A simple, convenient method of growing large quantities of Candida albicans chlamydospores on a cellulose dialysis membrane has been developed. Long, narrow, cylindrical suspensor cells bearing spherical to ovoid chlamydospores were observed. Ultrastructural observations showed the chlamydospore to have a bilayered cell wall made up of an outer electron-transport primary layer and an inner electron-dense secondary layer, a large portion of the total cell volume occupied by a single large vacuole and several smaller vacuoles, and cytoplasmic organelles typical of those observed in the yeast-like cell. There are structural similarities between the region of chlamydospore-suspensor cell connection and septa observed in budding yeast-like cells.
An insertion sequence (IS) element of Brucella ovis, named IS6501, was isolated and its complete nucleotide sequence determined. IS6501 is 836 bp in length and occurs 20-35 times in the B. ovis genome and 5-15 times in other Brucella species. Analysis of the junctions at the sites of insertion revealed a small target site duplication of four bases and inverted repeats of 17 bp with one mismatch. IS6501 presents significant similarity (53.4%) with IS427 identified in Agrobacterium tumefaciens, suggesting a common ancestral sequence. A long ORF of 708 bp was identified encoding a protein with a predicted molecular mass of 26 kDa and sharing sequence identity with the hypothetical protein 1 of A. tumefaciens and with the transposase of Mycobacterium tuberculosis. IS6501 is present in all Brucella strains we have tested. Restriction fragment length polymorphism of reference and field strains of two species (B. melitensis and B. ovis) was studied using either pulsed field gel electrophoresis (PFGE) on XbaI-digested DNA or hybridization of EcoRI-digested DNA using IS6501 as a probe. The genome of B. melitensis biovar 3 contains about 10 IS copies per genome and field strains of the same species could not be distinguished either by IS hybridization or by XbaI (PFGE) restriction patterns. In contrast, the number of IS copies in the B. ovis genome is around 30 and the different field strains can be differentiated by both methods.(ABSTRACT TRUNCATED AT 250 WORDS)
Synthetic oligonucleotides, which were designed according to amino acid sequences conserved between Rhodospirillum rubrum and Azospirillum brasilense DraT and DraG, respectively, were used to identify the corresponding genes of Rhodobacter capsulatus. Sequence analysis of a 1904 bp DNA fragment proved the existence of R. capsulatus draT and draG. These two genes were separated by 11 bp only, suggesting that R. capsulatus draT and draG were part of one transcriptional unit. In contrast to R. rubrum, A. brasilense and Azospirillum lipoferum, the R. capsulatus draTG genes were not located upstream of the structural genes of nitrogenase nifHDK but close to the dctP gene at a distance of about 1000 kb from the nifHDK genes. Deletion mutations in the draTG gene region were constructed and introduced into R. capsulatus wild-type and a nifHDK deletion strain. The resulting mutant strains were examined for post-translational regulation of the molybdenum and the alternative nitrogenase in response to ammonia and darkness. Under 'switch-off' conditions the modified (ADP-ribosylated) and the non-modified forms of component II of both the molybdenum and the alternative nitrogenase were detected in a draTG wild-type background by immunoblot analysis, whereas only the non-modified forms were present in the draTG deletion strains. Nitrogenase activity in these strains was followed by the acetylene reduction assay. In contrast to the wild-type, draTG mutants were not affected in nitrogenase activity in response to ammonia or darkness. These results demonstrated that the draTG genes are required for post-translational regulation of both the molybdenum and the heterometal-free nitrogenase in R. capsulatus.
The arom gene, encoding a single polypeptide that catalyses five consecutive steps of the pre-chorismate aromatic amino acid biosynthetic pathway, has been cloned from the opportunistic pathogen Pneumocystis carinii. There is a single open reading frame of 4788 bp which includes an intron of 45 bp that does not introduce a stop codon into the sequence. Thus, the derived amino acid sequence consists of 1581 residues, which is highly homologous to all fungal AROM proteins studied to date. These data support the view that P. carinii is a fungus and imply that its aromatic amino acid biosynthesis is conventionally organized.
Anaplasma marginale is a rickettsial parasite of bovine erythrocytes causing world-wide economic losses in livestock production. Despite its importance, little is known about this rickettsia at a molecular level because it has not been cultured in vitro, and there is no small-animal model. Although several genes have been cloned and sequenced, the gross genome structure of the organism has not yet been well characterized. We separated intact bovine erythrocytes from leucocytes, and determined the genome size of A. marginale by use of restriction endonuclease cleavage and pulsed-field gel electrophoresis (PFGE). A value of 56 mol% G+C was obtained for this genome by spectral analysis. Undigested A. marginale DNA failed to migrate under several different electrophoretic conditions, indicating a circular genome. Digestions of intact A. marginale DNA were performed using restriction endonucleases NotI, SfiI and PacI. Complete digestion with SfiI resulted in 12 distinct bands ranging in size from 14 to 170 kbp. Total size determined by addition of SfiI-digested fragments was approximately 1200 kbp. PacI cleaved the A. marginale genome from three different isolates into just three fragments, of 598, 557 and 97 kbp. Incomplete digestion produced a band measuring 1250 kbp. These results indicate that A. marginale has a circular genome between 1200 and 1260 kbp, with a G+C content of 56 mol%.
A plasmid designated pLPG36 was isolated from the naturally occurring Legionella pneumophila serogroup-1 and purified by CsCl buoyant density centrifugation. A restriction map of this 58 kb plasmid was constructed and provided the basis for cloning four BamHI fragments into the unique BamHI site of pUC18. The four recombinant plasmids were investigated for the mobilization function in Escherichia coli strains. Only one of these, pFLJ2, was mobilized by the IncP plasmids RP4, pRK231 and R702, but not by plasmids pSa, R40a, R387, pN3 or R16. The derivative plasmid pFLJ2 was mobilized more efficiently by R702 than by RP4 or pRK231. By genetic and deletion analysis, the mobilization region of pLPG36 was located to a 6 kb EcoRI fragment of the plasmid.
Multiple forms of beta-glucosidase (EC 3.2.1.21) of Sporotrichum thermophile were produced when the fungus was grown in a cellulose medium. One beta-glucosidase was purified 16-fold from 6-d-old culture filtrates by ion-exchange and gel-filtration chromatography. The purified enzyme was free of cellulase activity. It hydrolysed aryl beta-D-glucosides and beta-D-linked diglucosides. It was optimally active at pH 5.4, at 65-degrees-C. The apparent K(m) values for p-nitrophenyl beta-D-glucoside (PNPG) and cellobiose were 0.29 and 0.83 mm, respectively. Glucose, fucose, nojirimycin and gluconolactone inhibited beta-glucosidase competitively. At high (> 1 mm) substrate concentration, beta-glucosidase catalysed a parallel transglycosylation reaction. The transglycosylation product formed from cellobiose appeared to be a beta-linked tetramer of glucose. Admixtures of beta-glucosidase and cellulase components showed that the concept of cellobiose inhibition of cellulases was not valid for all components of the cellulase system of S. thermophile. Beta-Glucosidase supplementation also stimulated cellulose hydrolysis by cellulases when there was no accumulation of cellobiose in reaction mixture.
We investigated the usefulness of rRNA gene restriction fragment length polymorphism (RFLP) for grouping of the Borrelia isolates associated with Lyme disease or from ixodid ticks. Genomic DNA was digested with a restriction enzyme, blotted and hybridized with an rrl (23S rRNA) gene probe. The sizes of the restriction bands showed a good correlation with the genotypes recently proposed, and Borrelia isolates of diverse geographic origin formed four distinct DNA groups. Group I included all of the USA isolates and some European isolates; group II contained European isolates and Asian isolates; group III comprised European and Asian isolates; group IV included Japanese isolates and an eastern Russian isolate. Groups I, II and III corresponded to Borrelia burgdorferi, B. garinii and group VS461, respectively. The RFLPs of Japanese isolates were rather divergent and some of the isolates were quite distinct from the USA and European isolates. RFLP analysis of the rRNA genes and flanking regions using rrl gene probes as we reported here, may be useful in the taxonomic study of Borrelia.
A mutant (strain LLD-15) of Bacillus stearothermophilus strain LLD-R, that lacks L-lactate dehydrogenase activity was studied in anaerobic single-stage continuous culture using a defined minimal medium for its capacity to produce ethanol from sucrose at 70-degrees-C. Cultures were stable at neutral pH, dilution rates below 0.25 h -1 and sucrose concentrations below 15 g l-1, producing mainly ethanol, CO2, formate and acetate plus a little succinate. The data obtained were used to estimate the maintenance energy coefficient at 70-degrees-C and showed that the relative fluxes through the pyruvate-formate lyase (PFL) pathway and an anaerobic pyruvate dehydrogenase (PDH) pathway were about equal, though the later predominates at low dilution rates. At higher dilution rates or at acid pH, L-lactate became the major anaerobic product due to takeover by wild-type revertants. Reversion occurred only above a critical specific sucrose consumption rate of 4.2 g sucrose h -1 (g cells)-1 at pH 7, at which point pyruvate production was also seen. This can be attributed to saturation of the PFL pathway and PDH pathways by increasing glycolytic flux and consequent accumulation of intracellular pyruvate. The growth rate then declines, but a small population of revertant LLD-R cells would always be present in the bioreactor since the mutation in the L-lactate dehydrogenase gene has a significant reversion rate. These revertants have no selective advantage below the critical sugar uptake rate, but at higher rates they divert excess pyruvate to L-lactate, so their growth is unimpaired and they take over in the culture. At lower pH, growth restriction and reversion occur at low sucrose consumption rates due to difficulty in excreting acetic and formic acids against a pH gradient.
The mannoprotein components solubilized from the walls of Candida albicans blastoconidia following degradation of the glucan network with beta-glucanase (Zymolyase) have higher molecular masses than their probable precursors present in the supernatant of regenerating protoplasts. It therefore appears that the mannoproteins are released from the walls as part of supramolecular complexes. Immunological analysis using both polyclonal and monoclonal antibodies has demonstrated the probable relationship between molecules found in a mixed membrane preparation, those secreted by regenerating protoplasts, and those present in yeast cell walls. Some mannoproteins secreted by protoplasts incubated in the presence of tunicamycin had significantly increased mobility on SDS-PAGE, whereas others were not affected by the treatment. It is therefore possible that two types of mannoproteins are secreted by protoplasts: one carrying N-glycosylated chains (mannan) and one lacking them. All the proteins secreted in the presence of tunicamycin stained with Concanavalin A-peroxidase, demonstrating that they all, including the N-glycosylated ones, carried O-glycosylated sugar residues. Both classes of mannoproteins, secreted independently of each other, were found in the molecular complexes rendered soluble from the wall by Zymolyase digestion. Data obtained with a monoclonal antibody demonstrated the presence of a repeated epitope within one wall protein(s) detectable in a mixed membrane preparation and in the wall complexes released by Zymolyase.
A promoter-probe vector (pHX200) was constructed using the broad-host-range cosmid pLA2917 and a promoterless xylE gene of Pseudomonas as the reporter gene. Insertion of the cloned promoter fragment of the methanol dehydrogenase large subunit gene moxF (methanol oxidation) in front of the xylE gene in pHX200V-47 resulted in high-level expression of the xylE gene product--catechol 2,3-dioxygenase--in Methylobacterium organophilum XX. The specific activity of the enzyme was four times higher in methanol-grown M. organophilum XX culture than in succinate-grown culture. Interestingly, the insertion of the same fragment in the opposite orientation in front of the xylE gene (pHX200V-74) also led to elevated catechol 2,3-dioxygenase activity. This promoter activity was also methanol regulated. A total of 21 methanol-regulated promoter clones were identified that originate from three gene clusters (groups V, VI and VII) on the M. organophilum XX chromosome involved in methanol oxidation. Vector pHX200 and its derivatives were successfully mobilized into cells of three phylogenetically diverse methylotrophic bacteria: Methylophilus methylotrophus AS1, Methylobacterium extorquens AM1 and Methylobacterium sp. DM4. The reporter gene (xylE) was functionally expressed in all three bacteria with the aid of a proper promoter. Transcriptional fusions of methanol-regulated promoters with the xylE gene were mobilized into Mox- mutants of M. organophilum XX and M. extorquens AM1 to study the roles of methanol oxidation genes, especially regulatory genes. It appeared that vector pHX200 is an efficient promoter probe with wide host-range and an excellent tool for studies of structure and function of promoters/regulators.
One of the chitinases of Streptomyces lividans, chitinase C, was encoded by a 2 kb smaI-XhoI restriction fragment contained in the recombinant plasmid pEMJ7. DNA sequence analysis of this region revealed the presence of two open reading frames (ORF1 and ORF2) which had opposite orientations. Northern analysis showed that only the mRNA complementary to ORF1 was transcribed, and that this transcription was induced by chitin and repressed by glucose. ORF1 showed a codon distribution typical of Streptomyces. A sequence identical to that of the N-terminus of mature secreted chitinase C was found from amino acid residue 31 in the deduced amino acid sequence of ORF1 (619 amino acids), implying that ORF1 encodes a pre-protein of chitinase C. The pre-protein of chitinase C consisted of four discrete domains. The 30 amino acid N-terminal sequence, domain 1, was characteristic of a signal peptide. Domain 2 consisted of 105 N-terminal amino acids of mature chitinase C, and was similar to cellulose-binding domains of several cellulases. Domain 3 (94 amino acids) showed homology with type III homology units of fibronectin. Domain 4, a C-terminal 390 amino acid sequence, is probably the catalytic domain of the chitinase, since it exhibited identity with several other chitinolytic enzymes.
UNLABELLEDEach of the four main polar lipids from Phaeodactylum tricornutum UTEX 640, monogalactyosyldiacylglycerol(MGDG), digalactosyldiacylglycerol (DGDG), sulphoquinovosyldiacylglycerol (SQDG) and phosphatidylcholine(PC), was separated into its molecular species by reverse-phase HPLC, and the positional distribution of fatty acids in each species was determined. Numbers of separated peaks in each lipid class were: MGDG, 10; DGDG,7; SQDG, 10; and PC, 11. Eicosapentaenoic acid (EPA) was present in 20 of the 45 molecular species but predominated in the MGDG and DGDG classes, where 13 of the proposed structures contained EPA. EPA was always located in the sn-1 position except in two lipid species, MG5 and PC2, where it was present at both the sn-1 and sn-2 locations. The predominant polar lipid molecular species found in P. tricornutum UTEX 640 were(mg total fatty acids per g dry weight of biomass) 20:5-16:4-MG, 41-2; 20:5-16:1-DG, 21.0; 16:1-16:1-SQ,18-2; and 20:5-16: 1-MG, 18-0. [Structure indicates fatty acid at sn-1 position - fatty acid at sn-2 position -carbohydrate component at sn-3 position of the glycerol molecule.ABBREVIATIONS20: 5, EPA; 16: 4,hexadecatetraenoic acid ; 16 : 1, palmitoleic acid ; MG, monogalactose ; DG, digalactose ; SQ, sulphoquinovose.]
Fingerprinting by random amplification of polymorphic DNA (RAPD) was compared with existing molecular typing systems for Candida albicans. Fifteen isolates were chosen, including three from the same patient; these gave 14 distinct karyotypes by pulsed-field gel electrophoresis (PFGE) and 7 different DNA types by EcoRI-generated restriction fragment length polymorphisms (RFLPs). RAPD with primer I (5'GCTGGTGG3') gave 5 types, whereas primer II (5'GCGCACGG3') yielded 11 types. Combining the results from both primers, all isolates were unique by RAPD with the exception of the three from the same patient. RAPD provided a fast, economical and reproducible means of typing C. albicans with a level of discrimination approaching that of PFGE.
When grown on ethene Mycobacterium strain E3 produces epoxyalkanes from alkenes in an oxygen- and NADH-dependent reaction. The process of co-factor regeneration was studied by analysing the intracellular pools of NADH and storage material during the production of 1,2-epoxypropane from propene. With the depletion of NADH the production of l,2-epoxypropane stopped. NADH could be regenerated from the oxidation of added cosubstrate or from oxidation of storage material. Cells cultivated in chemostat culture under nitrogen limitation produced more 1,2-epoxypropane compared to cells cultivated under carbon limitation, due to their higher content of storage material. Addition of glucose to cells grown under carbon limitation stimulated the formation of 1,2-epoxypropane. The uptake of glucose resulted in the accumulation of storage material, which was utilized after depletion of the glucose. Glycogen and trehalose were the preferred forms of storage material used for co-factor regeneration. From the results it was concluded that formation and utilization of storage material play a crucial role in the process of co-factor regeneration in Mycobacterium strain E3.
As described previously, in Rhodococcus sp. (formerly Nocardia opaca) strains MR11 and MR22, the ability to grow as an aerobic hydrogen bacterium (the Aut character) is located on giant conjugative linear plasmids--on pHG201 (270 kb) in strain MR11 and on pHG205 (280 kb) in strain MR22. In an autotrophic transconjugant originating from MR22 a smaller plasmid, pHG207 (225 kb), was detected and shown to be a recombination product of the wild-type plasmids pHG204 and pHG205. A donor carrying pHG207 as the sole plasmid transferred the Aut marker at a 1000-fold frequency compared to the wild-type plasmid pHG205. Analysis of the plasmid ends revealed that plasmid pHG207 carries proteins at both ends; the proteins are linked to the 5' ends of the strands. The cloned end fragments of about 2 kb were sequenced and found to contain highly homologous sequences within the terminal 583 bp (left end part) and 560 bp (right end part). Several potential reading frames were detected, but database searching gave no indication about possible functions.
Brevibacterium linens can grow in media of relatively high osmotic strength (up to 3 M-NaCl). Optimal growth in minimal medium occurred with either added NaCl (0.5-1 M) or other osmolytes developing an equivalent osmotic pressure. Above 1 M-NaCl the growth rate slowed, but was enhanced by adding the osmoprotectant glycine betaine or its precursor choline (1 mM) to the media. Neither soluble carbohydrates nor ninhydrin-reacting compounds accumulated during osmotic treatment. However, a Dragendorff-positive compound, together with K+ ions, accumulated (7-fold) as a consequence of elevating the medium osmolality. Spectral characteristics of the purified compound were similar to those of synthesized 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine). In the absence of exogenously supplied osmoprotectants, a strict dependence between the intracellular ectoine content and the external salt concentration (up to 1 M-NaCl) was observed. From 1 to 2 M-NaCl, the ectoine content decreased, and this signalled decreased self-osmoprotection by this bacterium. Interestingly, at these salt concentrations, exogenous glycine betaine led to the greatest beneficial effect. Glycine betaine or choline added to the medium was actively taken up by the cells in an osmolality-dependent manner, and the resulting betaine accumulation caused a sharp decrease in intracellular ectoine content. Radiolabelling of ectoine occurred only when L-[U-C-14]glutamate was used as a precursor; de novo synthesis was dependent on the external osmolality and was strongly inhibited by exogenously supplied glycine betaine. These results imply that ectoine may play a major role in counteracting the effects of osmotic stress in media of osmotic pressure equivalent to equal-to-or-less-than 1 M-NaCl.
The phytopathogenicity of Erwinia chrysanthemi is chiefly supported by the production of pectate lyase isoenzymes, encoded by the pel genes. One of these enzymes, PelA, encoded by the pelA gene, seems to represent only a small part of the total pectate lyase activity, but is required for full bacterial pathogenicity. To study the regulation of pelA gene expression, a pelA::uidA gene fusion was constructed. Expression of this fusion was analysed under various growth conditions and in various genetic backgrounds. Whatever the culture conditions, the pelA gene was weakly expressed. Moreover, pelA expression seems not to be regulated by the pecS gene product, but essentially by the kdgR gene product. In many plant-associated bacteria, genes involved in pathogenicity are induced by certain plant compounds. In this work, we demonstrate that E. chrysanthemi pel genes are induced in the presence of plant extracts, but only in synergy with known pectate lyase inducers (KDG: 2-keto-3-deoxygluconate; DKII: 2,5-diketo-3-deoxygluconate). However, different pel genes did not exhibit the same sensitivity to plant signal molecules. Partial purification of inducing plant compounds suggested that plant extracts contain at least one molecule involved in pectate lyase induction. This compound is thermoresistant, and has a low molecular mass and a very hydrophilic behaviour.
The Clostridium thermocellum celI gene, coding for endoglucanase I (CelI), consists of an open reading frame (ORF) of 2640 nucleotides and codes for a protein of M(r) 98531. The ORF was confirmed as celI by comparing the N-terminal sequence of purified recombinant CelI with that deduced from the nucleotide sequence. CelI hydrolysed lichenan and carboxymethylcellulose, but was principally active against barley beta-glucan. It exhibited significant sequence identity with subfamily E2 endoglucanases, and by analogy with others in this group contains a catalytic domain of around 500 residues located in the N-terminal half of the protein. The C-terminal region of CelI was highly homologous with the cellulose-binding domain of the non-catalytic cellulosome subunit, S1. A repeated segment, previously shown to be highly conserved in xylanase Z and in other endoglucanases from C. thermocellum, was absent from CelI. Antiserum raised against purified recombinant CelI cross-reacted with proteins contained in the cellulosomes of two strains of C. thermocellu, suggesting that CelI is either a component of the cellulosome or is homologous to other cellulosome proteins. A second gene, located upstream of celI, consisted of an ORF of 1671 nucleotides, coding for a protein of M(r) 61042. Based on its homology with the Escherichia coli tar gene product, the polypeptide encoded by the second gene is tentatively identified as a sensory transducer.
