencing of the celZ gene encoding the extracellular endoglucanase Z of Erwinia chrysanthemi indicated the presence of an open reading frame encoding 428 amino acids. The mature protein appeared to be extended by a signal peptide of 43 amino acids; this sequence is unusually long and positively charged (+5). It was shown to function as a signal peptide by fusing it to a truncated phoA gene encoding Escherichia coli alkaline phosphatase. Comparison of the encoded sequence with those of the endoglucanases of Bacillus subtilis and alkalophilic Bacillus revealed the existence of a region of extensive homology occurring in all three proteins at about the same distance from the NH2‐terminal end. These regions may be involved in substrate binding and/or catalytic sites.

Transfer and expression of an Erwinia chrysanthemi cellulase gene in Zymomonas mobilis

SUMMARY: The cellulase gene from Erwinia chrysanthemi coding for endoglucanase Z was subcloned into a broad-host-range plasmid pGSS33 in Escherichia coli. The recombinant pNB20 was transferred into Zymomonas mobilis ATCC 10988 by mobilization using the helper plasmid RP4. Plasmid pNB20 was stably maintained in E. coli and Z. mobilis hosts. The endoglucanase gene celZ was expressed efficiently and the level of expression was higher in Z. mobilis than in E. coli. The specific activity of the enzyme was comparable to that of the parent strain of Er. chrysanthemi. The proteins produced by Z. mobilis and Er. chrysanthemi presented identical immunological and electrophoretic properties. Biosynthesis of endoglucanase occurred during the exponential growth phase of Z. mobilis and about 35% of the enzyme was released into the medium in the absence of detectable cell lysis. The endoglucanase appeared to be located in the periplasmic space in Z. mobilis.

UPTAKE OF INORGANIC CARBON IN THE CYANOBACTERIUM SYNECHOCYSTIS PCC6803 : PHYSIOLOGICAL AND GENETIC EVIDENCE FOR A HIGH-AFFINITY UPTAKE SYSTEM

Synechocystis PCC6803 displays two inorganic carbon‐uptake processes, a low‐affinity one (apparent Km: 300–400 µM) functional in cells grown under standard or limiting inorganic carbon concentrations, and one with a higher affinity (60±12 µM), detected only in cells adapted to limiting inorganic carbon conditions. A mutational and screening procedure allowed the isolation of a mutant deficient in the high‐affinity system, but only slightly impaired in its growth capacities. The mutated genomic region revealed two open reading frames (ORFs), possibly belonging to an operonic structure. A clone in which the downstream ORF, hatR (high‐affinity transport), had been inactivated showed a phenotype close to that of the original mutant. Inactivation of the other ORF, hatA, yielded a clone unable to grow in limiting inorganic carbon conditions. The deduced HatA protein showed no homology with any registered protein. It possessed three hydrophobic domains, including a putative signal peptide. Several hypotheses are considered as to its role. The deduced HatR protein, which possessed the features characteristic of the response regulators of the two‐component regulatory systems ubiquitous in bacteria, might be a regulator controlling the activity of the high‐affinity transport process. It would belong to the subclass of these molecules lacking the DNA‐binding domain.

Cloning in Escherichia coli of genes involved in the synthesis of proline and leucine in Desulfovibrio desulfuricans Norway

SummaryA library of Deusulfovibrio desulfuricans Norway genomic DNA was constructed in Escherichia coli with pBR322 as vector and plasmids able to complement the proA and leuB mutations of the host were screened. It was observed that all the plasmids studied were highly unstable, the insert DNA being rapidely lost under non-selective growth conditions. A 2.75 kb DNA fragment of D. desulfuricans Norway was found to complement E. coli ProA, ProB and ProC deficiencies. From the results of restriction analysis and Southern hybridization, it is proposed that the genes involved in proline and leucine biosynthesis are clustered on the chromosome of D. desulfuricans Norway.

Heterologous expression and regulation of the lysA genes of Pseudomonas aeruginosa and Escherichia coli.

SummaryThe Pseudomonas aeruginosa lysA gene encoding diaminopimelate decarboxylase (DAP-decarboxylase) was cloned into a broad host range vector. This gene complemented a lys mutation at the lys-12 locus of P. aeruginosa and a lysA defect in Escherichia coli. The P. aeruginosa DAP-decarboxylase was synthesized constitutively in P. aeruginosa as well as in E. coli, where the Pseudomonas lysA gene was poorly expressed. By contrast, the E. coli lysA gene was expressed well in P. aeruginosa and subject to lysine regulation when the E. coli LysR activator protein was provided. This indicates that the mechanism of transcriptional activation for the E. coli lysA gene is effective in the heterologous host.

A protein is involved in accessibility of the inhibitor acetazolamide to the carbonic anhydrase(s) in the cyanobacterium Synechocystis PCC 6803

A gene, zam (for resistance to acetazolamide), controlling resistance to the carbonic anhydrase inhibitor acetazolamide, is described. It has been cloned from a spontaneous mutant, AZAr-5b, isolated from the cyanobacterium Synechocystis PCC 6803, for its resistance to this drug (Bédu et al., Plant Physiol 93: 1312–1315, 1990). This mutant, besides its resistance to acetazolamide, displayed an absence of catalysed oxygen exchange activity on whole cells, suggestive of a deficiency in carbonic anhydrase activity. The gene was isolated by screening a genomic library of AZAr-5b, and selecting for the capacity to transfer the AZAr phenotype to wild-type cells. A system leading to forced homologous recombination in the host chromosome, using a platform vector, was devised in order to bypass direct selection difficulties. The putative encoded protein, 782 amino acids long, showed some homology with four eukaryotic and prokaryotic proteins involved in different cellular processes, one of them suppressing a phosphatase deficiency. The mutated allele of AZAr-5b showed an in-frame 12 nucleotide duplication, which should not interfere with translation, and might result from transposition of a mobile element. Integration into a wild-type genome of either the spontaneous mutated allele or one inactivated by insertional mutagenesis conferred the character of resistance, but not the deficiency in oxygen exchange, indicating that the two phenotypic aspects of AZAr-5b corresponded to two independent mutations. A working hypothesis explaining the phenotypes of the mutants is that the presence of the Zam protein would be necessary for the inhibitor to reach (one of) the two carbonic anhydrases present in this strain. This, however, would be a secondary action, the physiological role of the protein still being cryptic.

A lipopeptide‐encoding sequence upstream from the IysA gene of Pseudomonas aeruginosa

An open reading frame (ORF) of 141 bp was observed upstream from the Pseudomonas aeruginosa lysA gene. The translation product of this ORF contains a signal peptide with a lipoprotein box, fle‐Ala‐Ala‐Cys, at the predicted signal peptidase cleavage site. The Escherichia coli phoA gene without its signal sequence was fused in frame to this ORF in a broad host‐range plasmid. The resulting construct expressed a hybrid protein exhibiting alkaline phosphatase activity in phoA mutants of both E. coli and P. aeruginosa. This indicates that the ORF encodes a peptide, part of which acts as an export signal. The hybrid peptide was identified by immunoblotting with alkaline phosphatase antiserum. The accumulation of a precursor form was observed when P. aeruginosa cells carrying this gene fusion on a plasmid were treated with globomycin. Moreover, the mature form could be labelled with 2‐[3H]‐glycerol, indicating that lipidic residues may be linked to the hybrid protein. Taken together, these results strongly suggest that the ORF encodes a lipopeptide. We propose that the gene is called IppL.

Possible involvement of a L-Δ1-pyrroline-5-carboxylate (P5C) reductase in the synthesis of proline in Desulfovibrio desulfuricans Norway

A L-delta 1-pyrroline-5-carboxylate reductase activity has been detected in crude extracts of Desulfovibrio desulfuricans Norway. This P5C reductase activity is also found when a 2.5 kb D. desulfuricans DNA fragment is introduced into an Escherichia coli proC mutant. Although it restores growth of the proC mutant, the ProDd enzyme might be detrimental to the E. coli host since the plasmid carrying the cognate proDd gene is segregated at high rate by the cells but is stabilized by small deletions which lead to a loss of the P5C reductase activity.