- Volume 141, Issue 3, 1995

Ureases of the cyanobacteria Leptolyngbya boryana (Plectonema boryanum) PCC 73110 and AnabaenalNostoc PCC 7120 were purified more than 1500-fold to homogeneity by heat treatment and liquid chromatography, reaching specific activities of up to 350 U (mg protein)-1. Both enzymes had a molecular mass of 220 kDa, as shown by native PAGE, and consisted of three subunits (α, β, γ) with molecular masses of 66 kDa (α), 18 kDa (Leptolyngbya; β) or 14 kDa (AnabaenalNostoc; β) and 11 kDa (γ). The enzyme of Leptolyngbya exhibited maximum activity at pH 8.2 and 60 °C, and that of nabaenalNostoc at pH 8.5 and 65 °C; the K m was 0.25 mM urea for both organisms. Almost identical specific activities were observed in cells grown with urea, ammonia, nitrate or dinitrogen as the source of nitrogen. The ureases from both organisms were heat-stable; no loss of activity was observed during incubation at 70 °C for 15 min.

Summary: Albicidin antibiotics specifically block prokaryote DNA replication. The albicidin resistance gene (albB) cloned from a soil isolate of Alcaligenes denitrificans encodes a 23 kDa protein capable of detoxifying albicidin by reversible binding. This mechanism operates intracellularly to protect DNA replication in albicidin-sensitive Escherichia coli expressing the cloned resistance gene, which can be induced fivefold in the presence of 1·5 μg albicidin ml-1 in the surrounding medium. The coding region of 621 bp has regions with partial DNA sequence homology to an albicidin resistance gene (albA) from Klebsiella oxytoca, but with rearrangements and frame-shifts resulting in loss of protein homology. There is a short region of N-terminal homology between the albicidin resistance (Albr) proteins from A. denitrificans and K. oxytoca, although the two genes use different codons for shared amino acids. The N-terminal homology suggested a common functional domain; this was confirmed by deletion analysis, translational fusions and albicidin binding by a synthetic oligopeptide. Antibiotic binding provides a high level of albicidin resistance in E. coli. The gene appears to be a useful candidate for transfer to plants to protect plastid DNA replication from inhibition by albicidin phytotoxins involved in sugarcane leaf scald disease.

Insecticidal crystal proteins, or protoxins, of Bacillus thuringiensis are composed of two domains, an amino-terminal half essential for toxicity, and a carboxy-terminal half with an as yet unassigned function. To define the boundary of the two domains, sequential termination codons were introduced from the 3′-end of the DNA sequence encoding the toxic domain of the 1155-residue cry1A(b) gene product. The mutated and the intact genes were placed under the control of the Escherichia coli inducible promoter PrecA, and toxicity of the cell extracts was determined using silkworm larvae. Under non-induced conditions, in which the gene products accumulated to a limited degree, mutations encoding 606 amino acid residues or more were toxic, whereas those encoding 605 residues or less were non-toxic. Comparison of the toxicities and the levels of the toxic proteins suggested that the mutant proteins had comparable activity to that of the intact protoxin. Furthermore, the non-toxic protein seemed to be unstable inthe extracts. To investigate the roles of the non-toxic domain, the mutant proteins were overproduced in both E. coli and B. thuringiensis. The intact and the mutated genes carrying natural promoters were introduced into acrystalliferous B. thuringiensis. Upon induction of PrecA in E. coli, and upon sporulation in B. thuringiensis, there was a large accumulation of gene products which formed inclusion bodies. The inclusion bodies of the intact protoxin were active, whereas those of the mutant proteins were inactive. Inclusion bodies of the intact protein could be solubilized in alkali, whereas the mutant inclusion bodies were insoluble. Since solubilization under alkaline conditions in the insect midgut is considered to be the first step of toxic action, the non-toxic domain is required to direct the synthesis of inclusion bodies as an active soluble form.

Bacillus stearothermophilus α-amylase has a signal peptide typical for proteins exported by Gram-positive bacteria. There is only one signal peptidase processing site when the protein is exported from the original host, but when it is exported by Escherichia coli, two alternative sites are utilized. Site-directed mutagenesis was used to study the processing in E. coli. Processing sites for 13 B. stearothermophilus α-amylases carrying mutations in their signal peptide were determined. Processing of the signal peptide was remarkably tolerant to mutations, because switching between the alternative sites was possible. The length and the sequence of the region between the hydrophobic correspe and the cleavage site was crucial for determining the choice of the processing site. Some mutations more distal to the cleavage site also affected the site preference.

The streptococcal plasmid pMV158 replicates by a rolling circle mechanism, which involves the generation of single-stranded plasmid DNA intermediates. This plasmid has the unique feature of having two lagging-strand origins of replication. One of these origins, termed ssoU , is functional in Streptococcus pneumoniae and in Bacillus subtilis in an orientation-dependent manner. The other origin, ssoA, is only functional in the former host. RNA polymerase seems to be involved in the initiation of the conversion of single- to double-stranded plasmid DNA from both ssoA and ssoU. Mutational and deletion analyses have allowed us to define ssoA as being within a highly structured, non-coding 199 bp region. Within this region, two elements which are conserved in several rolling-circle replicating plasmids are located, the recombination site RSB and a 6 base consensus sequence. Both elements may play a role in the conversion of single- to double-stranded plasmid DNA.

Saccharomycopsis fibuligera is a dimorphic yeast, which is both saccharolytic and fermentative, that is used in the production of rice wine. It has a predominant diploid phase. When grown on solid agar S. fibuligera strains develop different morphological forms. In previous studies, intergeneric hybrids between S. fibuligera and a related yeast, Yarrowia lipolytica, were obtained which were able to utilize starch and tributyrin. A putative haploid mitotic segregant, N14i60 met, was obtained from the intergeneric hybrid between S. fibuligera 193 met and Y. lipolytica A his1. Auxotrophic mutants were readily isolated following UV mutagenesis of N14i60 met. The auxotrophic mutants and N14i60 have a similar morphology. Protoplast fusants were produced between the auxotrophic mutant A6 met lys1 arg1 and Y. lipolytica 21501-4 B lys5 leu2 ade1 xpr2 and between specific pairs of the auxotrophic mutant strains. Differences and similarities in the DNA banding patterns of these genetically different strains of S. fibuligera and Y. lipolytica were demonstrated and it was established that four distinct types consistent with their specific positions in the pedigree chart could be clearly distinguished. Furthermore, by a study of the patterns of hybridization signals for specific genes of S. fibuligera for an intergeneric hybrid and its mitotic segregants, genetic segregants with different characteristics were obtained. The segregants from the intergeneric hybrid and the protoplast fusants probably arose by a process of chromosomal assortment at mitosis. The intergeneric hybrid, the protoplast fusants and their mitotic segregants showed a similar karyotype. Together these studies provide an explanation for the basis of phenotypic differences of some of the yeast strains studied.

Clostridium chauvoei strain Okinawa produced spontaneous non-motile variants at an unusually high rate (approx. 10-4 per generation) under normal conditions without mutagen. Revertants of non-motile variants were detected at a rate of approximately 10-3. Biochemically, every variant correspresponded well with the parental strain. By transmission electron microscopy, three of nine non-motile variants of strain Okinawa were found to be flagellate, while the other six were found to be aflagellate. These phenotypes were confirmed by Western blot analysis using monoclonal antibodies directed against the flagella of C. chauvoei. Moreover, the parental flagellate strain and non-motile flagellate variants were significantly more virulent in mice than non-motile, aflagellate variants. Our results demonstrated that phase variation in motility and flagellation occurs in C. chauvoei, and that the flagella are associated with the full expression of virulence.

The mtr (multiple transferable resistance) system of Neisseria gonorrhoeae determines levels of gonococcal resistance to hydrophobic agents (HAs), including detergent-like fatty acids and bile salts that bathe certain mucosal surfaces. The genetic organization of the mtr system was determined and found to consist of the mtrR gene, which encodes a transcriptional regulator (MtrR), and three tandemly linked genes termed mtrCDE. The mtrCDE genes were organized in the same apparent transcriptional unit, upstream and divergent from the mtrR gene. The mtrCDE-encoded proteins of N. gonorrhoeae were analogous to a family of bacterial efflux/transport proteins, notably the MexABOprK proteins of Pseudomonas aeruginosa and the AcrAE and EnvCD proteins of Escherichia coli, that mediate resistance to drugs, dyes, and detergents. Inactivation of the mtrC gene resulted in loss of the MtrC lipoprotein and rendered gonococci hypersusceptible to structurally diverse HAs; this revealed the importance of the mtr system in determining HAR in gonococci. Further support for a role of the mtrCDE gene complex in determining levels of HAR in gonococci was evident when transformants bearing mutations in the mtrR gene were analysed. In this respect, missense and null mutations in the mtrR gene were found to result in increased levels of MtrC and HAR. However, high levels of MtrC and HAR, similar to those observed for clinical isolates, were associated with a single bp deletion in a 13 bp inverted repeat sequence that intervened the divergent mtrR and mtrC genes. We propose that the 13 bp inverted-repeat sequence represents a transcriptional control element that regulates expression of the mtrRCDE gene complex, thereby modulating levels of gonococcal susceptibility to HA.

Pulsed-field gel electrophoresis of the genomic DNA of penicillin-resistant strains of Streptococcus pneumoniae was carried out. Eleven clinical strains of serogroup 9 from different French towns and Paris hospitals were tested. The restriction enzymes Apa I and Sma I were used to digest intact chromosomes, and the fragments were resolved by field-inversion gel electrophoresis (FIGE). Five strains were similar using Apa I and SmaI. Four others were closely related when using Apa I, and five others were closely related when using SmaI. These results suggest that 10 of these strains are genetically related and have a clonal origin. The profile of the eleventh strain was completely different. Thus, in a given serotype the spreading of penicillin resistance can result from both clonal and independent events. Five strains had similar FIGE profiles to strains first isolated in Spain, suggesting that a resistant strain had spread from Spain to France.

Evidence for the participation of the glutamine transaminase-ω-amidase pathway and a glutaminase in the utilization of glutamine in Rhizobium etli has been obtained. The glutamine transaminase preferentially transaminates glyoxylate and pyruvate. Glutamine transaminase activity was similar under all growth conditions tested except on PY (rich medium) where it was low. Glutaminase activity was positively regulated by glutamine and negatively regulated by ammonium and by the carbon source. In R. etli bacteroids, glutamine transaminase was low, whereas glutaminase activity was high. Ammonium liberated from glutamine was assimilated by glutamine synthase, thus leading to the operation of a glutamine cycle that consumes ATP. Our results suggest that glutamine transaminase plays a biosynthetic role in the irreversible synthesis of glycine and alanine, whereas glutaminase plays a catabolic role in the degradation of glutamine to carbon skeletons and to maintain the optimal balance between glutamine and glutamate. The high glutaminase activity found in bacteroids indicates that the degradation of glutamine by this enzyme may play an important role during symbiosis between R. etli and Phaseolus vulgaris.

A miniature electrode was used to measure, for the first time, the time-dependent change in dissolved oxygen concentration of small-scale cultures of two actinomycete species at various aeration efficiencies in both complex and defined media. Erythromycin was produced in both oxygen-limited and oxygen-sufficient conditions in shaken flask and inclined tube cultures of Saccharopolyspora erythraea and a further, novel, secondary metabolite was produced only under oxygen limitation. In contrast, vancomycin was only produced in oxygen-sufficient cultures of Amycolatopsis orientalis. Similar results were obtained in batch bioreactor cultures. These findings indicate that oxygen limitation acts in an analogous manner to substrate limitation imposed by dissolved nutrients, stimulating secondary metabolite production in some cases and inhibiting it in others. The implications of these findings in screening programmes for novel secondary metabolites are discussed.

Stress tolerance of Saccharomyces cerevisiae was examined after exposure to heat and salt shock in the presence or absence of the protein synthesis inhibitor cycloheximide. Cells heat-shocked (37 °C for 45 min) in the absence of cycloheximide demonstrated increased tolerance of heat, freezing and salt stress. For cells heat-shocked in the presence of cycloheximide, heat and salt tolerance could still be induced, although at lower levels, while induction of freezing tolerance was completely inhibited. These results indicated that while heat shock proteins (hsps) may contribute to induced heat and salt tolerance they are not essential, although induction of freezing tolerance appears to require protein synthesis. Exposure of cells to salt shock (300 mM NaCI for 45 min) induced stress protein synthesis and the accumulation of glycerol, responses analogous to induction of hsp synthesis and trehalose accumulation in cells exposed to heat shock. Cells salt-shocked in the absence of cycloheximide showed a similar pattern of induced stress tolerance as with heat, with increased tolerance of heat, salt and freezing. Cells salt-shocked in the presence of cycloheximide continued to show induced heat and salt tolerance, but freezing tolerance could not be induced. These results lend support to the hypothesis that hsp synthesis is not essential for induced tolerance of some forms of stress and that accumulated solutes such as trehalose or glycerol may contribute to induced stress tolerance.

Control of the pH of liquid synthetic culture media made possible mycelial growth of both diploid and haploid strains ofUstilago maydis. Whereas at neutral pH the fungus grew as a homogeneous population of budding yeast-like cells (sporidia), at acid pH it developed in the mycelial form. Mycelial cells appeared branched and narrower than yeast cells. Cell morphology was affected by the carbon and nitrogen sources. When the culture medium was removed continuously or intermittently, very long, filamentous cells were formed. Colonies of haploid strains developed aerial mycelium (‘fuzz’ morphology) on acid solid medium. Nullb, bW, andbEmutants behaved in the same way as haploid wild-type strains. It is suggested that growth at low pH overcomes the control processes governed by heterologousbEandbWloci.

Summary: Strain ASV2, an unidentified Gram-negative bacterium newly isolated from activated sludge, was found to utilize arsonoacetate at concentrations up to at least 30 mM as sole carbon and energy source, with essentially quantitative extracellular release of arsenate. Cell-free conversion of arsonoacetate could not be obtained, but resting-cell studies indicated that the carbon-arsenic bond cleavage activity was inducible in the presence of arsonoacetate and was of limited substrate specificity, also breaking down arsonochloroacetate. The inorganic product of the reaction may be arsenite since an inducible arsenite-oxidizing activity was also found in arsonoacetate-metabolizing cells. This is the first report of a micro-organism capable of utilizing a compound containing the carbon-arsenic bond. The results indicate that the ability of bacteria to degrade arsonoacetate is not fortuitous and may be found in environments not previously exposed to organoarsenicals.

23Na NMR spectroscopy was used to determine free Na+ concentrations in a halotolerant bacterium, Brevibacterium sp., and Escherichia coli. The internal Na+ concentration of both strains depended little on the growth phases and was unchanged after 5 d storage at 2°C. In Brevibacterium sp. the level of intracellular sodium increased gradually at higher extracellular NaCI concentrations in both the presence and absence of yeast extract in the growth medium. E. coli cells accumulated a higher concentration of free Na+ than those of Brevibacterium sp. The change of Na+ concentration in both strains was inverse to that of growth rate. When appropriate amounts of osmoprotectants (proline, glycine betaine, or γ-aminobutyrate) were added with the NaCI, internal free Na+ levels in Brevibacterium sp. were lowered, but those of E. coli were unchanged. While addition of KCl to medium containing NaCI increased the intracellular level of free Na+, the total sodium concentration in the cells remained unchanged, indicating that sodium that had been bound or attached was made free in the cytosol. In Brevibacterium sp. grown in the presence of 0.5 M NaCI, free and bound sodium concentrations in the cytosol were estimated to be 0.14 and 0.23 μmol (mg protein)-1, respectively. As a result, visibility by 23Na NMR was 38%.

Summary: The secretion of several hemicellulolytic enzymes and endoglucanase was studied in S- and P-group strains of a wood-rotting pathogen, Heterobasidio annosum, using enzyme activity assays and isoelectric focusing. In liquid cultures supplemented with birch sawdust or pure xylan, both strains of the fungus produced xylanases, mannanases, endoglucanases and α-galactosidases. Low activities were detected in cultures containing only glucose as a carbon source. When S-strain cultures were provided with a cart matrix, protein secretion to the culture medium started earlier and was significantly increased compared to free cell cultures. This was not the case with the P-strain. In both strains of H. annosum the pH of the birch sawdust culture medium fell within 12 d to below 3.5, and then started to increase towards the end of the cultivation period. The increase in pH appeared to corresprelate with the formation of hemicellulolytic and endoglucanase activitie. The isoenzyme patterns of xylanase, mannanase and endoglucanase were typical for P- and S-strains. In P-strain cultures containing xylan, a xylanase with an alkaline pl value was induced which, however, was absent when the strain was grown with the birch sawdust substrate. In the P-strain the mannanase pattern at the early stage of growth was comparable to that of t S-strain at the later stages of growth on birch sawdust medium. Later in the growth of the P-strain, this pattern was replaced by mannanases with more acid pl values. The relative intensities of the individual endoglucanases varie between the P- and S-strains.

Summary: Two transposon Tn5-induced mutants of wild-type broad-host-range Rhizobium sp. GRH2 were isolated and found to harbour different alterations in surface polysaccharides. These mutants, designated GRH2-14 and GRH2-50, induced a few, empty nodules on Acacia and lost the ability to nodulate most host herbaceous legumes. Whereas mutant GRH2-14 produces an acidic exopolysaccharide (EPS) similar to the wild-type, the acidic EPS of mutant GRH2-50 lacks galactose and the pyruvyl and 3-hydroxybutyryl substituents attached to this sugar moiety. In addition, both mutants GRH2-50 and GRH2-14 were altered in smooth lipopolysaccharides (LPS). DNA sequence analyses of the correspresponding Tn5 insertions revealed that strain GRH2-50 was mutated in a DNA locus homologous to galE , and in vitro enzyme assays indicated that the UDPglucose 4-epimerase (GalE) activity was missing in this mutant strain. DNA hybridization studies showed that the GRH2-50 mutant DNA has homologous sequences within the different biovars of Rhizobium leguminosarum. However, no DNA homology to GRH2-14 altered DNA was found in those rhizobial strains, indicating that it represents a new chromosomal lps locus in Rhizobium sp. ( Acacia) involved in symbiotic development.

A large operon-type structure has been located between the gltA and citB loci on the Bacillus subtilis chromosome. On the basis of the analysis of the 25 kb sequenced so far, it potentially encodes at least three large proteins which contain structural motifs associated with the subunits of all characterized peptide synthases. The amino acid recognition specificity of this new peptide synthase is discussed in the light of sequence homology with other synthases.

Variations in trichothecene patterns of 26 Fusarium sporotrichioides isolates from different plant and geographic origins showed no corresprelation with electrophoretic karyotype polymorphisms. When intact chromosomes were examined, interisolate karyotype differences were observed only in the mini-chromosome range. Further polymorphisms were revealed in Notl-digested samples. By summing the Notl fragments the average genome size of F. sporotrichioides was estimated to be 20.4 Mb. Mini-chromosomes shared common sequences with the larger ones; however, clones (RMS-1 and RMS-2) specific to these structures have also been found. These clones contained no coding region and no promising similarities were observed when they were compared to sequences held at GenBank. Mini-chromosomes in F. sporotrichioides constitute a mosaic composed of dispersed repeats and unique sequences. This mosaic structure was maintained in all non-interbreeding, genetically isolated strains examined.