- Volume 142, Issue 4, 1996
Volume 142, Issue 4, 1996
- Review Article
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- Microbiology Comment
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- Antigens And Immunity
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A 28 kDa major immunogen of Chlamydia psittaci shares identity with Mip proteins of Legionella spp. and Chlamydia trachomatis - cloning and characterization of the C. psittaci mip-like gene
More LessChlamydia psittaci strain guinea-pig inclusion conjunctivitis (GPIC) produces a self-limiting ocular infection of guinea-pigs, and this condition is a representative animal model of ocular chlamydial disease. Convalescent guinea-pigs, which are resistant to reinfection, produce antibodies to several elementary-body proteins, including an uncharacterized antigen of 28 kDa. Convalescent guinea-pig sera were used to identify, from a lambda expression library, two overlapping GPIC genomic clones that produced the 28 kDa antigenic protein. Nucleotide sequence analysis revealed that the gene coding for the 28 kDa protein was similar to the mip (macrophage infectivity potentiator) genes from Legionella pneumophila and Chlamydia trachomatis. The GPIC gene and its product were accordingly designated mip and Mip, respectively. Analysis of the regions flanking mip identified three tightly linked open reading frames coding for predicted products with sequence similarity to asparagine tRNA ligase (AspS), rRNA methylase (SpoU), and thioredoxin (TrxA). The arrangement of these genes in GPIC was aspS-mip-spoU-trxA. Sequence analysis of PCR products produced using genomic DNA from an ovine abortion strain of C. psittaci and from C. trachomatis strain LGV-434 demonstrated that the arrangement of mip, spoU and trxA is common among these chlamydiae.
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Epitopes of Bordetella pertussis lipopolysaccharides as potential markers for typing of isolates with monoclonal antibodies
More LessThree hybridomas (P1P3, D7 and 60.5) producing monoclonal antibodies (mAbs) against Bordetella pertussis lipopolysaccharide (LPS) were established. All reacted with the LPS from a typical, vaccine strain of B. pertussis (1414), but not with that of a variant strain (A100). Two of these mAbs (P1P3 and 60.5) cross-reacted with a B. bronchiseptica LPS; only one (P1P3) reacted with a B. parapertussis LPS. ELISA reactivities with intact LPSs, and defined partial structures covalently linked to bovine serum albumin, were compared. mAb 60.5 bound to the terminal region of a distal trisaccharide consisting of N-acetylated amino sugars. D7 reacted with a substructure which can be modified in the B. parapertussis and B. bronchiseptica LPSs by addition of a polymeric O-chain. P1P3 bound to a nonacetylated glucosamine substituted with L-glycero-D-manno-heptose, present in the ‘core’ of the B. pertussis LPS. These mAbs may be useful for rapid typing of Bordetella in clinical isolates.
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- Biochemistry
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Polysaccharide lyases from gellan-producing Sphingomonas spp.
More LessA number of Sphingomonas strains capable of synthesizing the bacterial exopolysaccharide gellan and related polymers were shown to possess constitutive gellanase activity. In each case, the degradation of deacylated gellan was due to extracellular, eliminase-type enzymes (lyases) which cleave the sequence …ß-D-glucosyl 1,4-ß-D-glucuronosyl… in the tetrasaccharide repeat unit of the substrate polysaccharides. Deacetylated rhamsan was an alternative substrate but there was little or no action against most other polysaccharides with similar structures. Slight differences were found between the specificities of the lyases from different strains. Activities of gellan lyase preparations were generally low. As well as the extracellular ‘gellanase’ activity, all the bacteria possessed varying amounts of ß-D-glucosidase and ß-D-glucuronidase activities apparently located in the periplasm. The products from deacylated gellan and the chemically deacylated form of polysaccharide S194 (rhamsan gum), which is effectively a gentiobiosylated form of gellan, closely resembled those recently obtained by the authors from other, gellandegrading, non-gellan-producing bacteria. The enzymes had negligible activity against the natural, acylated gellan and rhamsan polysaccharides from bacteria now designated as strains of Sphingomonas.
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Bacteriophage receptors on Listeria monocytogenes cells are the N-acetylglucosamine and rhamnose substituents of teichoic acids or the peptidoglycan itself
More LessDifferent approaches were used to examine the function of teichoic acids (TA) as phage receptors among selected Listeria strains, and to identify and characterize specific receptor structures of host cells belonging to different serovars. This included successive removal of cell wall constituents, preparation and purification of TA, and GLC analysis of TA components. Adsorption of Listeria monocytogenes bacteriophages could be inhibited by polyvalent antisera, specific lectins and addition of purified TA. The results confirmed the necessity of TA in general and of rhamnose and glucosamine in particular for adsorption of Listeria phage A118, which is a temperate Siphovirus (morphotype B1), attacking predominantly serovars 1/2. Host binding of siphoviral phage A500 (predominantly lysing serovars 4b), was also dependent on cell wall TA. A phage-resistant L. monocytogenes strain was shown to lack glucosamine in its TA. These results support the view that TA substituents may play an important role not only in antigenicity of Listeria cells, but also in specificity of host recognition by two temperate Listeria phages. In contrast, the broad-host-range virulent phage A511 (Myovirus, morphotype A1) uses the listerial peptidoglycan as primary receptor. This corresponds well with the observation that A511 is capable of lysing the majority of L. monocytogenes strains.
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- Bioenergetics And Transport
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The cytochrome bd quinol oxidase in Escherichia coli has an extremely high oxygen affinity and two oxygen-binding haems: implications for regulation of activity in vivo by oxygen inhibition
More LessCytochrome bd is a respiratory oxidase in Escherichia coli and many other bacteria. It contains cytochromes b 558′ b 595 and d as redox centres, and is thus unrelated to the haem-copper super-family of terminal oxidases. The apparent affinities (K m) for oxygen uptake by respiring cells and membranes from a mutant lacking the alternative oxidase cytochrome bo’ were determined by deoxygenation of oxyleghaemoglobin as a sensitive reporter of dissolved oxygen concentration. Respiration rates were maximal at oxygen concentrations of 25-50 nM, but the kinetics were complex and indicative of substrate (i.e. oxygen) inhibition. K m values were in the range 3-8 nM (the lowest recorded for a respiratory oxidase), and K l values between 0.5 and 1.8 μM were obtained. Low temperature photodissociation of anoxic, CO-ligated membranes confirmed the absence of cytochrome bo’ and revealed a high-spin b-type cytochrome identified as cytochrome b 595 of the cytochrome bd complex. Photodissociation in the presence of oxygen revealed binding of a ligand (presumably oxygen) to cytochrome b 595 at a rate much greater than that of CO binding, and formation of the oxygenated form of cytochrome d. The results confirm that both high-spin haems in the cytochrome bd complex bind CO and demonstrate that oxygen can also react with both haems. Substrate inhibition of oxidase activity, in addition to transcriptional regulation of oxidase synthesis, may play a crucial role in the regulation of partitioning of electron flux between the cytochrome bd- and bo’-terminated respiratory pathways.
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Dissimilatory iron(III) reduction by Rhodobacter capsulatus
The photosynthetic proteobacterium Rhodobacter capsulatus was shown to be capable of dissimilatory Fe(III) reduction. Activity was expressed during anaerobic phototrophic and microaerobic growth with malate as the carbon source, but not during equivalent aerobic growth. A variety of Fe(III) complexes were demonstrated to act as substrates for intact cells and membrane fractions of strain N22DNAR+ using a ferrozine assay for Fe(II) formation. Rates of reduction appeared to be influenced by the reduction potentials of the Fe(III) complexes. However, Fe(III) complexed by citrate, which is readily reduced by Shewanella putrefaciens, was a poor substrate for dissimilation by R. capsulatus. The Fe(III)-reducing activity of R. capsulatus was located solely in the membrane fraction. The reduction of Fe(III) complexes by intact cells was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), suggesting the involvement of ubiquinol: cytochrome c oxidoreductases in the electron transport chain. Lack of sensitivity to myxothiazol plus data from mutant strains implies that the cytochrome bc 1 complex and cytochrome c 2 are not obligatory for dissimilation of Fe(III)(maltol)3. Alternative pathways of electron transfer to Fe(III) must hence operate in R. capsulatus. Using strain N22DNAR+, the reduction rate of Fe(III) complexed by nitrilotriacetic acid (NTA) was elevated compared to that of Fe(III)(maltol)3, and moreover was sensitive to myxothiazol. However, these differences were not observed in the absence of the electron donor malate. The governing factor for the reduction rate of Fe(III)(maltol)3 thus appears to be the limited Fe(III)-reducing activity, whilst the reduction rate of Fe(III) complexed by NTA is controlled by the flux of electrons through the respiratory chain. The use of mutant strains confirmed that the role of the cytochrome bc 1 complex in Fe(III) reduction becomes apparent only with the superior substrate. The energy-conserving nature of Fe(III) reduction by R. capsulatus was demonstrated by electrochromic measurements, with the endogenous carotenoid pigments being employed as indicators of membrane potential generation in intact cells. Using Fe(III)EDTA as electron acceptor, periods of membrane potential generation were directly proportional to the quantity of complex added, and were extended in the presence of HQNO. Fe(III)-dependent carotenoid bandshifts were abolished by addition of the protonophoric uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone.
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- Biotechnology
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A new, broad-substrate-specificity aminopeptidase from the dairy organism Lactobacillus helveticus SBT 2171
More LessAn aminopeptidase with a very broad substrate specificity was purified to homogeneity from Lactobacillus helveticus SBT 2171 by FPLC. The enzyme was purified 144-fold from a cell-free extract with a yield of 16%. The purified enzyme appeared as a single band on an SDS-PAGE gel. It had a molecular mass of 95 kDa and an isoelectric point of 4.9. The enzyme hydrolysed a large range of naphthylamide- and nitroanilide-substituted amino acids, as well as several di-, tri- and oligopeptides. It also exhibited significant prolineiminopeptidase-like activity, since it hydrolysed several proline-containing peptides. Prolyl-p-nitroanilide was hydrolysed with a low affinity (Michaelis-Menten constant 0.6 mM) and a V max of 2.5 μmol min-1 (mg protein)-1 while lysyl-p-nitroanilide was hydrolysed with a high affinity [K m 0.003 mM; V max 37.5 μmol min-1 (mg protein)-1]. The aminopeptidase activity, which was optimal between pH 6.0 and 8.0 and at 50 °, was very stable at 30 ° for more than 7 d. The activity lost by treatment with the thiol-blocking reagents could be restored with ß-mercaptoethanol, while Co2+ and Mn2+ restored the activity of the EDTA-treated enzyme. Immunological experiments with antibodies raised against the aminopeptidases from Lactococcus lactis and Lb. helveticus clearly showed that both aminopeptidases are at least immunologically different from each other.
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Bacterial production of trans-dihydroxycyclohexadiene carboxylates by metabolic pathway engineering
More LessHomochiral-cis-cyclohexa-3,5-diene-1,2-diols are important synthons. We found a way to produce trans-configured homochiral diols using recombinant Klebsiella pneumoniae 62-1. Transformation of this mutant (Phe- Trp- Tyr-) with plasmids carrying genes involved in chorismic and isochorismic acid metabolism leads to the production of either (+)-trans-(2S,3S)-2,3-dihydroxycyclohexa-4,6-dienecarboxylic acid or (-)-trans-(3R,4R)-3,4-dihydroxycyclohexa-1,5-dienecarboxylic acid, with a yield of 70 or 90 mg (l culture broth)−1, respectively. The metabolic shift from one diene to the other is caused by a change in activity of isochorismate hydroxymutase and/or isochorismatase which in turn results from growth under iron deficiency or overexpression of genes (entC and/or entB) involved in chorismate metabolism.
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- Development And Structure
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A bacteriophage of Rhodopseudomonas blastica
More LessA bacteriophage, øBHG1, was isolated from a small eutrophic pond from which its host, Rhodopseudomonas blastica, was originally obtained. It is a lytic bacteriophage specific for R. blastica which also causes non-specific lysis of Rhodobacter sphaeroides 8253. øBHG1 has an icosahedral head of 62 nm diameter and a short 39 nm tail. Caesium chloride density gradient centrifugation of infected cell lysates gave a single bacteriophage band at a density of 1.385 g cm-3, but also occasionally a second band was observed at a lower density. No differences were apparent between bacteriophage taken from either of the two bands. øBHG1 contained double-stranded DNA with a size of 48 kb and a G + C content of 50.6 mol%. The bacteriophage adsorbed to both photosynthetically and chemoheterotrophically grown R. blastica at an identical rate of 1.39 × 10-9 ml−1 min−1. One-step growth curves and kinetic studies of the bacteriophage under these physiological regimes showed no differences in the latent and rise periods and only slight changes in the burst size. Adsorption of this bacteriophage is cell-surface specific and attachment only occurs to the ‘older’ pole of the budding reproductive cell.
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A mutational analysis of Dictyostelium discoideum multicellular development
More LessWe have collected Dictyostelium mutants that arrest in development after aggregation, but before first finger formation. A total of 118 mutant strains were isolated and are referred to as mound (mnd) mutants. Nine complementation groups (mndA-mndl), containing 46 of the mutant strains, were defined by parasexual methods. A statistical analysis suggested that there are about 118 genes which, when mutated, confer the mound phenotype. Of these genes, about 60 are predicted to be mutated in our collection: the 9 assigned to complementation groups and another 51 unassigned mutants. mndA, G, H and I were assigned to linkage groups VII, IV, II and VI, respectively. Development of the mutant strains was characterized by terminal morphology, neutral red staining and expression of marker mRNAs for prespore and prestalk cells. Three broad classes were recognized. (1) Postaggregative mutants - those blocked early in multicellular development. They did not express any of the prestalk or prespore marker mRNAs and generally arrested as low mounds or ridges. (2) Pathway mutants - those blocked specifically in either prestalk or prespore differentiation. They expressed either prestalk or prespore marker mRNAs, but not both, and generally proceeded further morphologically than post-aggregative mutants. (3) Morphogenesis mutants - those apparently blocked in morphogenesis rather than cell differentiation. They expressed all the cell-type marker mRNAs tested. Most arrested as tight mounds lacking a tip and of defined upper size, but some mutants produced aberrant tips. The majority of mutants tested synergized with wild-type: 24/28 strains which cannot make spores when developed alone, were able do so when allowed to develop with an equal number of wild-type cells. We suggest that some of the morphogenesis mutants have a cytoskeletal defect which prevents first finger formation and that these mutants can be physically carried through development by the wildtype (synergy by ‘piggy-backing’).
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- Environmental Microbiology
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Stress resistance and recovery potential of culturable and viable but nonculturable cells of Vibrio vulnificus
More LessThe estuarine, human-pathogenic bacterium Vibrio vulnificus responds to low temperature by the formation of viable but nonculturable (VBNC) cells, while starvation at moderate temperatures allows for maintenance of culturability of this organism. Recovery of cold-incubated populations of V. vulnificus was restricted to the culturable fraction in slide cultures and most probable number assays. These populations, however, gave between 1.1- and 8-fold higher c.f.u. counts on soft agar plates than on ordinary agar plates, indicating that a small and variable fraction of the cell population was injured rather than nonculturable. Thus, the population of cold-incubated cells is composed of culturable, injured and nonculturable cells, with the numbers of the culturable and injured cells rapidly decreasing during cold incubation. Recovery of nonculturable cells of the organism, however, could not be obtained by any combination of temperature and nutrient shifts in any of the assays. VBNC cells of the organism were assessed with regard to their persistence and stress resistance in comparison to growing and starved cells. The sonication resistance of VBNC cells was initially similar to that of growing cells, but increased during prolonged cold incubation. The final resistance of cold-incubated VBNC cells was equal to the markedly increased resistance of starving cells, which also displayed increased resistance against exposure to ethanol and mechanical stress. Our results indicate that in spite of the apparent absence of recovery under a wide range of laboratory conditions, VBNC cells of V. vulnificus undergo changes at low temperature which potentially allow them to persist for extended periods.
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Direct evidence for the involvement of extracellular proteins in the adhesion of Azospirillum brasilense
More LessAdhesion of Azospirillum brasilense to glass and polystyrene was investigated by bringing the cells into contact with the support by sedimentation. Adhesion depended on time and temperature: lower adhesion densities were observed when the contact time was only 2 h or 6 h, as compared to 24 h, or when the test was performed at 4 -C, as compared to 30 °. The influence of cell physiology was further demonstrated by the effect of tetracycline, which inhibited adhesion. Scanning electron microscopy showed that cells produced extracellular material when left in contact with a support for 24 h. The surface elemental composition of cells and of polystyrene supports after cell adhesion and subsequent detachment was determined by X-ray photoelectron spectroscopy; this provided information on the relative concentrations of proteins and polysaccharides at the surface. The protein concentration at the surface of a cell sediment increased as a function of time at 30 °, correlating with an increase of adhesion density. A similar correlation between protein concentration and adhesion density was found when comparing exponentialphase cells with stationary-phase cells. The surface composition of polystyrene supports examined after cell detachment was found to be rich in proteins, indicating that proteins are the major constituent at the support surface. Lowering the contact time, or performing adhesion under unfavourable metabolic conditions (4 °) or in the presence of tetracycline, resulted in a decrease in protein concentration at the support surface, which was correlated with a decrease in adhesion density. The correlation between protein concentration at the cell surface or at the support surface and adhesion density, under different experimental conditions, provides a direct demonstration of the involvement of extracellular proteins in the adhesion of A. brasilense to inert surfaces.
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- Genetics And Molecular Biology
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Use of green fluorescent protein for detection of cell-specific gene expression and subcellular protein localization during sporulation in Bacillus subtilis
More LessWild-type and mutant forms of the gene encoding green fluorescent protein (GFP) from Aequorea victoria have been introduced into Bacillus subtilis as translational fusions to the prespore-specific and mother-cell-specific genes dacF and spoIVA. In both cases, the protein was readily detected by fluorescence microscopy, and its synthesis was correctly localized. The S65T substitution, which improves the quantum yield and rate of development of fluorescence, also produced a spectral shift that allowed the protein to be colocalized with DNA, after staining with 4′,6-diamidino-2-phenylindole. Three different translational fusions to the N-terminal region of GFP all produced active protein. Moreover, a full-length spoIVA-GFP fusion showed proper targeting to the surface of the spore, albeit at low temperature and in the presence of wild-type spoIVA protein. A mutation in the gfp gene which changes the light emitted by the protein from green to blue was found not to be useful because of the intrinsic autofluorescence of B. subtilis in the blue part of the spectrum.
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Two multifunctional peptide synthetases and an O-methyltransferase are involved in the biosynthesis of the DNA-binding antibiotic and antitumour agent saframycin Mx1 from Myxococcus xanthus
More LessSaframycin Mx1 is a DNA-binding antibiotic and antitumour agent produced by Myxococcus xanthus. It is a heterocyclic quinone, thought to be synthesized via the linear pepide intermediate AlaGlyTyrTyr. Analysis of 14.1 kb DNA sequence involved in saframycin production revealed genes for two large multifunctional peptide synthetases of 1770 and 2605 amino acids, respectively, and a putative O-methyltransferase of 220 amino acids. The three ORFs read in the same direction and are separated by short non-translated gaps of 44 and 49 bp. The peptide synthetases contain two amino-acid-activating domains each. The first domain lacks two of the most conserved ‘core’ sequences, and the last domain is followed by a putative reductase functionality, not previously seen in peptide synthetases. Complementation tests showed that antibiotic-nonproducing mutant strains lacking one of the peptide synthetases secrete a substrate, presumably a modified amino acid precursor, that can be used by O-methyltransferase-deficient mutant strains to synthesize saframycin Mx1.
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Streptomyces akiyoshiensis differs from other Gram-positive bacteria in the organization of a core biosynthetic pathway gene for aspartate family amino acids
More LessA partial Sau3Al digest of genomic DNA from Streptomyces akiyoshiensis was cloned in a Streptomyces-Escherichia coli shuttle vector, and the recombinant plasmids were used to transform E. coli CGSC 6212, which carries a mutation in the gene for aspartate semialdehyde dehydrogenase (Asd). One of 39000 transformants tested grew on LB medium lacking diaminopimelate. A 17 kb plasmid (pJV21) isolated from this strain conferred prototrophy when used to transform E. coli CGSC 6212. The gene responsible was located on a 2.2 kb DNA fragment by subcloning. Nucleotide sequencing and codon preference analysis of the subcloned insert and of the 3.3 kb insert in the Asd -complementing plasmid pJV36 located three complete and two incomplete open reading frames (ORFs). One of these (ORF3), encoding a polypeptide of 338 amino acids (M r 35484), was identified as the gene for Asd by comparing its sequence with database sequences of asd from other bacteria. The inability of pJV30, in which a segment of ORF3 had been deleted, to transform E. coli CGSC 6212 to prototrophy supported this assignment. Southern hybridization indicated that the sequenced region of the cloned DNA fragment represented a continuous segment of the S. akiyoshiensis chromosome. The deduced amino acid sequences of the ORFs adjacent to asd showed no similarity to sequences for aspartate kinase (Ask); also, transformation with plasmids containing asd and adjacent regions from the S. akiyoshiensis chromosome did not complement the ask mutant E. coli CGSC 5074. It is concluded that asd and ask in S. akiyoshiensis are not present in an operon, and thus are organized differently from these genes in the Gram-positive bacteria previously examined.
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Characterization of a prolinase gene and its product and an adjacent ABC transporter gene from Lactobacillus helveticus
More LessA prolinase (pepR) gene was cloned from an industrial Lactobacillus helveticus strain (53/7). Three clones, hybridizing with a gene probe specific for a peptidase shown to have activity against di- and tripeptides, were detected from a L. helveticus genomic library constructed in Escherichia coli . None of the three clones, however, showed enzyme activity against the di- or tripeptide substrates tested. One of the clones, carrying a vector with a 5.5 kb insert, was further characterized by DNA sequencing. The sequence analysis revealed the presence of two ORFs, ORF1 and ORF2 of 912 and 1602 bp, respectively, ORF2, located upstream of and in the opposite orientation to ORF1, had a promoter region overlapping that of ORF1. ORF1 had the capacity to encode a 35083 Da protein. When amplified by PCR, ORF1 with its control regions specified a 35 kDa protein in E. coli that was able to hydrolyse dipeptides, with highest activity against Pro-Leu, whereas from the tripeptides tested, only Leu-Leu-Leu was slowly degraded. By the substrate-specificity profile and protein homologies, the 35 kDa protein was identified as a prolinase. The activity of the cloned prolinase was inhibited by p-hydroxymercuribenzoate. Northern and primer-extension analyses of ORF1 revealed a 1.25 kb transcript and two adjacent transcription start sites, respectively, thus confirming the DNA sequence data. ORF2 had encoding capacity for a 59.5 kDa protein that showed significant homology to several members of the family of ABC transporters. Determination of the mRNA levels at different growth phases revealed that the pep gene and ORF2 are transcribed in L. helveticus at the exponential and stationary phases of growth, respectively. Furthermore, two ORF2 deletion constructs, carrying the intact pepR gene, showed that this upstream operon adversely affected PepR activity in E. coli, which explains the enzymic inactivity of the original clones.
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The [Ni-Fe] hydrogenase from the thermophilic bacterium Acetomicrobium flavidum
More LessBiochemical analysis of the soluble hydrogenase from the thermophilic organism Acetomicrobium flavidum revealed that the enzyme is an a2ß2 tetramer, with the a and ß subunits having a molecular mass of 50 kDa and 25 kDa, respectively. The most important biochemical properties of the enzyme are a specific activity of 77 μmol min-1 (mg protein)-1 a K m for methylviologen of 0.2 mM, a pH optimum of 7.5 and a T 50 of about 70 †C. In addition, the enzyme is remarkably stable to oxygen inactivation, retaining full activity after 24 h exposure to air. By using oligodeoxynucleotides designed on the basis of the N-terminal sequences of the two subunits, the corresponding genes have been isolated and sequenced. When compared to the other hydrogenases so far characterized, the A. flavidum hydrogenase appears to be a typical [Ni-Fe] enzyme. The hydrogenase was expressed in Escherichia coli at high levels in a soluble form but it was not active. The analysis of the recombinant large subunit showed that it was not post-translationally processed at its C-terminus.
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The Azotobacter vinelandii gene algJ encodes an outer-membrane protein presumably involved in export of alginate
More LessThe algJ gene from Azotobacter vinelandii was cloned using a labelled RNA probe representing the coding region of the algE gene from Pseudomonas aeruginosa. DNA sequencing revealed an ORF of 1452 bp encoding a protein of 484 amino acid residues with a calculated molecular mass of 54611 Da. An RNA probe corresponding to algE was also used for Southern hybridization of chromosomal DNA, which showed that algE-related DNA sequences are also present in the alginate-producing phytopathogen species Pseudomonas marginalis and Pseudomonas syringae pv. glycinea. The coding region of algJ was subcloned in the expression vector pT7-7, leading to a corresponding gene product with an apparent molecular mass of 54 kDa which could be identified in the outer membrane (OM) of Escherichia coli BL21(DE3). Additionally, a cross-reacting protein with the same molecular mass was also found in the OM of A. vinelandii using an anti-AlgE antiserum. The derived amino acid sequence of AlgJ shared approximately 52% identity with AlgE from P. aeruginosa. The hydrophilicity profile as well as the amphipathicity of regions in the amino acid sequence of AlgJ showed significant similarities to AlgE. Based on these data, a topological model of AlgJ was created with the aid of known structures of outer-membrane proteins. This model presents AlgJ as a ß-barrel containing 18 ß-strands inserted in the OM.
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Organization of ribosomal RNA genes from the footrot pathogen Dichelobacter nodosus
More LessSouthern hybridization analysis revealed that there were three rrn loci within the genome of Dichelobacter nodosus, the causative organism of ovine footrot. These loci (rrnA, rrnB and rrnC) were isolated on recombinant lambda clones, and comprised 16S, 23S and 5S rRNA genes closely linked in that order. Sequence and primer extension analysis revealed the presence of putative genes encoding tRNAlle and tRNAAla within the 16S-23S spacer region, as well as a number of potential regulatory features. These elements included a single promoter, which was mapped upstream of the 16S rRNA gene and which was similar to Escherichia coli consensus promoter sequences, an AT-rich upstream region, a GC-rich motif that may be involved in stringent control, leader and spacer antitermination sequences, sites for ribonuclease processing, and a putative factor-independent terminator sequence. Potential open reading frames (ORFs) were identified within the regions flanking the rrn loci, with identical copies of the 3′ terminal ORF present downstream of each rRNA operon. Determination of the complete sequence of the 5S rRNA gene, and derivation of the 5S rRNA secondary structure, further substantiated the 16S rRNA-based placement of D. nodosus within the gamma division of the Proteobacteria.
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Rickettsia prowazekii sigma factor σ73 can be overexpressed in Escherichia coli and promotes RNA polymerase binding and transcription
More LessThe sigma factor σ73 of the obligate intracytoplasmic bacterium Rickettsia prowazekii was overexpressed and purified from Escherichia coli . The rickettsial rpoD gene encoding σ73 was cloned into a Ndel-BamHI-cleaved pET15b vector under control of T7 transcription and translation signals. The recombinant plasmid encoded a 75 kDa fusion protein that was overproduced in E. coli BL21(DE3) and purified from inclusion bodies after solubilization with guanidine hydrochloride and using His. Bind metal chelation resin. The N-terminal His. Tag sequence of the 75 kDa fusion protein was removed by thrombin treatment to obtain R. prowazekii σ73T. The R. prowazekii σ73T as well as the 75 kDa fusion protein had the ability to bind to core DNA-dependent RNA polymerase of both R. prowazekii and E. coli and to stimulate their interaction with a rickettsial promoter.
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Analysis of the EHEC hly operon and its location in the physical map of the large plasmid of enterohaemorrhagic Escherichia coli O157:H7
More LessAlmost all clinical enterohaemorrhagic Escherichia coli (EHEC) O157: H7 isolates harbour a large virulence plasmid designated pO157. In this study, pO157 of EHEC O157:H7 reference strain EDL 933 was characterized at the molecular level. A restriction map was constructed by using seven restriction enzymes, with appropriate gel electrophoretic and hybridization methods. The molecular size of pO157 was determined to be 93.6 kb. By sequencing the DNA region extending in the 3′-direction of the previously described EHEC hlyC and hlyA genes, two further genes were discovered and analysed; these were termed EHEC hlyB and EHEC hlyD. The newly discovered genes together with the EHEC hlyC and hlyA genes constitute a typical RTX (Repeats in ToXin) determinant (EHEC hly operon) with the gene order CABD. The map position of the operon was determined by hybridization experiments. Analysis of a DNA fragment carrying the downstream flanking region of the EHEC hly operon revealed an open reading frame which was highly homologous to orf1 of RepFIB, a basic replicon of IncF plasmids. It was located close to the EHEC hly operon.
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Codon usage in the Mycobacterium tuberculosis complex
More LessThe usage of alternative synonymous codons in Mycobacterium tuberculosis (and M. bovis) genes has been investigated. This species is a member of the high-G + C Gram-positive bacteria, with a genomic G + C content around 65 mol%. This G + C-richness is reflected in a strong bias towards C- and Gending codons for every amino acid: overall, the G + C content at the third positions of codons is 83%. However, there is significant variation in codon usage patterns among genes, which appears to be associated with gene expression level. From the variation among genes, putative optimal codons were identified for 15 amino acids. The degree of bias towards optimal codons in an M. tuberculosis gene is correlated with that in homologues from Escherichia coli and Bacillus subtilis. The set of selectively favoured codons seems to be quite highly conserved between M. tuberculosis and another high-G + C Gram-positive bacterium, Corynebacterium glutamicum, even though the genome and overall codon usage of the latter are much less G + C-rich.
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Molecular study and overexpression of the Clostridium cellulolyticum celF cellulase gene in Escherichia coli
The CelF-encoding sequence was isolated from Clostridium cellulolyticum genomic DNA using the inverse PCR technique. The gene lies between cipC (the gene encoding the cellulosome scaffolding protein) and celC (coding for the endoglucanase C) in the large cel cluster of this mesophilic cellulolytic Clostridium species. Comparisons between the deduced amino acid sequence of the mature CelF (693 amino acids, molecular mass 77626) and those of other ß-glycanases showed that this enzyme belongs to the recently proposed family L of cellulases (family 48 of glycosyl hydrolases). The protein was overproduced in Escherichia coli using the T7 expression system. It formed both cytoplasmic and periplasmic inclusion bodies when induction was performed at 37 °. Surprisingly, the protein synthesized from the cytoplasmic production vector was degraded in the Ion protease-deficient strain BL21(DE3). The induction conditions were optimized with regard to the concentration of inductor, cell density, and temperature and time of induction in order to overproduce an active periplasmic protein (CelFp) which was both soluble and stable. It was collected using the osmotic shock method. The enzymic degradation of various cellulosic substrates by CelFp was studied. CelFp degraded swollen Avicel more efficiently than substituted soluble CM-cellulose or crystalline Avicel and was not active on xylan. Its activity is therefore quite different from that of endoglucanases, which are most active on CM-cellulose.
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The expression of the Acinetobacter calcoaceticus recA gene increases in response to DNA damage independently of RecA and of development of competence for natural transformation
Using the lacZ operon fusion technique, the transcriptional control of the Acinetobacter calcoaceticus recA gene was studied. A low (approximately twofold) inductive capacity was observed for compounds that damage DNA and/or inhibit DNA replication, e.g. methyl methanesulfonate, mitomycin C, UV light and nalidixic acid. Induction of the recA gene by DNA damage was independent of functional RecA. The presence of the recA promoter region on a multicopy plasmid had the same effect on recA transcription as the presence of DNA-damaging agents. Thus, recA expression in A. calcoaceticus appears to be regulated in a novel fashion, possibly involving a non-LexA-like repressor. Regulation of the recA gene in A. calcoaceticus appears not to be part of a regulon responsible for competence for natural transformation: in cells exhibiting extremely low transformation frequencies, the level of transcription of the recA gene was found to be comparable to the level found in cells in the state of maximal competence.
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recA-dependence of the response of Pseudomonas aeruginosa to UVA and UVB irradiation
More LessThe responses of the autochthonous soil and aquatic organism, Pseudomonas aeruginosa to UV radiation wavelengths (UVA, 320-400 nm, and UVB, 280-320 nm) has been investigated in this study. P. aeruginosa recA mutants were found to be more sensitive to both UVA and UVB radiation than were their isogenic RecA+ parents. Introduction of a low-copy-number plasmid containing the cloned wild-type P. aeruginosa recA gene restored UVA and UVB resistance to recA mutants. The concentration of RecA protein increased twofold 120 min after exposure to either UVA or UVB radiation, suggesting induction of expression of the recA gene by these wavelengths. In this study, we found that a functional RecA protein is required for activation of D3 prophage in lysogenic cells following exposure to UVB radiation. Prophage were not induced by exposure of their hosts to UVA radiation. Induction of damage-inducible (din) genes in response to UVA or UVB irradiation was also shown to be RecA dependent. These data indicate that the recA gene plays a role in the response of P. aeruginosa to exposure to wavelengths of UV radiation found in the solar spectrum.
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Two classes of ethidium-bromide-resistant mutants of Streptomyces lividans 66
More LessFive spontaneous mutants of Streptomyces lividans TK64 resistant to 5 or 15 μM ethidium bromide (EB) were isolated, and the corresponding mutations were mapped to two different chromosomal locations. Both types of mutations conferred unselected resistance to several basic dyes and norfloxacin. The strain with the low-level resistance exhibited wild-type levels of EB uptake and energy-dependent efflux, and the resistance mechanism is unclear. The highly resistant mutants, which additionally were resistant to phosphonium ions, had a reduced accumulation and an increased efflux of EB, reminiscent of a mammalian multidrug resistance efflux pump.
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- Pathogenicity And Medical Microbiology
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The outermost capsular arabinomannans and other mannoconjugates of virulent and avirulent tubercle bacilli
More LessIt has been shown that phagocyte mannose receptors play an important role in phagocytosis of virulent tubercle bacilli, but not of avirulent strains. Accordingly, we investigated the occurrence and structure of the outermost mannoconjugates of the capsule of five strains of the tubercle bacillus differing in their degrees of virulence. The extracellular and surface-exposed arabinomannan-containing polysaccharides were chemically characterized as being composed mainly of neutral fatty-acyl-free arabinomannans (AMs) possessing a reducing end consisting of mannose. Although no lipoarabinomannan (LAM) was detected, small amounts of acidic polysaccharides, exhibiting the same electrophoretic mobility as LAM, were identified as succinylated AMs (two to three residues per molecule) lacking the phosphatidylinositol anchor of LAM. AMs from the different strains shared the same structural features, notably the capping of a large portion of the arabinan segments with mannosyl residues. However, no correlation was observed between either the percentage of capping or the amount of AMs and the degrees of virulence of the strains. The occurrence and amounts of other mannoconjugates (phosphatidylinositol mannosides and the mannoseassociated 19 and 38 kDa lipoproteins) in the various tubercle bacilli were also examined. Although both classes of compounds were identified in all the examined strains, a correlation between the amounts of the glycoconjugates and the degrees of virulence of the strains could not be established. These data do not support the implication of these promising mannosylated molecules in the selective phagocytosis of virulent tubercle bacilli and indicate that the involvement of mannose receptors in phagocytosis of virulent M. tuberculosis needs to be re-investigated.
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Stress tolerance and pathogenic potential of a mannitol mutant of Cryptococcus neoformans
More LessCryptococcus neoformans produces large amounts of the acyclic hexitol mannitol in culture and infected animals, but the functional and pathogenic significance of mannitol production by this fungus is not known. We exposed C. neoformans H99 (Cn H99) to UV irradiation (1 × LD50) and screened survivors for mannitol production. A mutant, Cn MLP (Mannitol Low Producer), synthesized less mannitol from glucose (2.7 vs 8.2 nmol per 108 cells min−1 at 37 °) and contained less intracellular mannitol (1 vs 11 μmol per 106 cells at 37 °) than did Cn H99. Cn MLP and Cn H99 were similar with respect to carbon assimilation patterns, rates of glucose consumption, growth rates at 30 °, urease and phenoloxidase activities, morphology, capsule formation, mating type, electrophoretic karyotype, rapid amplification of polymorphic DNA (RAPD) patterns and antifungal susceptibility. However, Cn MLP was more susceptible than was Cn H99 to growth inhibition and killing by heat and high NaCl concentrations. Also, the LD50 values in mice injected intravenously were 3.7 × 106 c.f.u. for Cn MLP compared to 6.9 × 102 c.f.u. for Cn H99. Moreover, 500 c.f.u. Cn H99 intravenously killed 12 of 12 mice by 60 d, whereas all mice given the same inoculum of Cn MLP survived. Classical genetic studies were undertaken to determine if these differences were due to a single mutation, but the basidiospores were nonviable. These results suggest that the abilities of C. neoformans to produce and accumulate mannitol may influence its tolerance to heat and osmotic stresses and its pathogenicity in mice.
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Degradation of host protease inhibitors and activation of plasminogen by proteolytic enzymes from Porphyromonas gingivalis and Treponema denticola
More LessBacterial proteases may participate in the pathogenesis of periodontal diseases through their action on host proteins. In the present study, the ability of selected periodontopathogens, as well as two proteases isolated from Porphyromonas gingivalis and Treponema denticola, to degrade host protease inhibitors was evaluated. The activation of human plasminogen by the two bacterial proteases was also investigated. Proteolytic breakdown of host protease inhibitors (α-1-antitrypsin, antichymotrypsin, α2-macroglobulin, antithrombin III, antiplasmin and cystatin C) was evaluated by SDS-PAGE. The 80 kDa trypsin-like protease of P. gingivalis completely digested the six protease inhibitors under investigation, whereas the 95 kDa chymotrypsin-like protease of T. denticola was slightly less active, more particularly on α2-macroglobulin and cystatin C. When whole cells from a number of oral bacterial species were tested, the most significant degradation was obtained with P. gingivalis, T. denticola, Prevotella intermedia, Prevotella nigrescens and Capnocytophaga spp. Peptostreptococcus micros and Propionibacterium acnes had only some degradative activity on selected inhibitors, whereas three bacterial species, Actinobacillus actinomycetemcomitans, Bacteroides forsythus and Fusobacterium nucleatum, had no effect on the protease inhibitors. The 80 kDa protease of P. gingivalis demonstrated strong plasminogen activation, whereas no such activity was associated with the 95 kDa protease of T. denticola. This study indicates the high potential of some periodontal pathogens to destroy protease inhibitors and activate plasminogen. This may result in an uncontrolled degradation of periodontal tissues and a rapid progression of the disease.
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- Physiology And Growth
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Fatty acid adaptation in an Antarctic bacterium - changes in primer utilization
More LessThe fatty acid composition and temperature/growth characteristics of a psychrophilic bacterium, strain ACAM 456, isolated from Antarctic sea-ice is reported. The bacterium produced acyl components that may be grouped in three different carbon chain types: even-chain, odd-chain and iso-branched odd-chain. The proportions of these chain types varied according to growth temperature, and were manipulated by growth on L-serine, t-leucine or propionic acid as sole carbon sources. De novo fatty acid synthesis was investigated using sodium [1-14C]acetate, L-[U-14C]leucine and L-[U-14C]serine as radioactive precursors. Compared with a control culture, resuspension of midexponential phase cells in artificial seawater led to a change in the selection and/or intracellular availability of acyl chain primer molecules. The proportion of radiolabel incorporated into even-chain length components from cells declined, whereas the percentage of radiolabel present in odd-chain length components increased. An increase in incubation temperature augmented this effect, and also elicited a rise in the proportion of label present in branchedchain products. ACAM 456 manipulated the utilization of acyl chain primer molecules as an adaptive response to changes in environmental conditions. In particular, the regulation of odd-chain length fatty acids is described as a novel adaptational response.
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Primary metabolite kinetics of bacteriocin biosynthesis by Lactobacillus amylovorus and evidence for stimulation of bacteriocin production under unfavourable growth conditions
More LessTo optimize bacteriocin production processes, the relationships between growth, bacteriocin production and factors affecting the occurrence and intensity of the activity peak during the growth cycle must be understood. Amylovorin L471, a bacteriocin produced by Lactobacillus amylovorus DCE 471, displays primary metabolite kinetics with a peak activity during the midexponential phase. Because of this growth association, only conditions favouring a drastic increase in biomass improve the volumetric bacteriocin titre. Specific bacteriocin production is enhanced under unfavourable growth conditions such as low temperatures (30°), and the presence of potentially toxic compounds such as ethanol (1.0%, v/v) and oxygen (80%, v/v, air saturation). Whereas volumetric biomass formation and growth-associated bacteriocin production are dependent on the amount of glucose and nitrogen supplied, slow growth rates stimulate specific bacteriocin production. Bacteriocin inactivation can be ascribed to protein aggregation and adsorption phenomena. It may be overcome by switching the pH to 2.0 during the fermentation run after having reached the peak activity. Thus, manipulation of the cell environment can stimulate bacteriocin production. The latter can be induced by unfavourable growth conditions, so-called stress factors. The specific growth rate seems to play an important role in the control of bacteriocin production.
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Surface location of HPr, a phosphocarrier of the phosphoenolpyruvate: sugar phosphotransferase system in Streptococcus suis
More LessHPr is a low-molecular-mass phosphocarrier protein of the bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) found in the cytoplasm or associated with the inner surface of the cytoplasmic membrane. Treatment of Streptococcus suis cells with a Sorvall Omnimixer, a technique used to extract cell surface components, resulted in the extraction of a major protein with a molecular mass of 9 kDa. Several lines of evidence suggested that this protein was HPr: (i) the S. suis protein showed homology over the first 35 N-terminal amino acid residues with the HPrs of Streptococcus salivarius and Streptococcus mutans, including the signature sequence for the site of PEP-dependent phosphorylation; (ii) it cross-reacted with the S. salivarius anti-HPr antibody preparation; (iii) it could be phosphorylated by enzyme 1 at the expense of PEP, and by a membrane-associated kinase at the expense of ATP; and (iv) it possessed phosphocarrier activity when used as a source of HPr in an in vitro PTS assay. The data suggested that a portion of the cellular HPr is associated with the external cell surface in S. suis, a result that was confirmed by immunogold electron microscopy. The cellular HPr of S. suis consisted of two forms that could be distinguished by the presence or the absence of the N-terminal methionine. Amino acid sequence analysis indicated that the cell-surface-associated HPr of S. suis lacked the N-terminal methionine residue.
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Physiological and biochemical changes accompanying the loss of mucoidy by Pseudomonas aeruginosa
More LessPseudomonas aeruginosa M60, a mucoid strain, was grown in continuous culture (D 0-05 h−1) under ammonia limitation with glucose as the carbon source. Steady-state alginate production occurred for only 1-2 d under these conditions [q alginate 0.097 g alginate h−1 (g dry wt cells)−1], after which time the percentage of mucoid cells and the alginate concentration in the culture decreased in parallel and approached zero after approximately 10 d. These changes were accompanied by similar decreases in the activities of the alginate biosynthetic enzymes (represented by phosphomannomutase and GDP-mannose dehydrogenase) and by a large increase in the activity of the first enzyme of the ‘external’ non-phosphorylative pathway of glucose metabolism, glucose dehydrogenase. In contrast, the activities of other enzymes associated with this pathway (gluconate dehydrogenase, 2-ketogluconate kinase plus 2-ketogluconate-6-phosphate reductase) or with the ‘internal’ phosphorylative pathway of glucose metabolism (glucokinase and glucose-6-phosphate dehydrogenase) remained essentially unchanged. The loss of mucoidy and alginate production was accompanied by the appearance of low concentrations of intracellular polyhydroxyalkanoate (PHA) and of extracellular gluconate and 2-ketogluconate (partly at the expense of alginate production and partly as a result of increased glucose consumption). It is suggested that ammonia-limited, glucose-excess cultures of P. aeruginosa growing at low dilution rate are unable fully to regulate the rate at which glucose and/or its ‘external’ pathway metabolites are taken up by the cell, and therefore form copious amounts of alginate in order both to overcome the potentially deleterious osmotic effects of accumulating surplus intracellular metabolites and to consume the surplus ATP generated by the further oxidation of these metabolites. The loss of mucoidy invokes the use of an alternative, but analogous, strategy via which non-mucoid cells produce an osmotically inactive intracellular product (PHA) plus increased amounts of the extracellular metabolites gluconate and 2-ketogluconate via the low-energy-yielding and, under these conditions, largely dead-end ‘external’ metabolic pathway.
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The relationships between leukotoxin production, growth rate and the bicarbonate concentration in a toxin-production-variable strain of Actinobacillus actinomycetemcomitans
More LessActinobacillus actinomycetemcomitans, a Gram-negative periodontopathic bacterium, produces a leukotoxin belonging to the RTX family. The production of leukotoxin varies greatly among different strains of this species. In this paper the effects of growth rate and bicarbonate on the leukotoxin production by a toxin-production-variable strain (301-b) during growth in a chemostat were examined. When the bacterium was grown in anaerobic fructose-limited chemostat cultures (pH 7.0 and 37 °) at dilution rates (D) ranging from 0.04 to 0.20 h−1 in the absence and presence of 10 mM bicarbonate, it produced leukotoxin as a cluster of two polypeptides (M r 113000 and 120000) and complexed with nucleic acids on the bacterial cell surface. The relationship between leukotoxin production and specific growth rate was analysed by plotting the specific rate of leukotoxin production [q LT, in μg (mg dry wt)−1 h−1] against D. The plots were approximated to the linear relationships q LT 2.7D − 0.058 and q LT − 9.3D − 0.407 without and with bicarbonate, respectively. These relationships suggest that the apparent leukotoxin production is a result of both growth-rate-dependent production and growth-rate-independent decomposition. The cellular leukotoxin level was also followed after the change from chemostat to batch culture in the same fermenter. In batch culture leukotoxin production stopped immediately and the cellular toxin level rapidly decreased, suggesting toxin decomposition. From the slopes of the approximated linear relationships between q LT and D, a theoretical maximum leukotoxin yield (Y LT) was estimated as 2.7 and 9.3 μg (mg dry wt)−1 in the absence and presence of 10 mM bicarbonate, respectively. The increased Y LT value in the cultures containing bicarbonate indicated that the addition stimulated the efficiency of leukotoxin synthesis up to about threefold. Further increases of bicarbonate concentration to between 20 and 40 mM had no effect on the total leukotoxin production, but the amount of extracellular leukotoxin increased with higher bicarbonate concentrations.
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- Systematics
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Characteristics of Sulfobacillus acidophilus sp. nov. and other moderately thermophilic mineral-sulphide-oxidizing bacteria
More LessSeveral isolates of Gram-positive, acidophilic, moderately thermophilic, ferrous-iron- and mineral-sulphide-oxidizing bacteria were examined to establish unequivocally the characteristics of Sulfobacillus-like bacteria. Two species were evident: Sulfobacillus thermosulfidooxidans with 48-50 mol% G + C and Sulfobacillus acidophilus sp. nov. with 55-57 mol% G + C. Both species grew autotrophically and mixotrophically on ferrous iron, on elemental sulphur in the presence of yeast extract, and heterotrophically on yeast extract. Autotrophic growth on sulphur was consistently obtained only with S. acidophilus.
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Acidimicrobium ferrooxidans gen. nov., sp. nov.: mixed-culture ferrous iron oxidation with Sulfobacillus species
More LessA new species of ferrous-iron-oxidizing, moderately thermophilic, acidophilic bacteria, Acidimicrobium ferrooxidans, has been described. Two isolates of the species differed only in the tendency of one, previously known as strain TH3, to grow in filaments. The chromosomal DNA base composition is between 67 and 69 mol% G + C. The capacity of this species to fix CO2 from air was greater than that of iron-oxidizing thermoacidophiles of the genus Sulfobacillus, which required an enhanced CO2 concentration for optimum autotrophic growth. Under air, ferrous iron oxidation in mixed cultures of A. ferrooxidans with either Sulfobacillus thermosulfidooxidans or Sulfobacillus acidophilus was more extensive than in pure cultures of these three strains. The greater part of ferrous iron oxidation in mixed cultures probably resulted from activity of the Sulfobacillus species, which possess a greater tolerance of ferric iron, and which presumably grew mixotrophically utilizing organic compounds from A. ferrooxidans.
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