- Volume 143, Issue 3, 1997
Volume 143, Issue 3, 1997
- Review Article
-
- Microbiology Comment
-
- Antigens And Immunity
-
-
-
A phosphate-starvation-inducible outer-membrane protein of Pseudomonas fluorescens Ag1 as an immunological phosphate-starvation marker
More LessA phosphate-starvation-inducible outer-membrane protein of Pseudomonas fluorescens Ag1, expressed at phosphate concentrations below 0.08—0.13 mM, was purified and characterized. The purification method involved separation of outer-membrane proteins by SDS-PAGE and extraction of the protein from nitrocellulose or PVDF membranes after electrotransfer of proteins to the membranes. The N-terminal amino acid sequence of the purified protein, called Psi1, did not show homology to any known proteins, and in contrast to the phosphate-specific porin OprP of P. aeruginosa its mobility in SDS-PAGE was not affected by solubilization temperature. An antiserum against Psi1 recognized a protein of M r 55000 in four other P. fluorescens strains among 24 tested strains representing Pseudomonas rRNA homology group I, showing antigenic heterogeneity within this group. A method for immunofluorescence microscopy involving cell permeabilization was adapted to visualize cell-specific expression of Psi1 in P. fluorescens exposed to limiting amounts of phosphate. This approach should be useful for further exploration of Psi1 as a marker to study the availability of phosphate to P. fluorescens in natural environments.
-
-
- Biochemistry
-
-
-
Structure of the Clostridium stercorarium gene celY encoding the exo-1,4-�-glucanase Avicelase II
More LessThe nucleotide sequence of the celY gene coding for the thermostable exo-1,4-�-glucanase Avicelase II of Clostridium stercorarium was determined. The gene consists of an ORF of 2742 bp which encodes a preprotein of 914 amino acids with a molecular mass of 103 kDa. The signal-peptide cleavage site was identified by comparison with the N-terminal amino acid sequence of Avicelase II purified from C. stercorarium. The celY gene is located in close vicinity to the celZ gene coding for the endo-1,4-�-glucanase Avicelase I. The CelY-encoding sequence was isolated from genomic DNA of C. stercorarium with the PCR technique. The recombinant enzyme produced in Escherichia coli as a LacZ'-CelY fusion protein could be purified using a simple two-step procedure. The properties of CelY proved to be consistent with those of Avicelase II purified from C. stercorarium. Sequence comparison revealed that CelY consists of an N-terminal catalytic domain flanked by a domain of 95 amino acids with unknown function joined to a type III cellulose-binding domain. The catalytic domain belongs to the recently proposed family L of cellulases (family 48 of glycosyl hydrolases).
-
-
-
-
Determination of the pathway for rhamnose biosynthesis in mycobacteria: cloning, sequencing and expression of the Mycobacterium tuberculosis gene encoding α-D-glucose-1-phosphate thymidylyltransferase
The mycobacterial cell wall core consists of an outer lipid layer of mycolic acids connected, via arabinogalactan polysaccharide, to an inner peptidoglycan layer. An α-L-rhamnopyranosyl residue has been shown to be a key component linking the mycolated arabinogalactan to the peptidoglycan and, therefore, the biosynthesis of L-rhamnose (Rha) in mycobacteria was investigated as the first step of developing inhibitors of its biosynthesis. Biochemical assays were used to show that dTDP-Rha was synthesized in Mycobacterium smegmatis from α-D-glucose 1-phosphate (α-D-Glc-1-P) and dTTP by the same four enzymic steps used by Escherichia coli and other bacteria. PCR primers based on consensus regions of known sequences of the first enzyme in this series, α-D-Glc-1-P thymidylyltransferase (RfbA) were used to amplify rfbA DNA from M. tuberculosis. The entire rfbA gene was then cloned and sequenced. The deduced amino acid sequence revealed a 31362 Da putative protein product which showed similarity to RfbA proteins of other bacteria (59% identity to that found in E. coli). Sequencing of DNA flanking the rfbA gene did not reveal any of the other rfb genes required for dTDP-Rha biosynthesis. Therefore, the four Rha biosynthetic genes are not clustered in M. tuberculosis. The enzymic activity of the sequenced gene product was confirmed by transformation of E. coli with pBluescript KS(–) containing the rfbA gene from M. tuberculosis. Analysis of enzyme extracts prepared from this transformant revealed an 11-fold increase in α-D-Glc-1-P thymidylyltransferase activity.
-
- Development And Structure
-
-
-
High resolution X-ray micrography of live Candida albicans using laser plasma pulsed point X-ray sources
More LessElectron microscopy is still the most frequently used method for visualization of subcellular structures in spite of limitations due to the preparation required to visualize the specimen. High resolution X-ray microscopy is a relatively new technique, still under development and restricted to a few large synchrotron X-ray sources. We utilized a single-shot laser (nanosecond) plasma to generate X-rays similar to synchrotron facilities to image live cells of Candida albicans. The emission spectrum was tuned for optimal absorption by carbon-rich material. The photoresist was then scanned by an atomic force microscope to give a differential X-ray absorption pattern. Using this technique, with a sample image time of 90 min, we have visualized a distinct 152.24 nm thick consistent ring structure around cells of C. albicans representing the cell wall, and distinct ‘craters’ inside, one of 570.90 nm diameter and three smaller ones, each 400 nm in diameter. This technique deserves further exploration concerning its application in the ultrastructural study of live, hydrated microbiological samples and of macromolecules.
-
-
-
-
Pirellulosomes: a new type of membrane-bounded cell compartment in planctomycete bacteria of the genus Pirellula
More LessA distinct type of cellular organization was found in two species of the planctomycete genus Pirellula, Pirellula marina and Pirellula staleyi. Both species possess two distinct regions within the cell which are separated by a single membrane. The major region of the cell, the pirellulosome, contains the fibrillar condensed nucleoid. The other area, the polar cap region, forms a continuous layer surrounding the entire pirellulosome and displays a cap of asymmetrically distributed material at one cell pole. Immuno- and cytochemical-labelling of P. marina demonstrated that DNA is located exclusively within the pirellulosome; cell RNA is concentrated in the pirellulosome, with some RNA also located in the polar cap region.
-
-
-
Bacterial capsules: no barrier against Bdellovibrio
More LessBdellovibrio bacteriovorus 109J attached to both capsulated and noncapsulated Escherichia coli K29 cells. Electron microscopy revealed penetration of the thick polysaccharide capsule without any major disintegration of the neighbouring capsular matrix. The capsule remained intact during bdelloplast formation and lysis was unaffected by capsulation of the prey cell. This study shows that, in contrast to its effect on bacteriophage penetration and its protective activities against immune defence mechanisms, the capsule of E. coli does not serve as a barrier against invasion by B. bacteriovorus.
-
- Genetics And Molecular Biology
-
-
-
Activation of the Erwinia carotovora subsp. carotovora pectin lyase structural gene pnIA: a role for RdgB
More LessThe activation of pectin lyase (Pnl) production in Erwinia carotovora subsp. carotovora strain 71 occurs upon DNA damage via a unique regulatory circuit involving recA, rdgA and rdgB. In a similar Pnl-inducible system reconstituted in Escherichia coli, the rdgB product was found to activate the expression of pnIA, the structural gene for pectin lyase. The kinetic data presented here also show that transcription of pnIA followed that of rdgB in Er. carotovora subsp. carotovora, indicating a temporal order of gene expression. By deletion analysis we have localized the promoter/regulatory region within a 66 bp DNA segment upstream of the pnIA transcriptional start site. This region contains the -10 consensus sequence but not the sequences corresponding to the E. coli -35 region. For DNA-binding studies, rdgB was overexpressed in E. coli and a 14 kDa polypeptide was identified as the gene product. RdgB from crude extracts or a purified preparation caused an identical gel mobility shift of a 164 bp DNA segment containing the pnIA promoter/regulatory region. Utilizing DNase I protection assay the RdgB-binding site was localized between nucleotides -29 and -56, i.e. overlapping the position of the putative -35 box. The findings reported here, taken along with our previous observation that the rdgB product is required for pnIA expression, establishes that rdgB encodes a transcriptional factor which specifically interacts with the pnIA promoter/regulatory region.
-
-
-
-
The general secretion pathway of Erwinia carotovora subsp. carotovora: analysis of the membrane topology of OutC and OutF
More LessThe out gene cluster of Erwinia carotovora subsp. carotovora (Ecc) encodes the proteins of the type II or general secretory pathway (GSP) apparatus which is required for secretion of pectinase and cellulase. In this study, fusions between Ecc out genes and the topology probe blaM were constructed. The ability of Out protein domains to export BlaM across the cytoplasmic membrane in both Escherichia coli and the cognate host was utilized to confirm the computer-predicted cytoplasmic membrane topology of OutC and OutF. When outC was fused to blaM, the resulting phenotype suggested that the majority of OutC is targeted to the periplasm, typical of a type II bitopic conformation in the cytoplasmic membrane. In contrast, for the outF gene product, three transmembrane regions were identified which connect a large N-terminal cytoplasmic domain, a smaller periplasmic domain, and a large cytoplasmic loop. Fusions between blaM and outD and outE were used to further substantiate the locations of these gene products in the outer membrane and the cytoplasm respectively. The data derived suggest that a number of the Out apparatus components possess domains in the cytoplasm and/or the periplasm with potential for protein-protein interactions which facilitate the secretion of periplasmic enzyme intermediates across the outer membrane to the external milieu.
-
-
-
Overlapping promoters modulate Fnr- and ArcA-dependent anaerobic transcriptional activation of the focApfl operon in Escherichia coli
More LessThe recently identified P6A promoter of the anaerobically inducible focApfl operon of Escherichia coli overlaps the Fnr (fumarate-nitrate reduction regulator)-dependent P6 promoter. The Fnr-binding site of P6 and the -35 hexamer sequence of P6A are shared between the promoters. Inactivation of P6A, through introduction of a -10 hexamer mutation, resulted in enhanced anaerobic induction of operon expression. The dependence on the ArcA (aerobic respiration control regulator) and Fnr transcription factors for anaerobic induction was tested for several focA-lacZ and pfl-lacZ gene fusions. Anaerobic induction became more dependent on Fnr in derivatives lacking a functional P6A promoter compared with wild-type constructs. Moreover, aerobic expression of the focA gene was reduced by the p6A mutation, as was the dependence on ArcA for anaerobic induction. Inactivation of P6 severely reduced Fnr-dependent anaerobic induction, in accord with previous findings. Transcription analyses demonstrated that a mutation in the -10 hexamer sequence of either P6A or P6 did not adversely affect transcription from the remaining promoter. Taken together, these results indicate that the P6A promoter moderates the Fnr-dependent activation of P6 through competition for RNA polymerase binding.
-
-
-
Expression of the mau gene cluster of Paracoccus denitrificans is controlled by MauR and a second transcription regulator
The mau gene cluster of Paracoccus denitrificans constitutes 11 genes (10 are located in the transcriptional order mauFBEDACJGMN; the 11th, mauR, is located upstream and divergently transcribed from these genes) that encode a functional methylamine-oxidizing electron transport branch. The mauR gene encodes a LysR-type transcriptional activator essential for induction of the mau operon. In this study, the characteristics of that process were established. By using lacZ transcriptional fusions integrated into the genome of P. denitrificans, it was found that the expression of the mauR gene during growth on methylamine and/or succinate was not autoregulated, but proceeded at a low and constant level. The mauF promoter activity was shown to be controlled by MauR and a second transcriptional regulator. This activity was very high during growth on methylamine, low on succinate plus methylamine, and absent on succinate alone. MauR was overexpressed in Escherichia coli by using a T7 RNA polymerase expression system. Gel shift assays indicated that MauR binds to a 403 bp DNA fragment spanning the mauR-mauF promoter region. It is concluded from these results that the expression of the structural mau genes is dependent on MauR and its inducer, methylamine, as well as on another transcription factor. Both activators are required for high-level transcription from the mauF promoter. It is hypothesized that the two activators act synergistically to activate transcription: the effects of the two activators are not additive and either one alone activates the mauF promoter rather weakly.
-
-
-
Molecular analysis and expression of the extracellular lipase of Aeromonas hydrophila MCC-2
More LessThe structural gene encoding the extracellular lipase of Aeromonas hydrophila MCC-2 was cloned and found to be expressed in Escherichia coli using its own promoter. When the cloned gene (lip) was expressed in E. coli minicells, an 80 kDa protein was identified. Subcellular fractionation of E. coli carrying the lip gene indicated that the Lip protein was mainly associated with the membrane fraction. Nucleotide sequence analysis revealed that the gene is 2253 bp long, coding for a 79.9 kDa protein with an estimated pl of 10.36. The deduced protein contains two putative signal peptide cleavage sites; one is a typical signal peptidase cleavage site and the other bears a strong resemblance to known lipoprotein leader sequences. Radioactivity from [3H]palmitate was incorporated into the Lip protein when expressed in E. coli. The deduced protein contains a sequence of VHFLGHSLGA which is very well conserved among lipases. It shows 67% and 65% overall identity to the amino acid sequences of lipase from A. hydrophila strains H3 and JMP636, respectively, but shows little homology to those of other lipases. The Lip protein was purified to homogeneity from both A. hydrophila and recombinant E. coli. In hydrolysis of p-nitrophenyl esters and triacylglycerols, using purified enzyme, the optimum chain lengths for the acyl moiety on the substrate were C10 to C12 for ester hydrolysis and C8 to C10 for triacylglycerol hydrolysis.
-
-
-
Analysis of a Rhizobium leguminosarum gene encoding a protein homologous to glutathione S-transferases
More LessA novel Rhizobium leguminosarum gene, gstA, the sequence of which indicated that it was a member of the gene family of glutathione S-transferases (GSTs), was identified. The homology was greatest to the GST enzymes of higher plants. The Rhizobium gstA gene was normally expressed at a very low level. The product of gstA was over-expressed and purified from Escherichia coli. It was shown to bind to the affinity matrix glutathione-Sepharose, but no enzymic GST activity with 1-chloro-2,4-dinitrobenzene as substrate was detected. gstA encoded a polypeptide of 203 amino acid residues with a calculated molecular mass of 21990 Da. Transcribed divergently from gstA is another gene, gstR, which was similar in sequence to the LysR family of bacterial transcriptional regulators. A mutation in gstR had no effect on the transcription of itself or gstA under the growth conditions used here. Mutations in gstA and gstR caused no obvious phenotypic defect and the biological functions of these genes remain to be determined.
-
-
-
Primary and secondary structures of rRNA spacer regions in enterococci
More LessThe 16S-23S and 23S-5S rRNA spacer DNA regions (spacer regions 1 and 2, respectively) from Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus durans and Enterococcus mundtii were amplified by PCR. Their nucleotide sequences were established and a secondary structure model showing the interaction between the two spacer regions was built. Whereas lactococci and Streptococcus sensu stricto are characterized by a single type of spacer region 1, the enterococci show a high degree of variability in this region; thus the spacer regions 1 with and without tRNAAlawere characterized. However, as shown for lactococci and Streptococcus sensu stricto, the tRNAAlagene does not encode the 3'-terminal CCA trinucleotide. A putative antitermination signal is found downstream from the tRNAAlagene. Based on comparison with Lactococcus lactis and Streptococcus thermophilus, a double-stranded processing stem is proposed. In E. hirae, one of the three different types of spacer region 1 contains no tRNAAla, but displays a 107 nt insertion that forms a long stem-loop structure. A similar insertion (115 nt in length) was found in E. faecium and base compensatory mutations preserve the ability to form the long stem-loop structure. Such insertions may correspond to mobile intervening sequences, as found in the 23S rRNA coding sequences of some Gram-negative bacteria. The spacer regions 1 and 2 from the three subgroups of streptococci were compared, and except for the tRNAAlagene and the double-stranded processing sites, little similarity was found, which opens large possibilities for future development of DNA-based typing methods.
-
-
-
Reversion rates in a leuB auxotroph of Escherichia coli K-12 correlate with ppGpp levels during exponential growth
More LessTwo isogenic strains of Escherichia coli K-12 differing only in relA, as well as two spoT transductants of the relA -strain, were examined with respect to ppGpp levels and reversion rates of a leuB -allele under nine different conditions. A positive correlation was established between reversion rates and the steady-state concentration of ppGpp during exponential growth. The leuB genes from two leuB -strains (isogenic except for relA) were cloned and sequenced and found to contain a single mutation, namely, a C-to-T transition at nucleotide 857. This mutation resulted in a serine-to-leucine substitution at amino acid residue 286 of the LeuB protein. PCR products that encompassed the leuB lesion were generated from 53 revertants and then sequenced. Of these revertants, 36 were found to contain nucleotide substitutions that would result in a serine (wild type), valine or methionine at amino acid residue 286 of LeuB, and nearly all of them exhibited generation times similar to wild type. Seventeen of the analysed revertants were found to be suppressors that retained the encoded leucine at residue 286. The majority of the suppressor mutants exhibited generation times that were significantly longer than wild type.
-
-
-
Product formation and phosphoglucomutase activities in Lactococcus lactis: cloning and characterization of a novel phosphoglucomutase gene
More LessMaltose metabolism in Lactococcus lactis involves the conversion of -glucose 1-phosphate to glucose 6-phosphate, a reaction which is reversibly catalysed by a maltose-inducible and glucose-repressible -phosphoglucomutase (-PGM). The gene encoding -PGM (pgmB) was cloned from a genomic library of L. lactis using antibodies. The nucleotide sequence of a 5695 bp fragment was determined and six ORFs, including the pgmB gene, were found. The gene expressed a polypeptide with a calculated molecular mass of 24210 Da, which is in agreement with the molecular mass of the purified -PGM (25 kDa). A short sequence at the N-terminus was found to be similar to known metal-binding domains. The expression of -PGM in L. lactis was found to be induced also by trehalose and sucrose, and repressed by lactose in the growth medium. This indicates that -PGM does not serve solely to degrade maltose, but that it is also involved in the metabolism of other carbohydrates. The specific activity of a-PGM during fermentation was dependent on the maltose concentration in the medium. The maximum specific activity of -PGM increased by a factor of 4.6, and the specific growth rate by a factor of 7, when the maltose concentration was raised from 0.8 to 11.0 g I-1. Furthermore, a higher amount of lactate produced relative to formate, acetate and ethanol was observed when the initial maltose concentration in the medium was increased. The specific activity of -PGM responded similarly to -PGM, but the magnitude of the response was lower. Preferential sugar utilization and a- and -PGM suppression was observed when L. lactis was grown on the substrate combinations glucose and maltose, or lactose and maltose; maltose was the least-preferred sugar. In contrast, galactose and maltose were utilized concurrently and both PGM activities were high throughout the fermentation.
-
-
-
A novel fusidic acid resistance gene from Streptomyces lividans 66 encodes a highly specific esterase
More LessResistance to fusidic acid in Streptomyces lividans is due to secretion of an extracellular enzyme (FusH) that converts the steroid antibiotic into an inactive derivative. NH2-terminal and several internal amino acid sequences were prepared from the purified enzyme. Using one of the deduced oligonucleotides to probe a subgenomic DNA library, the fusH gene was cloned and sequenced. Sequence analysis located an ORF which, owing to the presence of two putative start codons, indicates a predicted protein with 520 or 509 amino acids. A signal peptide was identified by aligning the deduced amino acids with the N-terminal sequence determined for the mature enzyme. The C-terminal part of the deduced FusH contains a region of three tandemly repeated stretches of 50 amino acids, which is preceded and followed by amino acids showing high homology with the repeats. FusH was found to share a GDS motif with some deduced esterases. S. lividans transformants carrying fusH on a multicopy vector synthesized high levels of FusH. Purified FusH cleaved equally well an acetyl, a thioacetyl or a formyl group from the 16β-position of fusidic acid and its derivatives. However, a propionyl group at the 16β-position was attacked with difficulty and a 16β-acetyl group was not hydrolysed at all. These data indicate that FusH is a highly specific esterase. The fusH gene is widely distributed among streptomycetes that modify fusidic acid to its inactive lactone derivative.
-
-
-
Gene disruption and replacement in the rapamycin-producing Streptomyces hygroscopicus strain ATCC 29253
A system for gene disruption and replacement based on a streptomycete temperate phage vector was developed to introduce DNA in the rapamycin-producing Streptomyces hygroscopicus strain ATCC 29253. This will be useful in attempts to produce, through genetic manipulation, novel forms of the therapeutically important immunosuppressive drug rapamycin. Recombinant phages were constructed from the ?31 phage derivative KC515 (c + attP) carrying a thiostrepton or viomycin resistance gene along with segments of the S. hygroscopicus chromosome. Each of the cloned segments also contained the aphll neomycin/kanamycin resistance gene to enable gene replacement by loss of the phage-derived DNA. Specific deletion of the entire polyketide synthase (PKS) believed to govern rapamycin biosynthesis resulted in the loss of rapamycin production. In contrast, disruption or deletion of a region predicted to encode four PKS open reading frames, or another region predicted to encode another PKS plus a cytochrome P450 hydroxylase and ferredoxin, had no effect on the production of rapamycin or nigericin, a polyether antibiotic also produced by S. hygroscopicus. Therefore, S. hygroscopicus may have the capacity to produce polyketides additional to rapamycin and nigericin.
-
-
-
The recA gene from Clostridium perfringens is induced by methyl methanesulphonate and contains an upstream Cheo box
More LessThe recA gene from Clostridium perfringens was cloned using degenerate oligonucleotide primers designed from conserved regions of RecA proteins from other bacteria. The 1089 bp gene encoded a putative RecA protein with 69% amino acid sequence similarity to the RecA protein from Bacillus subtilis. The C. perfringens recA gene was induced by exposure to methyl methanesulphonate and complemented a recA mutant of Escherichia coli. A Cheo box was identified in the region upstream of the gene. Since this SOS-like operator site is conserved in many DNA-damage-inducible recA gene regions from Gram-positive bacteria, the results suggest that the regulation of the C. perfringens recA gene also involves the binding of a LexA-like protein to this site.
-
-
-
Molecular genetic analysis of the region containing the essential Pseudomonas aeruginosa asd gene encoding aspartate-β-semialdehyde dehydrogenase
More Lessasd mutants of Gram-negative and some Gram-positive bacteria have an obligate requirement for diaminopimelic acid (DAP), an essential constituent of the cell wall of these organisms. In environments deprived of DAP, for example mammalian tissues, they will undergo lysis. This was previously exploited to develop vaccine strains of Salmonella typhimurium and cloning vectors containing asd as an in vivo selectable marker. As a first step for development of such systems for Pseudomonas aeruginosa, the asd gene from wild-type strain PAO1 was cloned by a combined approach of PCR amplification from chromosomal DNA, construction of mini-libraries and by complementation of an Escherichia coli δasd mutant. The nucleotide sequence of a 2433 bp Smal-Nsil fragment was determined. This fragment contained the C-terminal 47 nucleotides of leuB, encoding 3-isopropylmalate dehydrogenase; asd, encoding aspartate-β-semialdehyde dehydrogenase (Asd); and orfA, whose product showed similarity to the Asd proteins from Vibrio spp. By subcloning, asd was localized to a 1.24 kb DNA fragment which in an E. coli T7 expression system strongly expressed a 40000 Da protein. The amino acid sequence was deduced from the DNA sequence. A comparison of the Asd proteins from P. aeruginosa, E. coli and Haemophilus influenzae revealed greater than 63% identity, demonstrating the conserved nature of Asd in Gram-negative bacteria, and defined the active-site-containing consensus sequence GGNCTVXMLMXXXLGLF as a possible signature motif. Chromosomal δasd mutants were isolated. They were auxotrophic for DAP, lysine, methionine and threonine, and lysed in the absence of DAP. Genetic analyses indicated that orfA probably is naturally frame-shifted and does not contribute to the Asd phenotype. By PFGE, the asd gene was mapped to between coordinates 1.89 and 2.15 Mbp, or 37-40 min, on the 5.9 Mbp P. aeruginosa chromosome.
-
-
-
Intraclonal mating in Trypanosoma brucei is associated with out-crossing
More LessTo examine whether mating can occur within as well as between clones of Trypanosoma brucei, we transformed three T. brucei subspecies stocks with heterologous genes conferring resistance to either hygromycin or Geneticin and carried out a series of inter- and intraclone matings in all possible double drug combinations. Double drug-resistant hybrids were recovered from three of the six out-crosses, but not from any of the three intraclone matings. However, further analysis of cloned progeny trypanosomes from one of the out-crosses using RFLP markers, molecular karyotyping and RAPD (random amplification of polymorphic DNA) produced unequivocal evidence that intraas well as interclone mating had occurred. The progeny of interclone mating were double drug-resistant and heterozygous at 9 of 13 loci examined. In contrast, the progeny of intraclone mating had no demonstrable input of genetic material from the hygromycin-resistant parent and were similar to the Geneticin-resistant parent for most markers, except for five loci which were heterozygous in the Geneticin-resistant parent but homozygous in these clones (aldolase, THT1 glucose transporter, procyclin, tubulin and cDNA 23). In addition, PFGE showed considerable karyotypic rearrangements in these clones and loss of genetic material was evident from RAPD and VSG (variant surface glycoprotein) gene fingerprint analysis. We conclude that intraclone mating can occur in trypanosomes, but only during out-crossing, suggesting that meiosis and/or fusion are triggered by a diffusible factor.
-
-
-
Identification of Mycobacterium paratuberculosis gene expression signals
More LessMycobacterium paratuberculosis promoter-containing clones were isolated from a genomic DNA library constructed in the transcriptional-translational fusion vector pYUB76. The promoter-containing DNA fragments were identified in the surrogate host Mycobacterium smegmatis by expression of the promoterless lacZ reporter gene of pYUB76. The expression signals exhibited a wide range of strengths, as indicated by their corresponding β-galactosidase activities. Eight clones were sequenced and characterized further. Predicted open reading frames and codon usage were identified by computer analysis. Database searching for related sequences using the BLAST method revealed no homologies. Transcriptional activity was measured by slot-blot hybridization with steady-state RNA isolated from lacZ + M. smegmatis clones. Primer extension analysis identified the transcription start sites within the cloned fragments. The promoter regions characterized in this study were used to establish a consensus promoter sequence for M. paratuberculosis. M. paratuberculosis consensus hexanucleotide sequences of TGMCGT and CGGCCS centred approximately 35 and 10 bp upstream from the transcription startpoints do not correspond to the consensus hexanucleotides of Escherichia coli promoters.
-
-
-
Characterization of Mycobacterium tuberculosis LexA: recognition of a Cheo (Bacillus-type SOS) box
More LessThe gene coding for the Mycobacterium tuberculosis homologue of LexA has been cloned and sequenced. Amino acids required for autocatalytic cleavage are conserved, whereas those important for specific DNA binding are not, when compared with Escherichia coli LexA. The transcriptional start site was mapped and a DNA sequence motif was identified which resembled the consensus Cheo box sequence involved in the regulation of DNA-damage-inducible genes in Bacillus subtilis. The M. tuberculosis LexA protein was overexpressed in E. coli and purified by means of a His tag. The purifed LexA was shown to bind to the Cheo box sequence found upstream of its own gene.
-
-
-
The Bacillus subtilis L-arabinose (ara) operon: nucleotide sequence, genetic organization and expression
More LessThe Bacillus subtilis L-arabinose metabolic genes araA, araB and araD, encoding L-arabinose isomerase, L-ribulokinase and L-ribulose-5-phosphate 4-epimerase, respectively, have been cloned previously and the products of araB and araD were shown to be functionally homologous to their Escherichia coli counterparts by complementation experiments. Here we report that araA, araB and araD, whose inactivation leads to an Ara- phenotype, are the first three ORFs of a nine cistron transcriptional unit with a total length of 11 kb. This operon, called ara, is located at about 256 on the B. subtilis genetic map and contains six new genes named araL, araM, araN, araP, araQ and abfA. Expression of the ara operon is directed by a strong sA-like promoter identified within a 150 bp DNA fragment upstream from the translation start site of araA. Analysis of the sequence of the ara operon showed that the putative products of araN, araP and araQ are homologous to bacterial components of binding-protein-dependent transport systems and abfA most probably encodes an a-L-arabinofuranosidase. The functions of araL and araM are unknown. An in vitro-constructed insertion-deletion mutation in the region downstream from araD allowed us to demonstrate that araL, araM, araN, araP, araQ and abfA are not essential for L-arabinose utilization. Studies with strains bearing transcriptional fusions of the operon to the E. coli lacZ gene revealed that expression from the ara promoter is induced by L-arabinose and repressed by glucose.
-
-
-
Characterization of the ftsH gene of Bacillus subtilis
More LessMembers of the AAA-protein family are found in both prokaryotes and eukaryotes. These ATPases are involved in a number of diverse activities ranging from protein secretion to cell cycle control. This paper reports the functional analysis of the Bacillus subtilis ftsH gene, which encodes a member of this protein family. In cells containing reduced levels of a truncated FtsH protein cell growth was impaired under certain nutritional conditions. In a hypersaline environment FtsH was required in increased amounts for the cells' recovery from osmotic stress. In the absence of FtsH the abundance of several of the major penicillin-binding proteins (PBP2A and 2B) in the cytoplasmic membrane was affected. Lastly, it has been established that FtsH is required for entry into the developmental life cycle.
-
-
-
The trp RNA-binding attenuation protein (TRAP) regulates the steady-state levels of transcripts of the Bacillus subtilis folate operon
More LessThe Bacillus subtilis folate operon contains nine genes. The first six genes are involved in the biosynthesis of folic acid and tryptophan and have been characterized previously. The 3-region of the folate operon contains three additional ORFs: orf3, potentially encoding a DNA-binding protein of 68 amino acids, orf4, encoding a protein of 338 amino acids with homology to the Orf1 of the E. coli fis operon, and a putative lysyl-tRNA synthetase gene (lysS). Four transcripts were identified which encode the first two, eight or all nine proteins or only the last protein LysS. The folate operon contains two promoters, one upstream of the first gene and the second preceding lysS. Transcription of the entire folate operon starts 33 bp upstream of the ATG codon of pab, the first gene of the operon. The mtrB-encoded trp RNA-binding attenuation protein (TRAP) dramatically reduces the steady-state levels of the folate operon transcripts encoding the first eight and all nine proteins, but only has a relatively small effect on the steady-state level of the 2.1 kb transcript encoding the first two genes of the operon, pab and trpG. In addition, transcription of the folate operon is regulated in a growth-phase-dependent manner. Transcripts were present in very low levels after mid-exponential phase, but were dramatically increased directly after transfer of the cells to fresh medium. These results indicate that transcription of the folate operon is regulated by TRAP and also depends on the growth phase of the culture.
-
- Pathogenicity And Medical Microbiology
-
-
-
Identification of a 95 kDa putative adhesin from Actinomyces serovar WVA963 strain PK1259 that is distinct from type 2 fimbrial subunits
The species Actinomyces serovar WVA963 is among the 20 bacteria most frequently isolated from human subgingival plaque. The interactions of this species with streptococci are inhibited by lactose, a function associated with type 2 fimbrial surface structures in Actinomyces naeslundii. Type 1 fimbriae mediate binding of cells to salivary proline-rich proteins. Specific polyclonal antisera against type 1 and type 2 fimbriae of A. naeslundii T14V revealed both types of fimbriae on Actinomyces serovar WVA963 strain PK1259. To investigate the role of type 2 fimbriae of strain PK1259 in Actinomyces-Streptococcus lactose-inhibitable coaggregations, spontaneous coaggregation-defective (Cog-) mutants that failed to coaggregate with streptococci were isolated; three were chosen for study. All three mutant strains synthesized type 1 fimbriae and a 59 kDa protein; mutant strains PK2415 and PK3092 synthesized type 2 fimbriae and a 57 kDa protein. In contrast, the Cog-strain PK2407 did not agglutinate with anti-type 2 antibodies or show the 57 kDa band, suggesting that the 57 kDa protein was the type 2 fimbrial subunit. Polyclonal antiserum raised against the Actinomyces serovar WVA963 strain PK2399, an antibiotic-resistant derivative of wild-type PK1259, blocked coaggregation between this strain and streptococci. Anti-PK2399 serum absorbed with mutant strain PK3092 bearing type 2 fimbriae retained its blocking ability. Surface sonicates of the parent and mutant strains were adsorbed to streptococcal cells and to lactose-agarose beads. Lactose eluates from both the streptococcal cells and the affinity beads were characterized by SDS-PAGE and corresponding immunoblots using anti-PK2399 serum absorbed with Cog-mutant PK3092. These blots revealed a 95 kDa putative adhesin in the parent strain PK2399 that was absent in the Cog-mutant strain PK3092. These results suggest the presence of a putative 95 kDa actinomyces adhesin distinct from the 57 kDa type 2 fimbrial subunit and that this adhesin mediates lactose-inhibitable coaggregation with streptococci.
-
-
- Physiology And Growth
-
-
-
Assay and properties of acetone carboxylase, a novel enzyme involved in acetone-dependent growth and CO2 fixation in Rhodobacter capsulatus and other photosynthetic and denitrifying bacteria
More LessThe photosynthetic bacterium Rhodobacter capsulatus is able to grow, in the presence of carbon dioxide, under anaerobic (photosynthetic) conditions with the solvents acetone or butanone as carbon source. The carboxylation of acetone to form acetoacetate is the most likely initial step in acetone metabolism. This paper describes an assay for acetone carboxylation, in which fixation of radiolabeled carbon dioxide from NaH14CO3 is measured in the presence of acetone, ATP, magnesium ions and acetyl-CoA. Acetone carboxylase activity was specifically induced by growth of R. capsulatus on acetone or butanone and was associated with a high-molecular-mass protein complex containing two major polypeptides, of 70 and 85 kDa. Partial purification of the activity was achieved by FPLC ion-exchange chromatography, which confirmed that the 70 and 85 kDa proteins were subunits of the enzyme and suggested that at least one additional protein (60 kDa) may be associated with carboxylase activity. N-terminal sequences of the two major subunits were not significantly similar to any other carboxylases in the databases and neither contained covalently bound biotin, indicating that the enzyme represents a novel type of carboxylase. Acetone carboxylase activity was also demonstrated in cell-free extracts of acetone-grown Rhodomicrobium vannielii and the denitrifying bacterium Thiosphaera pantotropha.
-
-
-
-
Dimethyl sulphoxide reduction with reduced sulphur compounds as electron donors by anoxygenic phototrophic bacteria
More LessThe marine purple non-sulphur bacterium Rhodovulum euryhalinum strain DSM 4868 reduced dimethyl sulphoxide (DMSO) to dimethyl sulphide (DMS) chemotrophically with sulphide as electron donor. The oxidation of sulphide was correlated with the formation of polysulphides. R. euryhalinum reduced DMSO phototrophically with sulphide as well, but the amount of DMSO reduced in relation to sulphide oxidized was lower. The marine green sulphur bacterium Chlorobium vibrioforme strain DSM 8327 reduced DMSO to DMS phototrophically with sulphide and thiosulphate as electron donors. The extent of DMSO reduction was much less in the dark. Eight strains of purple sulphur bacteria - marine, brackish water and freshwater isolates - and another marine green sulphur bacterium showed a very weak capacity for DMSO reduction with sulphide or thiosulphate as electron donors in the light and dark, respectively.
-
-
-
Cra-mediated regulation of Escherichia coli adenylate cyclase
More LessIn Escherichia coli, expression of certain genes and operons, including the fructose operon, is controlled by Cra, the pleiotropic catabolite repressor/activator protein formerly known as FruR. In this study we have demonstrated that cra mutant strains synthesize 10-fold less cAMP than isogenic wild-type strains, specifically when grown in fructose-containing minimal media. The glucose-specific IIA protein (IIAglc) of the phosphotransferase system, which activates adenylate cyclase when phosphorylated, is largely dephosphorylated in cra but not wild-type strains growing under these conditions. Dephosphorylation of IIAglcin cra strains apparently results from enhanced fructose operon transcription and fructose uptake. These conclusions were supported by showing that fructose-grown cra strains possess 2·5-fold higher fructose-1-phosphate kinase activity than fructose-grown wild-type strains. Moreover, artificially increasing fructose operon expression in cells transporting fructose dramatically decreased the activity of adenylate cyclase. The results establish that Cra indirectly regulates the activity of adenylate cyclase by controlling the expression of the fructose operon in cells growing with fructose as the sole carbon source.
-
-
-
Influence of Bacillus subtilis phoR on cell wall anionic polymers
More LessIn Bacillus subtilis the Pho regulon is controlled by a sensor and regulator protein pair, PhoR and PhoP, that respond to phosphate concentrations. To facilitate studies of the Pho regulon, a strain with an altered PhoR protein was isolated by in vitro mutagenesis. The mutation in this strain (phoR12) leads to the production of a PhoR sensor kinase that, unlike the wild-type, is functionally active in phosphate-replete conditions. The lesion in phoR12 was shown to be a single base change that results in an Arg to Ser substitution in a region of PhoR that is highly conserved in histidine sensor kinases. While a phoR-negative mutant was unable to induce the synthesis of cell wall teichuronic acid under phosphate-limited conditions, the phoR12 mutant showed a relative increase in teichuronic acid and a decrease in teichoic acid, even under phosphate-replete conditions. The latter suggests that some or all of the genes required for teichuronic acid synthesis are members of the Pho regulon.
-
-
-
Identification of vegetative proteins for a two-dimensional protein index of Bacillus subtilis
Twenty-three of the most prominent spots which are visible on two-dimensional (2-D) protein gels of Bacillus subtilis crude extracts were selected as marker spots for the construction of a 2-D protein index. N-terminal sequencing of the corresponding proteins resulted in the identification of enzymes involved in glycolysis, TCA cycle, pentose phosphate cycle, amino acid metabolism, nucleotide biosynthesis and translation. Using computer analysis of the 2-D protein gels, most of these metabolic enzymes were found to be synthesized at a reduced rate after different stresses and glucose starvation. Such an approach permits a rapid and global evaluation of the regulation of different branches of metabolism in response to various physiological conditions.
-
-
-
Specific and general stress proteins in Bacillus subtilis � a two-dimensional protein electrophoresis study
A computer-aided analysis of high resolution two-dimensional polyacrylamide gels was used to investigate the changes in the protein synthesis profile in B. subtilis wild-type strains and sigB mutants in response to heat shock, salt and ethanol stress, and glucose or phosphate starvation. The data provided evidence that the induction of at least 42 general stress proteins absolutely required the alternative sigma factor sGB. However, at least seven stress proteins, among them ClpC, ClpP, Sod, AhpC and AhpF, remained stress-inducible in a sigB mutant. Such a detailed analysis also permitted the description of subgroups of general stress proteins which are subject to additional regulatory circuits, indicating a very thorough fine-tuning of this complex response. The relative synthesis rate of the general stress proteins constituted up to 40% of the total protein synthesis of stressed cells and thereby emphasizes the importance of the stress regulon. Besides the induction of these general or rather unspecific stress proteins, the induction of stress-specific proteins is shown and discussed.
-
-
-
Growth temperature dependence of channel size of the major outer-membrane protein (OprF) in psychrotrophic Pseudomonas fluorescens strains
More LessThe outer-membrane (OM) permeability of the psychrotrophic bacterium Pseudomonas fluorescens strain MFO for the -lactam mezlocillin is increased at the optimum growth temperature (28 C) compared to low growth temperatures (8 C). In an attempt to explain this phenomenon, OM protein content was studied in cultures grown at both temperatures. No significant difference in proportion or composition was found, suggesting that a change in the structure and function of porins could be responsible for the differential permeability. The major OM protein OprF of two psychrotrophic P. fluorescens strains, MFO and OE 28.3, was purified from cultures grown at 8 C and 28 C in order to reincorporate them in solvent-free lipid bilayers. From cultures grown at the same temperature, OprF displayed very similar channel-forming properties for both strains. Decreasing the growth temperature induced a threefold reduction of the major conductance values (250270 pS in 1 M NaCl for 28 C cultures and 8090 pS in 1 M NaCl for 8 C cultures). The trypsin digestion kinetics showed a very different reactivity for these porins between cultures grown at 8 C and 28 C. This may indicate that the pore structure of OprF is modified depending on the growth temperature, as suggested by its functional behaviour.
-
- Systematics
-
-
-
Classification of Enterobacteriaceae by minimization of stochastic complexity
More LessA new method for classifying bacteria is presented and applied to a large set of biochemical data for the Enterobacteriaceae. The method minimizes the bits needed to encode the classes and the items or, equivalently, maximizes the information content of the classification. The resulting taxonomy of Enterobacteriaceae corresponds well to the general structure of earlier classifications. Minimization of stochastic complexity can be considered as a useful tool to create bacterial classifications that are optimal from the point of view of information theory.
-
-
Volumes and issues
-
Volume 170 (2024)
-
Volume 169 (2023)
-
Volume 168 (2022)
-
Volume 167 (2021)
-
Volume 166 (2020)
-
Volume 165 (2019)
-
Volume 164 (2018)
-
Volume 163 (2017)
-
Volume 162 (2016)
-
Volume 161 (2015)
-
Volume 160 (2014)
-
Volume 159 (2013)
-
Volume 158 (2012)
-
Volume 157 (2011)
-
Volume 156 (2010)
-
Volume 155 (2009)
-
Volume 154 (2008)
-
Volume 153 (2007)
-
Volume 152 (2006)
-
Volume 151 (2005)
-
Volume 150 (2004)
-
Volume 149 (2003)
-
Volume 148 (2002)
-
Volume 147 (2001)
-
Volume 146 (2000)
-
Volume 145 (1999)
-
Volume 144 (1998)
-
Volume 143 (1997)
-
Volume 142 (1996)
-
Volume 141 (1995)
-
Volume 140 (1994)
-
Volume 139 (1993)
-
Volume 138 (1992)
-
Volume 137 (1991)
-
Volume 136 (1990)
-
Volume 135 (1989)
-
Volume 134 (1988)
-
Volume 133 (1987)
-
Volume 132 (1986)
-
Volume 131 (1985)
-
Volume 130 (1984)
-
Volume 129 (1983)
-
Volume 128 (1982)
-
Volume 127 (1981)
-
Volume 126 (1981)
-
Volume 125 (1981)
-
Volume 124 (1981)
-
Volume 123 (1981)
-
Volume 122 (1981)
-
Volume 121 (1980)
-
Volume 120 (1980)
-
Volume 119 (1980)
-
Volume 118 (1980)
-
Volume 117 (1980)
-
Volume 116 (1980)
-
Volume 115 (1979)
-
Volume 114 (1979)
-
Volume 113 (1979)
-
Volume 112 (1979)
-
Volume 111 (1979)
-
Volume 110 (1979)
-
Volume 109 (1978)
-
Volume 108 (1978)
-
Volume 107 (1978)
-
Volume 106 (1978)
-
Volume 105 (1978)
-
Volume 104 (1978)
-
Volume 103 (1977)
-
Volume 102 (1977)
-
Volume 101 (1977)
-
Volume 100 (1977)
-
Volume 99 (1977)
-
Volume 98 (1977)
-
Volume 97 (1976)
-
Volume 96 (1976)
-
Volume 95 (1976)
-
Volume 94 (1976)
-
Volume 93 (1976)
-
Volume 92 (1976)
-
Volume 91 (1975)
-
Volume 90 (1975)
-
Volume 89 (1975)
-
Volume 88 (1975)
-
Volume 87 (1975)
-
Volume 86 (1975)
-
Volume 85 (1974)
-
Volume 84 (1974)
-
Volume 83 (1974)
-
Volume 82 (1974)
-
Volume 81 (1974)
-
Volume 80 (1974)
-
Volume 79 (1973)
-
Volume 78 (1973)
-
Volume 77 (1973)
-
Volume 76 (1973)
-
Volume 75 (1973)
-
Volume 74 (1973)
-
Volume 73 (1972)
-
Volume 72 (1972)
-
Volume 71 (1972)
-
Volume 70 (1972)
-
Volume 69 (1971)
-
Volume 68 (1971)
-
Volume 67 (1971)
-
Volume 66 (1971)
-
Volume 65 (1971)
-
Volume 64 (1970)
-
Volume 63 (1970)
-
Volume 62 (1970)
-
Volume 61 (1970)
-
Volume 60 (1970)
-
Volume 59 (1969)
-
Volume 58 (1969)
-
Volume 57 (1969)
-
Volume 56 (1969)
-
Volume 55 (1969)
-
Volume 54 (1968)
-
Volume 53 (1968)
-
Volume 52 (1968)
-
Volume 51 (1968)
-
Volume 50 (1968)
-
Volume 49 (1967)
-
Volume 48 (1967)
-
Volume 47 (1967)
-
Volume 46 (1967)
-
Volume 45 (1966)
-
Volume 44 (1966)
-
Volume 43 (1966)
-
Volume 42 (1966)
-
Volume 41 (1965)
-
Volume 40 (1965)
-
Volume 39 (1965)
-
Volume 38 (1965)
-
Volume 37 (1964)
-
Volume 36 (1964)
-
Volume 35 (1964)
-
Volume 34 (1964)
-
Volume 33 (1963)
-
Volume 32 (1963)
-
Volume 31 (1963)
-
Volume 30 (1963)
-
Volume 29 (1962)
-
Volume 28 (1962)
-
Volume 27 (1962)
-
Volume 26 (1961)
-
Volume 25 (1961)
-
Volume 24 (1961)
-
Volume 23 (1960)
-
Volume 22 (1960)
-
Volume 21 (1959)
-
Volume 20 (1959)
-
Volume 19 (1958)
-
Volume 18 (1958)
-
Volume 17 (1957)
-
Volume 16 (1957)
-
Volume 15 (1956)
-
Volume 14 (1956)
-
Volume 13 (1955)
-
Volume 12 (1955)
-
Volume 11 (1954)
-
Volume 10 (1954)
-
Volume 9 (1953)
-
Volume 8 (1953)
-
Volume 7 (1952)
-
Volume 6 (1952)
-
Volume 5 (1951)
-
Volume 4 (1950)
-
Volume 3 (1949)
-
Volume 2 (1948)
-
Volume 1 (1947)