- Volume 142, Issue 8, 1996

Sequence analysis of the cloned 16S rRNA genes of Clostridium paradoxum DSM 7308T revealed the presence of 15 different sequences in variable region I ( Escherichia coli positions 73–97) of the 16S rDNA. The majority of the cloned genes contained intervening sequences (IVSs), which varied in length from 120–131 nt, and were present in the DNA obtained from single colonies of C. paradoxum. The absence of IVSs in the mature rRNA was demonstrated by Northern hybridization and sequence analysis of the 16S rRNA reverse transcriptase (RT)-PCR product. This finding was supported by the failure of oligonucleotide probes specific for certain IVSs to hybridize to the RT-PCR product obtained from C. paradoxum. Alterations in culture conditions (temperature, pH, salt) or culture age did not lead to expression of RNA containing IVSs, as indicated by the size of RT-PCR products. Hybridization of the restriction-enzyme-digested genomic DNA of C. paradoxum with probes derived from the IVSs demonstrated that the 16S rRNA genes containing different IVSs are located at different sites on the chromosome.

Acyl carrier protein (ACP) plays a crucial role in bacterial fatty acid synthesis. Cloning genes encoding ACPs from Gram-negative bacteria in Escherichia coli is difficult due to adverse effects of the cloned gene on host cell viability, and we were unsuccessful in cloning the full length ACP gene (acpP) from Azospirillum brasilense using conventional methods. Therefore, ACP from A. brasilense was purified to homogeneity and a part of the acpP gene was cloned using the polymerase chain reaction (PCR) technique with two primers, one designed from the N-terminal amino acid sequence of the purified ACP and the other from the highly conserved amino acid sequence of bacterial ACPs. The nucleotide sequence of the gene was obtained by cloning and sequencing inverse PCR products containing the acpP region generated by two oppositely oriented internal primers designed from the partial acpP gene sequence using restriction-enzyme-digested, self-circularized chromosomal DNA fragments as templates. Characterization of the purified ACP and analysis of the derived amino acid sequence of the acpP gene of A. brasilense revealed that: (a) the mature ACP, composed of 78 amino acids, is a highly expressed protein (about 2·0–3·0 × 104 molecules per cell), (b) compared to E. coli ACP, it has a more compact structure and contains significantly more hydrophobic amino acid residues and (c) the potential mRNA sequence of the ACP gene has some structural features typical of a stable mRNA.

The study of the intra-specific phylogenetic structure of Leuconostoc oenos is essential to understand the participation of several strains in malo-lactic fermentation (MLF). RFLP of the PCR-amplified 16S–23S rDNA intergenic spacer region (ISR) was performed in Leuc. oenos and other related species. The RFLP patterns with seven endonucleases were identical for the 37 Leuc. oenos strains, but differed from those obtained for all other species tested. This method could provide an invaluable insight for molecular identification of the wine leuconostocs. The RFLP relationships of members of the genera Leuconostoc and Weissella were highly similar to those previously reported by 16S and 23S rRNA sequencing studies. The 16S–23S rDNA ISR was sequenced in five strains of Leuc. oenos. A single tRNAAla was detected. The ISR sequence seems to be identical in the two rRNA (rrn) operons found in Leuc. oenos and no significant sequence variation was observed between strains that revealed relative differences as previously shown by PFGE. Results from the present study demonstrated that Leuc. oenos is phylogenetically a very homogeneous species (according to DNA-DNA hybridization studies) and sustain that this species is different from the genus Leuconostoc. The extremely conserved ISR of these organisms suggests that Leuc. oenos strains currently isolated and characterized must have spread with the transfer of viticulture rather than coming from indigenous populations.

The four enzymes needed to convert 3-hydroxybenzoate to pyruvate and fumarate via the gentisate pathway, as well as a putative positive regulator protein, were encoded on an 8 kb SphI fragment of Klebsiella pneumoniae DNA. The five genes were clustered in the order regulator-gentisate dioxygenase-fumarylpyruvate hydrolase-3-hydroxybenzoate monooxygenase-maleylpyruvate isomerase (mhbRDHMI), with the catabolic genes transcribed in the dioxygenase to isomerase direction. 2-Hydroxybenzoate was found to be a non-metabolizable inducer analogue for the mhb genes, supporting the view that gentisate rather than maleylpyruvate was the physiological inducer. The plasmid pNDR20 encoding the full gentisate catabolic pathway endowed Escherichia coli with the ability to grow on 3-hydroxybenzoate but the host cell appeared to be responsible for substrate uptake.

The outer membrane of a cyanobacterium (Synechococcus sp. strain PCC 7942) contains only a few major proteins. A gene encoding one of them, somA, was cloned and characterized. Based on the nucleotide sequence, SomA was predicted to comprise 531 amino acids with a calculated molecular mass of 57136 Da. The deduced amino acid sequence of SomA shares similarities with two bacterial cell-surface proteins, the S-layer protein of Thermus thermophilus and the flagellin of Campylobacter coli. The predicted amino acid sequence of SomA revealed also that it contains a signal peptide-like sequence at its N terminus. This signal peptide-like sequence was capable of mediating protein translocation across the cytoplasmic membrane into the outer membrane of Escherichia coli, provided that this sequence was fused to the E. coli outer-membrane protein, OmpF. The signal peptide-like sequence was cleaved upon the translocation of the SomA::OmpF protein. We suggest that SomA is synthesized as a precursor and that its N-terminal 24 amino acid sequence is a cleavable signal peptide involved in protein targeting into the outer membrane. To our knowledge, this is the first example of cleavable signal peptides for proteins transported into the outer membrane of cyanobacteria.

The non-haem chloroperoxidase gene (cpoF) from the pyrrolnitrin producer Pseudomonas fluorescens BL914 was cloned using an oligonucleotide derived from part of the N-terminal amino acid sequence of chloroperoxidase (CPO-P) from Pseudomonas pyrrocina as a probe. Based on the overexpression of cpoF in Escherichia coli and the stabilty of CPO-F against higher temperatures and proteases, the enzyme was purified to homogeneity. Partial characterization of the enzyme showed that it belongs to the class of bacterial non-haem CPOs. To investigate the role of CPO-F in pyrrolnitrin biosynthesis, the cpoF gene was inactivated by insertion of a kanamycin cassette. Exchange of the chromosomal cpoF gene against the disrupted copy had no influence on pyrrolnitrin production demonstrating that CPO-F was not involved in pyrrolnitrin biosynthesis.

Protein C (OprC) of the outer membrane of Pseudomonas aeruginosa forms small channels, as assayed by the liposome swelling method. We report here that OprC functions as a channel-forming and copper-binding protein. OprC purified to homogeneity formed a channel in planar lipid bilayers with an ion conductance of about 200 pS in 1 M NaCl. Cloning and sequencing of the gene encoding OprC revealed that it specified a polypeptide comprising 723 and 668 amino acid residues for the precursor and mature polypeptides (M r 73372), respectively. The amino acid sequence of OprC showed the highest degree of similarity with that of NosA of Pseudomonas stutzeri (65% sequence identity) which conveys Cu2+ to intracellular acceptor(s). OprC showed high copper-binding activity (K d = 2·6 μM) in aqueous solution containing surfactant. The expression of OprC appeared to be repressed by exogenous Cu2+ and derepressed by anaerobiosis in the presence of nitrate. These results suggest that OprC might be involved in copper utilization.

Nutrients are major determinants of ribosomal protein (rp-) gene transcription in Saccharomyces cerevisiae . In order to investigate the molecular mechanisms underlying this nutritional control, yeast mutants that display defects in the glucose upshift response of rp-gene transcription were isolated. Interestingly, although growth of these mutants on glucose-containing medium was severely affected an initial increase in rp-gene transcription by nutritional upshift was still observed. However, at later time points, rp-mRNA levels decreased strongly. Various other types of severe growth limitation also did not prevent the initial upshift in transcription. The results suggest that the glucose upshift response of rp-gene transcription comprises two phases: an initial, transient response independent of the actual growth potential, and a sustained response which is dependent on growth and requires both glucose and adequate nitrogen sources. Previously, it was found that protein kinase A (Pka) mediates the initial upshift response, without the need for regulation of Pka activity by cAMP. The present data substantiate that, besides the RAS/adenylate cyclase pathway, an alternative pathway through Pka regulates rp-gene transcription. In addition, evidence is presented that the sustained response does not require Pka activity. Based on these results, taken together, a model is proposed in which rp-gene transcription is dynamically regulated by multiple signal transduction pathways.

Saccharomyces cerevisiae has a limited life span, measured by the reproductive capacity of the individual cell. Several genes that are differentially expressed during the yeast life span have been isolated. One of these genes, LAG2, has been characterized for its role in longevity. LAG2 is preferentially expressed in young cells. It encodes a predicted 680 amino acid protein with a putative transmembrane helix. The sequence does not show significant similarity to any other DNA or protein sequences in the databases. Deletion of LAG2 in a haploid strain did not affect growth, but it resulted in a 50% decrease in the mean and maximum life span. When LAG2 was overexpressed, the mean and maximum life span of the yeasts was extended by about 36% and 54%, respectively. These results indicate that this is a longevity-assurance gene in yeast.

A low-fibronectin-binding mutant of Staphylococcus aureus strain 879R4SSp generated by transposon Tn918 mutagenesis is attenuated in a rat endocarditis model (J. M. Kuypers & R. A. Proctor, 1989, Infect Immun 57, 2306–2312). PCR and Southern hybridization analysis with primers and probes, respectively, for the fnbA and fnbB genes of strain 8325–4 showed that strain 879R4SSp possesses a single fnb gene which is homologous to fnbA. This was confirmed by sequencing 41 bp of 5′ non-coding and 237 bp of 5′ coding DNA, which showed 97% base identity to fnbA. Southern hybridization and sequencing showed that Tn918 was inserted 41 bp 5′ to fnbA in the mutant 879R4SSp/1536, between the promoter and initiation codon. Reduced adherence of the mutant to surface-bound fibronectin correlated with lower expression of a 180 kDa wall-associated fibronectin-binding protein.

In chemostat culture, the microaerophilic, CO2 requiring, gingival-plaque-associated bacterium Capnocytophaga gingivalis responded to the addition of glucose (1–6 g l−1) by doubling its growth rate and increasing its biomass yield fivefold. The data suggest that the glucose is catabolized by a fully aerobic route. Rather than repressing hydrolytic enzymes which might be associated with pathogenic properties, glucose enhanced the specific activity of aminopeptidase, trypsin-like protease, acid and alkaline phosphatase and α-glucosidase in comparison with a control culture grown in a tryptone/thiamin medium. Thus, the supply of glucose could be of importance in maximizing the pathogenic potential of this organism.

A large cluster of virulence genes encoding proteins involved in Vibrio cholerae accessory colonization factor (ACF) expression and toxin-coregulated pilus (TCP) biogenesis is flanked by sequences that resemble bacteriophage attachment (att) half-sites. Adjacent to the atfL-like site is a gene (int) that encodes a protein related to the integrase family of site-specific recombinases. The putative vibrio integrase appears to be most closely related to the Escherichia coli cryptic prophage (CP4-57) integrase protein (52% identity, 73% similarity). Genomic analysis of numerous V. cholerae strains (01, non-01 and 0139) revealed that only vibrios capable of causing epidemic Asiatic cholera possess the TCP-ACF colonization gene cluster in association with the integrase. The fact that the integrase gene is absent in avirulent strains suggests that epidemic strains of V. cholerae obtained the TCP-ACF colonization gene cluster via horizontal transfer.

The adhesion of Candida albicans to polystyrene and the effect of three monoclonal antibodies (mAbs) reactive with C. albicans cell wall surface antigens on this process was assessed in vitro with several C. albicans strains. In the absence of mAbs, adhesion of C. albicans to polystyrene increased in parallel with germ-tube formation. However, the growth of the strains in the yeast phase at 25 or the use of an agerminative mutant inhibited adhesion to polystyrene. Serotype A and B strains showed similar kinetics of adhesion to polystyrene and no statistically significant differences in germination or adhesion were observed when strains from the two serotypes were compared. The three mAbs had different effects on both germination and adhesion of C. albicans. mAb 3D9 showed no influence on either germination or adhesion to polystyrene in two C. albicans strains. mAb B9E decreased both adhesion (45·6%) and filamentation (52·6%), and mAb 21E6 decreased filamentation (34·0%) but enhanced adhesion by 23·3%. This enhancement was also observed with the agerminative mutant and it was dose-dependent. It was not related to the binding capacity of the MAb to polystyrene nor to an increase in cell surface hydrophobicity of the antibody-treated cells. In conclusion, both growth phases of C. albicans can adhere to polystyrene, although the conditions for this process seem to be different in each phase. The two types of adhesion of C. albicans to polystyrene might have a role in the colonization of medical implants. The disparate effects shown by mAbs directed against cell wall mannoproteins of C. albicans on the adhesion of the fungus to polystyrene should be taken into consideration when designing strategies to block the adhesion of C. albicans to plastic materials with mAbs.

Group A streptococci bind the serine protease plasmin with high affinity. Previously, a 41 kDa protein was identified as a candidate plasmin receptor protein (Plr) from group A streptococcal strain 64/14. The plr gene encoding Plr was cloned and the deduced amino acid sequence of Plr had significant similarity to glyceraldehyde-3-phosphate dehydrogenases (GAPDHs). In this study we have isolated cytoplasmic GAPDH of streptococcal strain 64/14. This enzyme was examined, on both structural and functional levels, for its relatedness to the Plr of strain 64/14 purified from mutanolysin extract and to recombinant Plr. We report here that no differences were detected between streptococcal Plr and cytoplasmic GAPDH on the basis of antibody reactivity, plasmin-binding activity, GAPDH activity, N-terminal amino acid sequence, peptide map analysis by V8 protease digestion and amino acid composition analysis. Furthermore, the plr gene appears to be present as a single copy in group A streptococci.

Escherichia coli possesses regulatory mechanisms that coordinate cell growth with the synthesis of essential macromolecules (protein, RNA and DNA). While fundamental differences have been identified in the growth habit and chromosome structure of E. coli and Streptomyces, little is known about these regulatory mechanisms in filamentous bacteria. This paper reports on the relationship between the macromolecule content of S. coelicolor A3(2) and its specific growth rate. The protein, RNA and DNA contents (g per 100 g biomass) of S. coelicolor A3(2) grown in steady-state continuous culture over a range of specific growth rates (0·025–0·3 h−1) were 31–45, 10–22 and 3·5–4·5% (w/w), respectively. This composition is qualitatively similar to that of other microorganisms. Changes in the macromolecular content of S. coelicolor A3(2) and E. coli B/r with specific growth rate appear to be essentially similar. However, the data indicate that the RNA content of S. coelicolor A3(2), grown under the conditions used, exceeds that of E. coli grown at the same specific growth rate. The data also suggest that overlapping rounds of replication are not a feature of DNA synthesis in S. coelicolor A3(2). This may be a function of the organism’s low maximum specific growth rate. Alternatively, it may be a consequence of regulatory mechanisms which act to inhibit the initiation of DNA synthesis in a linear chromosome which is already undergoing replication.

m-Aminophenylboronic acid (APBA) inhibited the germination, growth and sporulation of Streptomyces griseus NRRL B-2682 in an age- and concentration-dependent manner in submerged and solid cultures. When added to cells or cell extracts it irreversibly inhibited NAD+-glycohydrolase and ADP-ribosyltransferase activity. ADP-ribosyltransferase was more sensitive, but inhibition was not complete, even in the presence of 10 mM APBA. The in vivo effects of the inhibitor correlated with its in vitro effect on ADP-ribosylation and on the profile of ADP-ribosylated endogenous proteins. The physiological importance of ADP-ribosyltransferase was supported by the observation that APBA strongly inhibited the growth of a non-sporulating and NAD+-glycohydrolase-negative mutant of the parental strain. The resistance of S. griseus NRRL B-2682 strains able to grow in the presence of APBA was due to permeability factors. A comparison of the ADP-ribosylated protein profiles of S. griseus NRRL B-2682 grown under various conditions showed similarities, but also specific differences. The results suggest that the ADP-ribosyltransferase of S. griseus NRRL B-2682 is an indispensable enzyme for growth and differentiation of the strain. It may regulate the activity of key enzymes or developmental proteins by responding to intra- and extracellular conditions.

In streptomycetes, the branched-chain amino acids leucine, isoleucine and valine may serve as precursors for commercially important polyketides, and it is of interest to investigate whether the availability of these amino acids affects the production of the secondary metabolites derived from them. This paper reports studies on end-product control in the model organism Streptomyces coelicolor of the enzymes acetohydroxy acid synthase (AHAS) and isopropylmalate synthase (IPMS), mediating steps in the pathways to isoleucine-valine and leucine respectively. Specific activities of both enzymes were similarly affected when minimal medium was supplemented with the amino acids singly or in combination. Isoleucine alone caused a 2- to 3-fold increase, while all three amino acids caused a 5- to 8-fold decrease. Growth of an ilv auxotroph in media with limiting isoleucine gave enzyme specific activities 4- to 6-fold higher than in unsupplemented minimal medium. Spontaneous mutants were obtained by growing S. coelicolor on minimal medium containing 4-azaleucine. At least four patterns of end-product control were found among the mutants, one of which showed high constitutive levels of both enzymes (7- and 15-fold above unsupplemented minimal medium values for AHAS and IPMS respectively). It is concluded that the variation in specific activities of the two enzymes under different physiological and genetic conditions spans a range of around 50 to 100, and that S. coelicolor has molecular mechanisms capable of producing this response.