- Volume 140, Issue 12, 1994

Outer-membrane protein (OMP) profiles of two serotype A1 isolates of Pasteurella haemolyticawere compared by SDS-PAGE and Western blotting with bovine convalescent serum after growth (a) in vitrounder iron-sufficient and -deficient conditions, (b) in vivoin the lungs of experimentally infected calves and (c) in vivoin diffusion chambers implanted into the peritoneal cavities of calves. Lung-grown bacteria differed from iron-sufficient in vitro-grown bacteria in having enhanced expression of the previously recognized 71, 77 and 100 kDa iron-regulated proteins, reduced expression of 18, 31, 39.5 and 50 kDa proteins, and expression of a 19 kDa protein. Differences were also apparent in the Western blot profiles of OMPs of in vitro- and lung-grown bacteria. These included the apparent lack of recognition of the 100 kDa protein in the lung-grown bacteria, but not in the in vitro-grown bacteria, and more intense staining of a 47 kDa protein in in vitro-grown bacteria, but not in lung-grown bacteria. The OMP profiles of the chamber-grown bacteria resembled those of the lung-grown bacteria in that expression of the 18, 19, 31 and 39.5 kDa proteins was similar. These similarities demonstrated that the chamber-grown bacteria had adapted to the in vivoenvironment, and that growth conditions within the chambers resembled, but not perfectly, those within the lungs. For example, expression of the three iron-regulated OMPs was very low in the chamber-grown bacteria compared to the lung-grown bacteria. The OMP profiles of bacteria grown in vitroin newborn calf serum closely resembled those of lung-grown bacteria, suggesting that in vivogrowth may be partly reproduced in vitroby growing the bacteria in newborn calf serum.

In Corynebacterium glutamicum L-lysine is synthesized simultaneously via the succinylase and dehydrogenase variant of the diaminopimelate pathway. Starting from a strain with a disrupted dehydrogenase gene, three different-sized DNA fragments were isolated which complemented defective Escherichia colimutants in the succinylase pathway. Enzyme studies revealed that in one case the dehydrogenase gene had apparently been reconstituted in the heterologous host. The two other fragments resulted in desuccinylase activity; one of them additionally in succinylase activity. However, the physical analysis showed that structural changes had taken place in all fragments. Using a probe derived from one of the fragments we isolated a 3.4 kb BamHl DNA fragment without selective pressure (by colony hybridization). This was structurally intact and proved functionally to result in tenfold desuccinylase overexpression. The nucleotide sequence of a 1966 bp fragment revealed the presence of one truncated open reading frame of unknown function and that of dapEencoding N-succinyl diaminopimelate desuccinylase (EC 3.5.1.18). The deduced amino acid sequence of the dapEgene product shares 23% identical residues with that from E. coli.The C. glutamicumgene now available is the first gene from the succinylase branch of lysine synthesis of this biotechnologically important organism.

Streptomyces coelicolorATCC 10147 produced catalases whose electrophoretic mobility varied depending on the growth phase in liquid culture. Polyacrylamide gel electrophoresis of cell extracts resulted in six catalase activity bands, which were designated Cat1 to Cat6. Of these. Cat4 appeared during all growth phases, whereas Cat1 appeared only during the stationary phase. Catalase-deficient mutants were screened by the H2O2bubbling test following NTG mutagenesis. In all the non-bubbling mutants tested, the Cat4 activity band significantly decreased or disappeared, suggesting that Cat4 is the major catalase. Cat4 was purified to electrophoretic homogeneity and some of its properties analysed. The enzyme has a native molecular mass of 225 kDa, as determined by gel permeation column chromatography, and consists of four identical subunits of 57 kDa, as determined by SDS-PAGE. The enzyme contains 2.6 molecules of protohaem IX per tetramer, as indicated by the absorption spectrum. It was not reducible by sodium dithionite and exhibited no peroxidase activity with o-dianisidine as the substrate. All these characteristics, as well as inhibitor studies, indicate that the major vegetative catalase in S. coelicolor, unlike E. colivegetative catalase, is a member of the typical monofunctional catalases found in eukaryotes and some bacteria.

The 81 kDa protein (designated OpcPO) which forms a diffusion pore in the outer membrane of Burkholderia(formerly Pseudomonas) cepaciahas a unique characteristic in that when the purified protein is heated it yields a major 36 kDa protein (designated OpcP1) and a minor 27 kDa protein (designated OpcP2). Moreover, incubation of OpcPO in citrate buffer at pH 3.0 produced an unusual dissociation into 72 kDa and 27 kDa proteins. For the characterization of OpcPO and its derivatives, OpcP1 and OpcP2 from purified OpcPO were isolated by preparative SDS-PAGE. Reconstitution of OpcPO using purified preparations of OpcP1 and OpcP2 indicated that these derivatives were not proteolytic fragments of OpcPO. Moreover, immunoblot assays with murine polyclonal antisera specific for OpcP1 and OpcP2 yielded the following results: (i) OpcP1 and OpcP2 are immunologically distinguishable proteins; (ii) the unusual dissociation of OpcPO in citrate buffer at pH 3.0 resulted in the release of OpcP2 from OpcPO, and the resulting 72 kDa protein was probably an oligomer of OpcP1; (iii) purified OpcP1 itself produced two additional 53 kDa and 72 kDa proteins spontaneously following elution from the bottom of the SDS-PAGE gel. From these findings, it was concluded that OpcPO is formed by the non-covalent association of OpcP2 with an oligomer of OpcP1 that has the ability to self-assemble.

Nostocsp. strain ATCC 29133 (PCC 73102; Nostoc29133) is a symbiotically-competent, facultatively heterotrophic, diazotrophic cyanobacterium with the capacity to differentiate specialized cells such as heterocysts, akinetes and hormogonial filaments. We have optimized several methods for physiological and molecular genetic analysis of Nostoc29133. By use of a Tn5derivative, Tn5-1063 (KmrBmrSmr), delivered by conjugation from Escherichia coli, antibiotic-resistant mutants of Nostoc29133 were generated at a frequency of approximately 1 × 10−6, 0.4% of which expressed a nitrogen fixation (heterocyst) defective phenotype. Mutant strain UCD 328 was isolated after co-culture of 86 Nostoc29133::Tn5-1063 clones with the symbiotic plant partner, Anthoceros punctatus; strain UCD 328 expressed a symbiotic phenotype of increased frequency of hormogonia-dependent infection. The transposon and flanking genomic DNA was recovered from strain UCD 328, the mutation and phenotype reconstructed by homologous recombination in Nostoc29133, and the transposition site identified from a Nostoc29133 genomic library. Transposon mutagenesis has thus provided the means for isolation and identification of developmental and symbiotic-specific genes of Nostoc29133.

Exponentially growing Saccharomyces cerevisiaecells are more sensitive to oxidants such as hydrogen peroxide and superoxides than stationary phase cells. Using disruption mutations in the genes encoding the two S. cerevisiaesuperoxide dismutases, we show that the principal mechanism of toxicity of redox-cycling compounds, such as menadione and plumbagin, is via the production of superoxide anions. Using two-dimensional polyacrylamide gel electrophoresis we have compared the pattern of protein expression in cells labelled with L-[35S]methionine and stressed with either H2O2or menadione. Three groups of proteins were evident: those whose levels are elevated by both H2O2and menadione, and those specifically induced by either H2O2or menadione. Experiments with promoter fusions demonstrated that one of the heat inducible forms of HSP70 (SSA1) was inducible with H2O2. Furthermore, induction of the yeast H2O2-responsive TRX2promoter by menadione required the metabolism of menadione.

Auxotrophic derivatives of three strains of the pathogenic yeast Candida albicansof different origins, including 1006 derived from CBS5736, A5153 derived from FC18 and NARA2 derived from NUM961, were used in spheroplast fusion experiments. The DNA content of the prototrophic fusion product obtained following fusion between strains 1006 and A5153 approximated to the sum of those of the parents, but was variable when NARA2 was used as the parent for fusion. Chromosome-sized DNA molecules of the fusion derivatives were separated by pulsed-field gel electrophoresis to examine whether either or both of the chromosome-sized DNA molecules of each parent were transferred into the fusion derivatives. In the fusion derivatives obtained following fusion between strains 1006 and A5153, nearly the full complement of chromosomes was shown to be transferred, but partial transfer of chromosomes occurred in the fusion derivatives that were obtained following fusion between strains NARA2 and A5153. Results indicated that chromosome loss also occurred when these two strains were fused. Variations in the size of R chromosomes, the rDNA-containing chromosomes, were observed in all fusion derivatives tested, indicating high-frequency recombination between R chromosomes during the fusion process.

Polymerase chain reaction (PCR) was used to generate DNA encoding a 60 kDa stress protein of Mycobacterium paratuberculosisusing primers complementary to sequences at the 5′ and 3′ ends of 60 kDa stress protein genes (encoding the ‘65 kDa antigens’) of M. lepraeand M. tuberculosis.The predicted PCR product of 1.8kb contained the entire coding sequence of an M. paratuberculosis60kDa stress protein, with non-coding regions of 124bp and 1bp at the 5′ and 3′ ends, respectively. DNA encoding the entire ORF for the 60kDa stress protein, as well as thrombin and Factor Xa proteolytic cleavage sites, was ligated into the bacterial expression vector pGEX-2T and used to transform Escherichia colistrain JM83. Transformed bacteria, induced by IPTG, expressed an 85 kDa fusion protein comprising glutathione S-transferase (GST) and M. paratuberculosis60 kDa stress protein. This fusion protein was purified by adsorption to glutathione-agarose beads and shown to cross-react in Western blot analysis with an anti-mycobacterial 60 kDa stress protein monoclonal antibody. Recombinant M. paratuberculosis60 kDa stress protein was liberated from GST by proteolytic cleavage with either thrombin or Factor Xa enzyme. Authenticity of liberated recombinant stress protein was confirmed by N-terminal amino acid sequencing.

The dsbAgene of Escherichia coliencodes a periplasmic enzyme which catalyses disulfide bond formation. Analysis of its surrounding DNA region showed that it is preceded by an open reading frame, orfA, of 984 nucleotides. The intergenic region (19 nucleotides) carries no typical transcription termination signals. dsbAis transcribed from two promoters, the first (P1) lies in the distal part of orfA, and the second (P2) just upstream from orfA. Using a plasmid-borne dsbA:: TnphoAfusion and an orfA:: ω insertion, each promoter was shown to contribute equally to dsbAtranscription. The disruption of the single chromosomal copy of orfAby ω more drastically reduced the amount of DsbA in the periplasmic space. Such a reduction of the DsbA pool, however, did not change the activities of the AppA, Agp and PhoA periplasmic phosphatases, which all require disulfide bond formation, even when the enzymes were produced from multicopy recombinant plasmids. Thus, in a wild-type strain, DsbA is far from being in limiting amounts for physiological requirements. The orfAgene product was identified as a weakly expressed 39 kDa cytoplasmic protein, but it is not involved in the overall mechanism of disulfide bond formation.

Transcription start sites and processing sites of the Streptomyces coelicolorA3(2) rrnAoperon have been investigated by a combination of in vivoand in vitrotranscription analyses. The data from these approaches are consistent with the existence of four in vivotranscription sites, corresponding to the promoters P1-P4. The transcription start sites are located at −597, −416, −334 and −254 relative to the start of the 16S rRNA gene. Two putative processing sites were identified, one of which is similar to a sequence reported earlier in S. coelicolorand other eubacteria. The P1 promoter is likely to be recognized by the RNA polymerase holoenzyme containing σhrdB, the principal sigma factor in S. coelicolor.P2 also shares homology with the consensus for vegetative promoters, but has a sequence overlapping the consensus −35 region that is also present in the −35 regions of P3 and P4. The −35 sequence common to P2, P3 and P4 is not similar to any other known consensus promoter sequence. In fast-growing mycelium, P2 appears to be the most frequently used promoter. Transcription from all of the rrnApromoters decreased during the transition from exponential to stationary phase, although transcription from P1 and P2 ceased several hours before that from P3 and P4.

Streptomyces clavuligerusproduces the β-lactam antibiotics penicillin N, O-carbamoyldeacetycelcephalosporin C and cephamycin C. We characterized a wild-type DNA region which restores antibiotic formation to a mutant strain named NP1, previously shown to exhibit depressed activities for two early enzymes of cephalosporin synthesis, δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthase (IPNS). L-Lysine ε-aminotransferase (LAT) assays and α-AAA feeding experiments suggested that strain NP1 is a latmutant. NP1 recovered LAT, ACVS and IPNS activities when transformed with the cloned region. DNA sequencing showed that this region encodes the entire LAT gene (lat), required for the conversion of L-lysine to the β-lactam precursor L-α-aminoadipic acid (α-AAA), as well as the upstream half of the ACVS gene (pcbAB). The activities of ACVS and IPNS appear to depend upon LAT expression. Gene fusions constructed to investigate promoter activities in the cloned region support a model of interdependence in the expression of the genes for LAT, ACVS and IPNS (pcbC).

Hybridizing fragments in the genomic DNA of Streptomyces venezuelaeISP5230, which produces the jadomycin group of angucycline antibiotics, were detected by probing with actlDNA from Streptomyces coelicolorA3(2). The hybridizing regions were isolated from a 16.5 kb insert of S. venezuelae DNA recovered from a genomic library cloned in aλ replacement vector. Subcloning and sequencing of a 4.8 kb segment of the insert, containing regions hybridizing to actIIIas well as actl, identified five open reading frames (ORFs). The deduced polypeptide products of the ORFs closely resemble in sequence the components of streptomycete type-II polyketide synthases (PKSs): the ORF1 product corresponds to the ketoacyl synthase, and the ORF2 product to a polypeptide closely related to the ketoacyl synthase and involved in determining chain length; the ORF3 product matches the acyl carrier protein; ORF4 encodes a bifunctional cyclase/dehydrase; and ORF5 encodes a ketoreductase. Integration into the chromosomal DNA of a plasmid containing a segment of the ORF2-ORF4 region severely depressed jadomycin B biosynthesis; since the integrant showed no change in growth or spore pigmentation, the cloned PKS genes are presumed to encode enzymes in the pathway for jadomycin biosynthesis.