- Volume 139, Issue 4, 1993
Volume 139, Issue 4, 1993
- Biochemistry
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Characterization of the rnc-97 mutation of RNAaseIII: a glycine to glutamate substitution increases the requirement for magnesium ions
More LessThe rnc-97 mutation of the Escherichia coli double-stranded-RNA-specific ribonuclease III (RNAaseIII) was previously isolated by virtue of the lethal expression of RNAaseIII in Saccharomyces cerevisiae. Here we show that rnc-97 is a single point mutation causing the substitution of glycine 97 by glutamic acid. The mutation eliminates the lethal phenotype of RNAaseIII expression in yeast and reduces fourfold the effect of RNAaseIII expression on bacteriophage gy1 propagation in E. coli. Mutant RNAaseIII-G97E and wild-type RNAaseIII were purified according to published procedures. The apparent molecular masses of the two enzymes on SDS polyacrylamide gels are the same but they differ in pI (6·85 for RNAaseIII-G97E and 7·3 for RNAaseIII). Whereas the two enzymes (under standard assay conditions) do not show a great difference in activity towards double-stranded RNA and defined single-stranded RNAaseIII substrates, they differ dramatically (20-fold or more) under conditions of Mg2+ limitation. The hypothesis that limitation of Mg2+ ions in vivo is responsible for the phenotypes of the rnc-97 mutation in S. cerevisiae and E. coli is discussed.
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The sexual agglutinins in Chlamydomonas eugametos are sulphated glycoproteins
More LessSexual adhesion in the green alga Chlamydomonas eugametos is mediated by hydroxyproline-containing glycoproteins (agglutinins). The agglutinins were metabolically labelled with [35S]sulphate. The sulphur-containing amino acids methionine and cysteine were not detectably labelled; little or no sulphated tyrosine was detected. This indicated that the radioactive label had been largely or exclusively incorporated into sulphated sugar residues. Acid hydrolysis released essentially all radioactivity as free sulphate. Short periods of hydrolysis released sulphated oligosaccharides. β-Elimination released about 20–25% of the sulphated saccharides, indicating that these are O-linked to serine or threonine or both. The remaining sulphated sugar chains are presumably O-linked to hydroxyproline. It is proposed that the sulphated sugars of the agglutinins play a role in sexual adhesion.
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Sensitivity of elongation factor Tu (EF-Tu) from different bacterial species to the antibiotics efrotomycin, pulvomycin and MDL 62879
More LessThe sensitivity of elongation factor Tu (EF-Tu) from different species of bacteria to the EF-Tu-binding antibiotics efrotomycin, pulvomycin and MDL 62879 was tested by measuring the effect of these antibiotics on cell-free protein synthesis systems. EF-Tu from four different Gram-negative species was sensitive to all three antibiotics. Among Gram-positive bacteria, EF-Tu of Bacillus subtilis, Staphylococcus aureus and Enterococcus faecalis was resistant to efrotomycin and less sensitive to pulvomycin than EF-Tu of Gram-negative bacteria. EF-Tus from streptococci were significantly less sensitive than EF-Tus from Gram-negative bacteria to both efrotomycin and pulvomycin. All of the EF-Tus were sensitive to MDL 62879. The same sensitivity pattern emerged from GDP exchange assays, performed with partially purified EF-Tu from different bacterial species and pure Escherichia coli EF-Ts. These results suggest that the site of action of MDL 62879 is more conserved among bacterial species than those of efrotomycin and pulvomycin. Heterogeneity of EF-Tus from different bacterial species was also reflected in differences in their apparent molecular masses estimated by SDS-PAGE. EF-Tus from the Gram-positive species had higher molecular masses than those from all but one of the Gram-negative species.
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Properties of the cold-labile NAD+-specific glutamate dehydrogenase from Bacillus cereus DSM 31
More LessNicotinamide-adenine-dinucleotide-specific glutamate dehydrogenase (NAD-GDH; EC 1.4.1.3) from Bacillus cereus DSM 31 was enriched 260-fold. The molecular mass was determined by gel filtration to be 270 kDa (± 25 kDa). The enzyme was highly specific for the coenzyme NAD(H) and catalysed both the formation and the oxidation of glutamate. Apparent K m values of 7.7 mM for glutamate and 0.56 mM for NAD+ during oxidative deamination were measured. Both in crude cell-free extracts and in enriched preparations the enzyme was extremely unstable, especially at low temperatures. The loss of activity in the cold was found to be due to the dissociation of the holoenzyme into catalytically inactive subunits of molecular mass 48 kDa (±5 kDa), indicating that the native enzyme has a hexameric structure. The activity was restored under certain conditions, and no instability of the enzyme in the cold was observed in undisrupted cells.
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Purification and characterization of an alkaline isoamylase from an alkalophilic strain of Bacillus
More LessAlkaline isoamylase (glycogen 6-glucanohydrolase, EC 3.2.1.68) activity was detected in the culture medium of an alkalophilic strain of Bacillus sp., designated KSM-3309, which was isolated from a soil sample. This novel enzyme was purified to homogeneity from the culture filtrate by precipitation with ammonium sulphate, chromatography on DEAE-cellulose and DEAE-Bio-Gel A, and gel filtration on Sephacryl S-200. The purified enzyme had a pH optimum of approximately 9·0, and displayed maximum catalytic activity at 55 °C. The enzyme had a molecular mass of 65 kDa, as determined by both SDS-polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200. The isoelectric point was 4·2. This enzyme cleaved the branching points of both amylopectin and glycogen, and incubation of the enzyme with these glucans caused large increases in coloration of the iodine reagent. Amylose, pullulan and maltose were practically insensitive to the enzyme. The enzyme activity was inhibited by Hg2+ ions and by N-bromosuccinimide, but the thiol inhibitors iodoacetate, 4-chloromercuribenzoate and N-ethylmaleimide had either no effect or a slightly inhibitory effect. β-Cyclodextrin, an inhibitor of pullulanase, was not inhibitory.
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Characterization of the 6-aminohexanoate-dimer hydrolase from Pseudomonas sp. NK87
More LessThe DNA base sequence of the Pseudomonas sp. NK87 gene (P-nylB) for 6-aminohexanoate-dimer hydrolase (P-EII), a xenobiotic-compound-degrading enzyme, was determined. It has an open reading frame of 1188 bp, initiated by ATG and terminated by TAG, and coding for 396 amino acids. The base sequence of the open reading frame has 53% sequence similarity to that of the gene for the same enzyme of Flavobacterium sp. KI72 (F-nylB) and 35% sequence similarity with respect to the deduced amino acid sequence. The P-EII enzyme was purified from an Escherichia coli clone in which the P-EII gene was highly expressed. The P-EII enzyme was inhibited by a serine protease inhibitor, diisopropyl fluorophosphate, as was the F-EII enzyme. Double reciprocal plots obtained from various concentrations of 6-aminohexanoate-dimer indicated that the k cat value of the P-EII enzyme (9.2 s−1) was approximately half that of the F-EII enzyme (19 s−1), and the P-EII enzyme had higher affinity toward this substrate (K m for P-EII, 0·6 mm; K m for F-EII, 15 mm). The P-EII enzyme had a temperature optimum of 48 °C, and a pH optimum of 7·5. It is speculated that since the P-nylB and F-nylB genes are more diverged from each other than the corresponding nylA genes, the latter may have evolved more recently.
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Diketocamphane enantiomer-specific ‘Baeyer-Villiger’ monooxygenases from camphor-grown Pseudomonas putida ATCC 17453
More LessPseudomonas putida ATCC 17453 grew with either (+)- or (−)-camphor as sole carbon source. Enantiomer-specific ‘biological Baeyer-Villiger’ monooxygenases were synthesized irrespective of the camphor isomer used for growth. The two enzymes are probably the products of separate genes but showed many similarities. Each consisted of two electrophoretically indentical subunits, bound flavin mononucleotide (FMN) non-covalently and accepted electrons from an induced NADH dehydrogenase which interacted with the FMN bound to the oxygenating component. They showed minor differences in M r with 3,6-diketocamphane 1,6-monooxygenase being the smaller enzyme. Isoelectric focussing showed the two enzymes to have different acidic pI values. Polyclonal antibodies raised against 3,6-diketocamphane 1,6-monooxygenase also cross-reacted with 2,5-diketocamphane 1,2-monooxygenase and its subunits.
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- Development And Structure
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Gradients in the expression of cell surface glycoproteins in a simple tissue, the Dictyostelium discoideum slug
More LessGradients in diffusible morphogens have long been postulated to have a role in the establishment of polarity and pattern formation in developing animal embryos. In the cellular slime mould Dictyostelium discoideum, several morphogens have been described and their interactions and location in the multicellular ‘slug’ stage are being studied. Here we report that two cell surface antigens, which identify prespore and prestalk cells, respectively, are present at different levels on the surface of cells depending on their position within the slug. This is the first evidence of the existence of gradients in cell surface molecules in Dictyostelium discoideum tissues. The level of prespore glycoprotein PsA on the surface of prespore cells increased progressively along the length of the slug, so that the rear cells had the highest surface density of this glycoprotein. Since the pspA gene, which encodes PsA, is cAMP-regulated, this raises the possibility that expression of PsA reflects an underlying morphogenetic cAMP gradient. The level of the MUD9 antigen, which is prestalk-enriched, decreased on the surface of prestalk cells towards the rear of the slug. As this correlated with a decrease in size of these cells along the length of the slug, it is possible that the surface density of this antigen is approximately constant. We have also identified a population of prestalk-like cells that lack both the PsA glycoprotein and the MUD9 antigen. These cells primarily occurred in the posterior of the slug and could represent the population of cells that form the basal disc.
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Ultrastructure of the surface film of bacterial colonies
More LessThe structure of the surface of colonies of various Gram-negative and Gram-positive bacteria was examined by transmission electron microscopy. The results indicate that bacterial colonies in the course of their development produce a film which becomes thicker with increased duration of growth. The basic part of the film is an elementary membrane, which is a stable structure with a large surface area. The inner and outer surfaces of the film membrane are covered by amorphous layers. These layers are thicker in the surface film of Gram-negative bacterial colonies than in those of Gram-positive bacteria. Membrane vesicles from the bacterial colonies take part in the formation of the surface film. The presence of the film on the surface of the colonies of different bacteria suggests that this structure may play an important role.
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Protein composition of rhapidosomes isolated from Aquaspirillum itersonii
More LessRhapidosomes are tubular microstructures composed of proteins that are found in a variety of bacteria and algae. These structures, which are resistant to disruption by many denaturing agents, have potential application as a biomaterial and may serve as a new model for the study of self assembly. When rhapidosomes were purified and analysed by SDS-PAGE the presence of three proteins with molecular masses of 53, 29 and 18 kDa was revealed. However, lysozyme treatment of the rhapidosome preparations containing the three proteins resulted in the selective release of the 18 kDa protein from the rhapidosome complex. N-Terminal sequence analysis and amino acid composition analysis were performed on all three proteins. The amino acid composition of the 18 kDa protein closely matched the amino acid composition of protein H (a peptidoglycan-associated protein from Pseudomonas aeruginosa). These results suggest that the 18 kDa protein may be a contaminating peptidoglycan-associated protein and not part of the rhapidosome structure. Western blot analysis using antisera raised against rhapidosomes revealed that the 53 kDa component protein does not react with the antisera. We speculate that the 53 kDa protein may form the inner core of the rhapidosomes, whilst the 29 kDa protein forms the outer sheath of these structures.
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Induction of myxospores in Stigmatella aurantiaca (myxobacteria): inducers and inhibitors of myxospore formation, and mutants with a changed sporulation behaviour
More LessIn a screening programme, many new compounds were found that induced myxospore formation in Stigmatella aurantiaca strain Sg a1. The most efficient compounds were indole and indole derivatives, e.g. 3-methylindole. With an inducing concentration of 0·07 mm, this compound was about 2000 times more efficient than glycerol. Indole compounds, which have also been found as natural metabolites of myxobacteria, are thus candidates for auto-inducers within the fruiting bodies. Mutants were isolated that were resistant to particular inducers. They often showed cross-resistance to many other inducers. By the sporulation response of three different mutants, the inducers could be classified into four groups. Strains resistant to inducers of, e.g. group I were still sensitive to inducers of groups II and III, and vice versa. This classification was supported by newly discovered inhibitors of induced sporulation. These inhibitors, oxindole and pyrrole, were specific for inducers of group I. The grouping is further supported by differences in the kinetics of myxospore formation. We postulate that S. aurantiaca has three inducer-specific, independent receptors.
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- Environmental Microbiology
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Factors affecting competence in a high frequency of transformation marine Vibrio
More LessNatural plasmid transformation may be a mechanism for the horizontal transfer of non-conjugative plasmids in the marine environment, yet there are few marine model systems available for the study of this process. Using multimers of IncQ/P4 plasmids and a filter transformation assay, we have measured the effects of nutrients, salinity, temperature, as well as the development and maintenance of competence for genetic transformation in the high frequency of transformation (HFT) marine Vibrio strain WJT-1C. Transformation frequency was proportional to the amount of DNA used from 0·1 to 1·0 μg DNA and was saturated at concentrations greater than 1·0 μg. Competence began in the early-exponential phase and reached a maximum at the onset of stationary phase. Once attained, competence was maintained in both spent and nutrient-free media for at least 10 d. Thus, the establishment and maintenance of competence was unique compared to previously described transformation systems. Temperatures ranging from 4 to 33 °C had no significant effect on the maximal transformation frequency of WJT-1C, but at 37 °C the transformation frequency was reduced. However, temperature did affect the rate of the transformation process. Salinities in the range 12 to 50% had no significant effect on the transformation frequency but transformation frequencies were lower at 6% and 63%. Cells were transformed equally well in nutrient-free media or rich media. The ability of this marine HFT Vibrio strain to develop competence under a wide spectrum of conditions and to maintain the competent state indicates that natural plasmid transformation could occur in conditions found in tropical and subtropical estuaries.
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Inhibition of the cellulolytic activity of Neocallimastix frontalis by Ruminococcus flavefaciens
More LessA study was made of the antagonistic effect of Ruminococcus flavefaciens on the cellulolytic activity of Neocallimastix frontalis. An extracellular factor inhibiting the cellulolytic activity of the fungus was detected in the bacterial supernatant. The antagonistic factor, which precipitated with ammonium sulphate at 40% saturation, was temperature-sensitive and was destroyed at temperatures above 60 °C. After separation by anion-exchange chromatography, sequential precipitation, dialysis and SDS-PAGE, two protein species of 100 and 24 kDa were identified as being involved in this antagonistic effect. It is not known whether the proteins are two subunits of a single protein or represent two different proteins. The inhibitory factor, which is not a bacterial cellulase, did not affect fungal growth, but it inhibited the activity of the fungal cellulases.
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- Genetics And Molecular Biology
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A partial physical map for the chromosome of alkalophilic Bacillus sp. strain C-125
More LessBacillus sp. strain C-125 has been chosen as a model alkalophilic bacterium to understand how adaptation to growth at high pH is achieved. To aid genetic analysis, we have started characterization of its genome. By using the two infrequently-cutting restriction endonucleases, AscI and Sse8387I, in conjunction with pulsed-field electrophoretic techniques, the size of the genome was found to be 3·7 Mb. Southern blot analysis of single, double and partial digests of Bacillus sp. strain C-125 DNA, using AscI-linking clones, gene probes and purified Bacillus sp. strain C-125 restriction fragments, allowed a putative chromosome map to be constructed.
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Analysis of the site for second-strand initiation during replication of the Streptomyces plasmid pIJ101
More LessThe indigenous plasmid pIJ101 is the parent of many cloning vectors used in Streptomyces. One early pIJ101 derivative, pIJ702, has been particularly widely used. pIJ702 lacks sti:cop/korB and accumulates single-stranded DNA (ssDNA). The 1.2 kb BclI-BclI sti:cop/korB and 0.7 kb SpeI-BclI sti regions were isolated from pIJ101 and cloned into pIJ702 at the PstI site in both orientations. No ssDNA was detected in constructs containing sti present in its correct orientation with respect to the basic replicon, with or without cop/korB. Constructs which contained sti in the reverse orientation did accumulate ssDNA. Thus, sti is only active as the site for second-strand synthesis in its natural orientation. Furthermore, sti inserted in either orientation into the structurally unstable pIJ702-pUC8 shuttle vectors prevented them from rearranging in S. lividans. The sti function was defined to a 0.53 kb SpeI-SacII fragment and the probable site for second-strand initiation (ssi) was identified.
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Multiple domain structure in a chitinase gene (chiC) of Streptomyces lividans
More LessOne 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.
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Location of the genes for anthranilate synthase in Streptomyces venezuelae ISP5230: genetic mapping after integration of the cloned genes
More LessThe anthranilate synthase (trpEG) genes in Streptomyces venezuelae ISP5230 were located by allowing a segregationally unstable plasmid carrying cloned S. venezuelae trpEG DNA and a thiostrepton resistance (tsr) marker to integrate into the chromosome. The integrated tsr was mapped by conjugation and transduction to a location close to tyr-2, between arg-6 and trpA13. A genomic DNA fragment containing trpC from S. venezuelae ISP5230 was cloned by complementation of a trpC mutation in Streptomyces lividans. Evidence from restriction enzyme analysis of the cloned DNA fragments, from Southern hybridization using the cloned trp DNA as probes, and from cotransduction frequencies, placed trpEG at a distance of 12-45 kb from the trpCBA cluster. The overall arrangement of tryptophan biosynthesis genes in the S. venezuelae chromosome differs from that in other bacteria examined so far.
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Recombination at the coagulase locus in Staphylococcus aureus: plasmid integration and amplification
More LessThe integrating plasmid pCOA18, comprising pUC18 linked to a mutated coagulase (coa) gene from Staphylococcus aureus, and constructed by substituting coa sequences with a tetracycline (Tc)-resistance marker (∆coa::Tcr), was transformed into Staphylococcus aureus RN4220, where it underwent recombination with the chromosomal coa locus. Allele-replacement mutants were recovered at a low frequency directly after transformation. The majority of transformants carried pCOA18 integrated in the chromosome by a single Campbell-type recombination event. The majority of integrants contained tandem repeats of pCOA18 and expressed high levels of resistance to Tc (> 30 μg ml−1) compared to the single-copy integrants and allele-replacement mutants (15 μg ml−1). Transduction of a single-copy integrant to a Coa+ recipient allowed the cointegrant to be resolved and allele-replacement recombinants to be selected. In addition, growth of a single-copy integrant on high concentrations of Tc (> 30 μg ml−1) selected for amplified derivatives at a frequency of 10−5. It was estimated that up to 19 copies of pCOA18 could occur in a tandem array in the chromosome.
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Ribosome assembly in three strains of Escherichia coli with mutations in the rpmB,G operon
More LessThe rpmB,G operon of Escherichia coli codes for the synthesis of ribosomal proteins L28 and L33. In one mutant strain (TP28), these two proteins are made at about half their normal rates, ribosome assembly is greatly perturbed and precursor particles accumulate. The mutation in strain TP28 is in a Shine-Dalgarno sequence in the leader region of the rpmB,G messenger RNA. Another mutant, strain AM108, makes neither protein because it has the mobile element IS1 inserted into the rpmB coding sequence. Surprisingly, ribosome assembly in this strain is virtually normal with respect to growth rate. Strain AM90, which fails to make protein L33, has the element IS3 inserted into rpmG and also shows no major defects in ribosome assembly.
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Cloning and characterization of a nitrite reductase gene from Alcaligenes faecalis and its expression in Escherichia coli
The gene (nir) encoding the copper-containing nitrite reductase (NIR) of a denitrifying bacterium, Alcaligenes faecalis S-6, was cloned by a synthetic oligonucleotide-probing method. The nucleotide sequence of the cloned DNA fragment revealed the primary structure of the NIR precursor containing the N-terminal signal sequence for secretion. A nucleotide sequence, possibly recognized by a transcriptional regulator resembling FNR was found upstream of the structural gene. When the cloned gene was expressed in Escherichia coli under the control of the lac promoter at 37 °C, NIR was produced as large inclusion bodies and little activity was detected. When cultivation was at 20 °C, most of the NIR was detected in the soluble fraction and a significant portion of the protein was translocated into the periplasmic space, accompanied by removal of its signal sequence.
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Molecular comparison of the IncX plasmids allows division into IncX1 and IncX2 subgroups
More LessWe have investigated molecular relationships and evolution of plasmids classified genetically to incompatibility (Inc) group X, in particular by comparison of plasmids from the pre-antibiotic era (PAE) with contemporary R-plasmids. On the basis of restriction analysis, R6K, the best-described and ‘prototype’ plasmid of the IncX group, exhibited little similarity with the other plasmids in this Inc group. Other contemporary IncX R-plasmids exhibited a substantial degree of interrelationship, and were also related to PAE IncX plasmids. When the origin of plasmid replication of R6K was used as a replicon probe, R6K was the only plasmid tested which exhibited homology. Other contemporary and PAE IncX plasmids exhibited homology with the origin of plasmid R485. These data suggest that the IncX group should be subdivided. R485 may be regarded as representative of the major subgroup present before and after the advent of antibiotic selection pressure. Plasmids of this subgroup, IncX1, possess an internal region which yields five characteristic EcoRV fragments. R6K may be regarded as representative of subgroup IncX2, of which it is presently the sole well-described member. The antibiotic resistances encoded by contemporary IncX R-plasmids are due to insertion of identifiable transposons in progenitor plasmids identical to the R485 subgroup of PAE IncX plasmids.
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Identification of methanol-regulated promoter sequences from the facultative methylotrophic bacterium Methylobacterium organophilum XX
More LessA 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.
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Cyclohexadienyl dehydratase from Pseudomonas aeruginosa is a periplasmic protein
More LessThe gene encoding cyclohexadienyl dehydratase from Pseudomonas aeruginosa, designated pheC, was cloned in Escherichia coli and sequenced recently by Zhao et al. (Journal of Biological Chemistry 267, 2487–2493, 1992). N-Terminal sequencing of the purified cyclohexadienyl dehydratase yielded a run of 11 residues which matched perfectly with the deduced amino acid residues 26–36. This showed that a 25 residue peptide was cleaved from the N-terminus of a preprotein formed in E. coli. The amino acid composition of the 25 residue peptide was typical of signal sequences for periplasmic proteins. Most or all of the cyclohexadienyl dehydratase was released from P. aeruginosa and E. coli carrying the pheC gene following spheroplast formation, osmotic shock or chloroform treatment. The location of the enzyme in the periplasm of both E. coli and P. aeruginosa was confirmed by Western blotting analysis using antibody prepared against PheC. Electron microscopy using immunogold labelling showed an apparent localization of cyclohexadienyl dehydratase at the polar regions of the periplasmic space in E. coli.
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Immunological relatedness of the LamB proteins among members of Enterobacteriaceae
More LessWe have studied the immunological relatedness of LamB proteins from a wide range of enterobacterial species, using antibodies directed against denatured Escherichia coli K12 and Klebsiella pneumoniae LamB proteins (LamBE.c. and LamBK.p., respectively), and anti-peptide antibodies directed against 10 distinct loops of LamB from E. coli K12 predicted to protrude either side of the outer membrane. We have shown that a protein immunologically related to LamBE.c. and LamBK.p. was present in all members of Enterobacteriaceae tested. A protein recognized by several anti-peptide antibodies was identified in E. coli, Shigella sonnei, Salmonella typhimurium and Kleb. pneumoniae, as well as in two Citrobacter species, two Enterobacter species and Kluyvera ascorbata. The recognition patterns obtained with the anti-peptide antibodies were in agreement with the LamB protein sequence data available. They indicated that the cell surface and also the periplasmic loops of LamB are subject to great antigenic variability.
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- Physiology And Growth
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Motility and chemotaxis of a Xanthobacter sp. isolated from roots of rice
More LessChemotaxis and substrate-regulated motility of the rice isolate Xanthobacter sp. JW-KR2 was studied. Growth on alcohols produced motile cells possessing several peritrichous flagella while growth in the presence of organic acids, such as succinate, repressed flagellar synthesis, leading to nonmotile cells. Addition of 2·5 mm-adenosine 3′,5′-cyclic monophosphate (cAMP) to the culture resulted in motile cells even when succinate was the sole carbon source. Chemotaxis assays using microcapillary pipettes revealed a positive response to 1-propanol, 2-propanol, 1-butanol, 2-butanol and 1-pentanol but not to methanol, ethanol, isoamyl alcohol, hexanol, sugars, Casamino acids, tricarboxylic acid (TCA) cycle intermediates, butyrate, propionate, acetone or rice root exudates. The presence of TCA cycle intermediates in the chemotaxis assay inhibited chemotaxis towards butanol. Although the direct role of mobility and chemotaxis in the Xanthobacter-rice interaction is uncertain, one possibility is that Xanthobacter uses alcohols as a signal to move towards microaerobic zones, such as the rice rhizosphere, where carbon and energy sources such as H2, CO2, organic acids, alcohols and other anaerobic metabolites are present.
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A metabolic study of Buchnera, the intracellular bacterial symbionts of the pea aphid Acyrthosiphon pisum
More LessCells of the bacterium Buchnera were isolated from embryos of the pea aphid Acyrthosiphon pisum, with an intact perisymbiont membrane (the insect membrane which surrounds each bacterial cell inside the aphid). The bacterial preparations respired aerobically, consuming oxygen at an average rate of 24 nmol (mg protein)−1 min−1. The bacteria took up a range of carboxylic acids and the amino acids glutamate and aspartate from an external concentration of 0·5 mm at rates of 1–10 nmol (mg protein)−1 h−1; glucose was taken up at 0·17 nmol (mg protein)−1 h−1. Glutamate uptake was proportional to its external concentration, at all concentrations tested between 15 μm and 10 mm. Saturable systems for the uptake of succinate and aspartate were identified. The kinetic constants were: K m 0·79 mm, V max 12·6 nmol (mg protein)−1 min−1 for succinate; and K m 0·22 mm, V max 3·3 nmol (mg protein)−1 min−1 for aspartate. Succinate uptake was not inhibited by the uncoupler CCCP and was markedly stimulated by ATP, suggesting that its transport is not linked to a proton-motive force but is dependent on an energized membrane and possibly mediated by a co-transport system involving another ion.
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Transport kinetics, cation inhibition and intracellular location of accumulated caesium in the green microalga Chlorella salina
More LessCaesium accumulation by Chlorella salina, from buffer (pH 8.0) supplemented with 50 μm-CsCl and 137Cs, continued for approximately 15 h and displayed first-order kinetics, indicating a single rate-limiting transport process. Efflux of Cs+ from Cs+-loaded cells occurred in two distinct phases: a rapid initial loss, representing approximately 11% of total cellular Cs+, corresponded to release from the cell surface, whereas a second, slower, phase of efflux corresponded to loss from the cytoplasm and vacuole. Analysis of subcellular Cs+ compartmentation revealed that most Cs+ was accumulated into the vacuole of C. salina, with lesser amounts being associated with the cell surface or located in the cytoplasm. Uptake of Cs+ into the vacuole was correlated with a stoichiometric exchange for K+. However, no loss of K+ from the cell surface or cytoplasm was evident nor was Cs+ or K+ associated with insoluble intracellular components. Calculated values for the Cs+ flux across the vacuolar membrane were approximately equal to, or higher than, values for total cellular influx. Cs+ influx obeyed Michaelis-Menten kinetics over the lower range of external Cs+ concentrations examined (0.01−0.25 mM) and a single transport system with a K m ~ 0.5 mm was evident. The effects of other monovalent cations on Cs+ influx implied that K+ and Rb+ were competitive, and NH+ 4 non-competitive/uncompetitive inhibitors of Cs+ uptake. The order of inhibition was Rb+ > K+ > NH+ 4. We propose that a single, relatively non-selective, rate-limiting transport system for Cs+ influx is located on the cytoplasmic membrane of C. salina, while a more permeable vacuolar membrane facilitates transport of Cs+ into the vacuole.
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The need for consistent nomenclature and assessment of growth phases in diauxic cultures of Saccharomyces cerevisiae
More LessThe need for consistent nomenclature and accurate assessment of late growth phases in diauxic yeast cultures is highlighted by the substantial variation of stress tolerance in Saccharomyces cerevisiae after the exhaustion of the initial fermentable carbon source. At present, a wide variety of assessment methods and confused terminology exists in the literature, leading to difficulties in the interpretation and comparison of published results. A method based on the depletion of ethanol accumulated during the respiro-fermentative growth phase is suggested as suitable for assessing subsequent growth phases and reporting results. Consistent application of nomenclature for growth phases is recommended to assist the interpretation of published experimental results. It is suggested that the phases of growth in diauxic batch culture should be referred to using the terms (1) initial lag phase, (2) respirofermentative phase, (3) diauxic lag phase, (4) respiratory phase, (5) stationary phase, and (6) death phase.
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Phototoxicity of halogenofluorescein derivatives in Dictyostelium discoideum amoebae: comparison of 2′,4′,5′,7′-tetrabromofluorescein- and 4′,5′-diiodofluorescein dextran
More LessThe halogenated fluorescein derivatives: 2′,4′,5′,7′-tetrabromofluorescein isothiocyanate dextran (Br4FD) and 4′,5′-diiodofluorescein isothiocyanate dextran (I2FD), were found to be efficient photosensitizers for the production of singlet oxygen. The singlet oxygen quantum yields were determined by reaction with acceptors in aqueous solution. Their comparison showed that the singlet oxygen quantum yield of Br4FD was threefold higher than that of I2FD. Br4FD was more resistant to photobleaching than I2FD. Both derivatives were internalized by fluid-phase pinocytosis in amoebae of the cellular slime mould Dictyostelium discoideum. Subsequently, illumination of cells led to a dose-dependent loss of viability consistent with a role for singlet oxygen generated inside the endosomal compartments in the mechanism of photoinjury. Br4FD showed a three-fold higher efficiency than I2FD for photoinduced cytotoxicity.
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- Systematics
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Comparison of the fatty acid profiles of Borrelia, Serpulina and Leptospira species
More LessFatty acid methyl ester (FAME) derivatives were examined as a means of characterizing Borrelia burgdorferi isolates and distinguishing them from other spirochaetes. Analysis was performed using a gas liquid chromatography column in conjunction with Microbial Identification System (mis) software. Reproducible FAME profiles were produced which distinguished Borrelia species, Serpulina hyodysenteriae and Leptospira icterohaemorrhagiae. Furthermore, the FAME profiles of four recognized Borrelia species (including two American isolates of Borrelia burgdorferi, B31 and JD1) were distinct from one another and from the BSK II medium in which they were grown. The results confirm previous reports that FAME profiles of bacteria represent a diagnostic phenotypic property and suggest that they may have applications in the chemotaxonomic classification of Borrelia species.
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