- Volume 144, Issue 4, 1998
Volume 144, Issue 4, 1998
- Genetics And Molecular Biology
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Oligopeptide permease in Borrelia burgdorferi: putative peptide-binding components encoded by both chromosomal and plasmid loci
More LessTo elucidate the importance of oligopeptide permease for Borrelia burgdorferi, the agent of Lyme disease, a chromosomal locus in B. burgdorferi that encodes homologues of all five subunits of oligopeptide permease has been identified and characterized. B. burgdorferi has multiple copies of the gene encoding the peptide-binding component, OppA; three reside at the chromosomal locus and two are on plasmids. Northern analyses indicate that each oppA gene is independently transcribed, although the three chromosomal oppA genes are also expressed as bi- and tri-cistronic messages. Induction of one of the plasmid-encoded oppA genes was observed following an increase in temperature, which appears to be an important cue for adaptive responses in vivo. The deduced amino acid sequences suggest that all five borrelial OppA homologues are lipoproteins, but the protease-resistance of at least one of them in intact bacteria is inconsistent with outer-surface localization. Insertional inactivation of a plasmid-encoded oppA gene demonstrates that it is not essential for growth in culture.
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Tandem organization and highly disparate expression of the two laccase genes lcc1 and lcc2 in the cultivated mushroom Agaricus bisporus
More LessTwo non-allelic laccase genes (lcc1 and lcc2) in Agaricus bisporus have been mapped to the same cosmid clone and are close together, in tandem. The intergenic region consists of 1562 bp between the stop codon of lcc1 and the start codon of lcc2. Differences between the 5′ non-coding regions of the two genes suggest the potential for their differential regulation. By employing competitive RT-PCR and specific primer pairs that discriminate between lcc1 and lcc2, it has been shown that the level of lcc2 mRNA is approximately 300 times higher than that of lcc1 mRNA in malt extract liquid cultures; in compost cultures lcc2 mRNA is almost 7000 times more abundant than lcc1 mRNA.
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Molecular analysis of Physarum haemagglutinin l: lack of a signal sequence, sulphur amino acids and post-translational modifications
More LessThe cDNAs encoding haemagglutinin I from plasmodia of Physarum polycephalum have been cloned using PCR protocols. The composite haemagglutinin I cDNA sequence, derived from several overlapping clones from PCR fragments, spans 408 nt and the 315 bp ORF encodes a polypeptide of 104 aa without a typical signal sequence. The putative molecular mass deduced from the amino acid sequence (10760.76 Da) corresponds exactly to that determined by electrospray ionization MS (10759.86±.15 Da), suggesting that haemagglutinin I is not subject to post-translational modification. Haemagglutinin I lacks sulphur amino acids and has a β-sheet as the major secondary structure. Expression of the coding sequence in Escherichia coli yielded a product that exhibits the same sugar-binding specificity as natural haemagglutinin I. The deduced amino acid sequence shows little similarity to that of any known lectins and thus apparently represents a novel type of lectin.
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Identification of a CAP (adenylyl-cyclase-associated protein) homologous gene in Lentinus edodes and its functional complementation of yeast CAP mutants
The adenylyl-cyclase-associated protein, CAP, was originally identified in yeasts as a protein that functions in both signal transduction and cytoskeletal organization. This paper reports the identification of a cDNA and genomic DNA that encodes a CAP homologue from the mushroom Lentinus edodes. The L. edodes cap gene contains eight introns and an ORF encoding a 518 amino acid protein. The L. edodes CAP is 35.5% and 40.9% identical at the amino acid level with Saccharomyces cerevisiae CAP and Schizosaccharomyces pombe CAP, respectively. The C-terminal domain shows greater homology (39-46% identity) with yeast CAPs than does the N-terminal domain (27-35% identity). Southern blotting and Northern blotting results suggest that L. edodes cap is a sinμle-copy gene and uniformly expressed. Expression of the L. edodes CAP in both Schiz. pombe and Sacch. cerevisiae complemented defects associated with the loss of the C-terminal domain function of the endogenous CAP. By using a yeast two-hybrid assay, an interaction was demonstrated between the L. edodes CAP and Schiz. pombe actin. This result and the functional complementation test indicate that CAP from L. edodes has a conserved C-terminal domain function.
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D-Amino-acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217
The complete nucleotide sequence of the DAO1 gene encoding D-amino-acid oxidase (DAAO) in the yeast Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217 has been determined. The primary structure of DAAO was deduced from the nucleotide sequence of a cDNA clone that covered the entire amino acid coding sequence. Comparison of cDNA and genomic sequences of DAO1 revealed the presence of five introns. Because this is the first gene of strain ATCC 26217 that has been cloned so far, the nucleotide sequences of these introns were compared to those from other fungi. Upstream of the structural gene there was a stretch of C + T-rich DNA similar to that found in the promoter region of a number of yeast genes. The cDNA gene, which encoded a protein of 368 amino acids (molecular mass 40 kDa), was overexpressed in Escherichia coli under the control of the strong lipoprotein promoter. Interestingly, a significant fraction (13-62%) of the total DAAO activity was recovered in its apoenzyme form, the percentage depending on the culture conditions. This fact allowed a rapid purification of the recombinant DAAO by affinity chromatography. The high level of expression achieved in E. coli and the possibility of modifying its catalytic properties by protein engineering provide a new model for the study of this enzyme.
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- Pathogenicity And Medical Microbiology
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Candida dubliniensis: phylogeny and putative virulence factors
More LessCandida dubliniensis is a recently identified species which is implicated in oral candidosis in HIV-infected and AIDS patients. The species shares many phenotypic characteristics with, and is phylogenetically closely related to, Candida albicans. In this study the phylogenetic relationship between these two species was investigated and a comparison of putative virulence factors was performed. Four isolates of C. dubliniensis from different clinical sources were chosen for comparison with two reference C. albicans strains. First, the distinct phylogenetic position of C. dubliniensis was further established by the comparison of the sequence of its small rRNA subunit with representative Candida species. The G dubliniensis isolates formed true unconstricted hyphae under most induction conditions tested but failed to produce true hyphae when induced using N-acetylglucosamine. Oral G dubliniensis isolates were more adherent to human buccal epithelial cells than the reference C. albicans isolates when grown in glucose and equally adherent when grown in galactose. The G dubliniensis isolates were sensitive to fluconazole, itraconazole, ketoconazole and amphotericin B. Homologues of seven tested G albicans secretory aspartyl proteinase (SAP) genes were detected in G dubliniensis by Southern analysis. In vivo virulence assays using a systemic mouse model suggest that G dubliniensis is marginally less virulent than C. albicans. These data further confirm the distinct phenotypic and genotypic nature of G dubliniensis and suggest that this species may be particularly adapted to colonization of the oral cavity.
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The Staphylococcus aureus and Staphylococcus epidermidis transferrin-binding proteins are expressed in vivo during infection
More LessStaphylococci express a 42 kDa cell-wall-associated protein which functions as a receptor for the mammalian iron-binding glycoprotein transferrin. To determine whether this transferrin-binding protein (TBP) is expressed during infection, Staphylococcus aureus and Staphylococcus epidermidis were grown in vivo in chambers implanted intraperitoneally in rats. SDS-PAGE and Western blotting of cell wall proteins prepared from staphylococci recovered directly from the chambers revealed the presence of both the TBP and bacterial-surface-associated rat transferrin. To obtain evidence for the in vivo expression of the staphylococcal TBPs in humans, sera and human peritoneal dialysate (HPD) from non-infected patients undergoing continuous ambulatory peritoneal dialysis (CAPD) and sera from healthy human volunteers were screened for anti-TBP antibodies. Western immunoblots revealed that three out of ten samples from the latter group, seven out of ten HPD samples and ten of ten CAPD patient serum samples contained antibodies to the TBP of both S. aureus and S. epidermidis. To gain further insights into the appearance of TBP antibodies, HPD samples were collected over time from CAPD patients whose HPD samples taken immediately after catheter insertion lacked anti-TBP antibodies. In two of these patients, each of whom experienced an episode of peritonitis due to S. epidermidis or Staphylococcus hominis, antibodies to the TBP appeared in the HPD collected immediately post-infection. To determine whether such TBP antibodies were capable of blocking interactions between transferrin and its staphylococcal receptor, HPD immunoglobulin fractions were purified using protein A-Sepharose beads. In competition assays, these immunoglobulins blocked the binding of 125l-labelled transferrin both to whole bacteria and to the isolated 42 kDa TBPs of S. aureus and S. epidermidis. These provide evidence to show that staphylococcal TBPs are expressed in vivo during infection.
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The rrs (16S)-rrl (23S) ribosomal intergenic spacer region as a target for the detection of Haemophilus ducreyi by a heminested-PCR assay
More LessThe intergenic spacer region between the rrs and rrl ribosomal RNA genes of Haemophilus ducreyi was analysed and the DNA sequence was used for the selection of specific PCR primers. A highly sensitive and specific heminested-PCR assay for the identification of H. ducreyi was developed. The assay showed a sensitivity of 96% on genital ulcer specimens from patients with clinically diagnosed chancroid, compared with a sensitivity of 56% for culture methods. These results indicate that this PCR assay has the potential to become an accurate and easy reference method for the detection of H. ducreyi.
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Carbapenems as inhibitors of OXA-13, a novel, integron-encoded β-lactamase in Pseudomonas aeruginosa
More LessA clinical Pseudomonas aeruginosa strain, PAe391, was found to be resistant to a number of antibiotics including ticarcllin, piperacillin, cefsulodin and amikacin, and a disk diffusion assay showed evidence of pronounced synergy between imipenem and various β;-lactam antibiotics. Cloning and nucleotide sequence analysis revealed the dicistronic arrangement of an aac(6’)-lb variant and a novel bla OXA-type gene between the intl and qacEδ1 genes typical of integrons. In PAe391, this integron was apparently chromosome-borne. The β;-lactamase, named OXA-13, displayed nine amino acid changes with respect to OXA-10: I in position 10 of OXA-10 to T (I10T), G20S, D55N, N73S, T107S, Y174F, E229G, S245N and E259A. OXA-13 (plapp = 8.0) showed poor catalytic activity against penicillins as well as cephalosporins, but was efficient in hydrolysing some penicillinase-resistant β;-lactams, such as cefotaxime and aztreonam. It was efficiently inhibited by imipenem (K iapp = 11 nM), and formed a stable complex. While the K iapp value of meropenem was similar (16 nM), the corresponding complex was less stable.
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- Physiology And Growth
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Effects on Bacillus subtilis of conditional expression of the accBC operon encoding subunits of acetyl coenzyme A carboxylase, the first enzyme of fatty acid synthesis
More LessA Bacillus subtilis strain was constructed in which the operon accBC, encoding the biotin carboxyl carrier protein (BCCP) and biotin carboxylase (BC) subunits of acetyl-CoA carboxylase (ACC), was placed under the control of the IPTG-inducible promoter spac. A reduction in the levels of BCCP resulted in a decrease of de novo fatty acid synthesis and in the total content of membrane fatty acids. This strain was dependent upon the presence of IPTG for a normal growth phenotype. Growth was specifically restored by supplying exogenous long branched-chain fatty acids in the medium, indicating that the inducer-dependent phenotype was specifically related to a conditional block in fatty acid biosynthesis. The strain showed a strong decrease in sporulation frequency when it was induced to sporulate in an IPTG-free medium. Germination and outgrowth were both delayed in spores of the mutant obtained in the absence of IPTG.
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The cadmium-stress stimulon of Escherichia coli K-12
More LessThe influence of cadmium on stress protein production in Escherichia coli K-12 (strain MG1655) was analysed using two-dimensional polyacrylamide gel electrophoresis and the gene-protein database of E. coli K-12. Cadmium (273 μM) caused complete but transient inhibition of growth accompanied by the synthesis of cadmium-induced proteins (CDPs). It was found that some CDPs induced during the growth-arrested phase belong to the heat-shock, oxidation stress, SOS and stringent response regulons, while others are general stress inducible proteins (e.g. H-NS, UspA). In addition, trigger factor, adenylate kinase, W-protein, the cold shock protein G041.2, and seven unknown proteins whose synthesis is not known to be controlled by a global regulator, were identified as immediate responders to cadmium exposure. The rate of synthesis of most of the immediate responders to cadmium exposure decreased when the growth of the cells resumed. However, seven CDPs, including those encoded by argl, tyrA and xthA, maintained a high production rate during growth in the presence of cadmium. Two of the unidentified proteins were N-terminally sequenced by Edman degradation. The N-terminal amino acid sequence of one of these proteins (designated F023.3) matches the E. coli open reading frame o216. This ORF is similar to the N-terminal third of the copper-binding protein amine oxidases (encoded by maoA) of both E. coli and Klebsiella pneumoniae (K. aerogenes). The other N-terminally sequenced protein (designated C044.6) matches perfectly the product of the metK gene, S-adenosylmethionine synthetase I. In comparison to untreated cells, cadmium-stressed cells were found to recover more rapidly during subsequent stress conditions, such as ethanol, osmotic, heat shock, and nalidixic acid treatment. The role of the CDPs is discussed in view of their physiological assignments in the cell.
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Phytophthora cactorum can synthesize substances needed for sexual reproduction but requires a stress factor to trigger the process
More LessPhytophthora cactorum grown on basal agarose medium or in liquid basal medium produced oospores after being transferred to water agarose. The numbers of oospores produced under such conditions depended on the age of the culture prior to exposure to nutrient deprivation. When the concentration of basal medium used for cultivation of P. cactorum was increased, the numbers of oospores produced after being transferred to water agarose was also increased. P. cactorum grown on basal agarose medium also produced oospores when its mycelial growth was restricted after reaching the edge of Petri plates. In 5 cm plates oospore formation occurred in the third week, whereas in 9 cm and 14 cm plates oospores appeared in the fourth week. Most oospores were formed near the edge of the plates. The non-saponifiables extracted from mycelia of P. cactorum grown in liquid basal medium were stimulatory to oospore formation by P. cactorum and Phytophthora parasitica, whereas the saponifiables were stimulatory to P. cactorum only. Extracts from culture filtrate and basal medium were not stimulatory to oospore formation by either fungus. When the non-saponifiables were fractionated by Florisil columm chromatography, only fraction 1 was not active. Fractions 2, 3 and 4 were stimulatory to oospore formation by both P. cactorum and P. parasitica. These results support the hypothesis that P. cactorum, and possibly other pythiaceous fungi as well, can synthesize substances needed for sexual reproduction but requires a stress factor to trigger the process.
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Lack of correlation between trehalose accumulation, cell viability and intracellular acidification as induced by various stresses in Saccharomyces cerevisiae
More LessA pma1-1 mutant of Saccharomyces cerevisiae with reduced H+-ATPase activity and the isogenic wild-type strain accumulated high levels of trehalose in response to a temperature upshift to 40 éC and after addition of 10% ethanol, but only modest levels in response to a rapid drop in external pH and after addition of decanoic acid. There was, however, no correlation between the absolute levels of trehalose in the stressed cells and their viability. All these treatments induced a significant decrease in intracellular pH, and surprisingly, this decrease was very similar in both strains, indicating that intracellular acidification could not be the triggering mechanism for trehalose accumulation in response to stress. A careful investigation of metabolic parameters was carried out to explain how trehalose accumulated under the four different stress conditions tested. No single and common mechanism for trehalose accumulation could be put forward and the transcriptional activation of TPS1 was not unequivocally related to trehalose accumulation. Another finding was that a pma1-1 mutant exhibited a two- to threefold greater capacity to accumulate trehalose than the isogenic wild-type. This enhanced disaccharide synthesis could be attributed to a twofold higher trehalose-6-phosphate synthase activity, together with a fourfold higher content of intracellular UDP-Glc. In addition, this mutant showed 1.5-fold higher levels of ATP compared to the wild-type. The various stress treatments studied showed that a drop in intracellular pH does not correlate with trehalose accumulation. It is suggested that plasma membrane alteration could be the physiological trigger inducing trehalose accumulation in yeast.
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- Genome Analysis
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A 35.7 kb DNA fragment from the Bacillus subtilis chromosome containing a putative 12.3 kb operon involved in hexuronate catabolism and a perfectly symmetrical hypothetical catabolite-responsive element
The Bacillus subtilis strain 168 chromosomal region extending from 109† to 112° has been sequenced. Among the 35 ORFs identified, cotT and rapA were the only genes that had been previously mapped and sequenced. Out of ten ORFs belonging to a single putative transcription unit, seven are probably involved in hexuronate catabolism. Their sequences are homologous to Escherichia coli genes exuT, uidB, uxaA, uxaB, uxaC, uxuA and uxuB, which are all required for the uptake of free D-glucuronate, D-galacturonate and β-glucuronide, and their transformation into glyceraldehyde 3-phosphate and pyruvate via 2-keto-3-deoxygluconate. The remaining three ORFs encode two dehydrogenases and a transcriptional regulator. The operon is preceded by a putative catabolite-responsive element (CRE), located between a hypothetical promoter and the RBS of the first gene. This element, the longest and the only so far described that is fully symmetrical, consists of a 26 bp palindrome matching the theoretical B. subtilis CRE sequence. The remaining predicted amino acid sequences that share homologies with other proteins comprise: a cytochrome P-450, a glycosyltransferase, an ATP-binding cassette transporter, a protein similar to the formate dehydrogenase α-subunit (FdhA), a protein similar to NADH dehydrogenases, and three homologues of polypeptides that have undefined functions.
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