- Volume 143, Issue 1, 1997
Volume 143, Issue 1, 1997
- Review Article
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- Microbiology Comment
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- Biochemistry
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Enterobactin Synthase Polypeptides of Escherichia Coli are Present in an Osmotic-Shock-Sensitive Cytoplasmic Locality
More LessThe terminal reactions in the synthesis of the siderophore enterobactin (Ent) by Escherichia coli require the EntD, E, F and B/G polypeptides. The idea that these molecules form a complex (Ent synthase) that is membrane-associated was re-evaluated. In vitro results provided no evidence in support of the proposal: (i) Ent synthase activity occurred normally under conditions where membrane was either absent or disrupted by high concentrations of neutral detergents, and (ii) immunoprecipitation experiments conducted on extracts engaged in Ent synthesis failed to detect any association among the Ent polypeptides. However, Western blot analyses showed that EntE, F and B/G were released from cells by osmotic shock and freeze/thaw treatment but not by conversion of cells to spheroplasts. These results demonstrated that EntE, F and B/G belong to the Beacham group D class of proteins. The shockability of a given group D Ent protein was unaffected by the absence of either EntB/G or EntD and, for EntB/G, the N-terminus was sufficient for release by osmotic shock. The behaviour of group D proteins is generally attributed to their association (partial, loose or transient) with cytoplasmic membrane; therefore, the results are indirect evidence that Ent synthase interacts with membrane in vivo. At the very least, the data indicate that EntE, F and B/G are compartmentalized in E. coli and, because other biosynthetic enzymes for siderophores and surfactants are related to these Ent proteins, suggest that this entire protein class may be sequestered in vivo.
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Malic Enzyme is a Major Source of NADPH for Lipid Accumulation by Aspergillus Nidulans
More LessMalic enzyme was shown to be a major source of NADPH for the synthesis of storage lipid by Aspergillus nidulans. A previously produced mutant lacking malic enzyme activity (acuK248) accumulated only half the lipid (12%, w/w, of cell dry weight) accumulated by strains of A. nidulans possessing malic enzyme. When cultivated under conditions designed not to promote the production of storage lipid all three strains of A. nidulans studied contained equivalent amounts of lipid (5%, w/w, of cell dry weight). Thus malic enzyme did not appear to be limiting the synthesis of metabolically active lipid (e.g. membrane lipids). No evidence of a role for malic enzyme in the desaturation of fatty acids was found.
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A Mechanism for Production of Hydroxyl Radicals by the Brown-Rot Fungus Coniophora Puteana: Fe(III) Reduction by Cellobiose Dehydrogenase and Fe(II) Oxidation at a Distance from the Hyphae
More LessIn timber infested by brown-rot fungi, a rapid loss of strength is attributed to production of hydroxyl radicals (HO.). The hydroxyl radicals are produced by the Fenton reaction [Fe(II)/H2O2], but the pathways leading to Fe(II) and H2O2 have remained unclear. Cellobiose dehydrogenase, purified from cultures of Coniophora puteana, has been shown to couple oxidation of cellodextrins to conversion of Fe(III) to Fe(II). Two characteristics of brown rot are release of oxalic acid and lowering of the local pH, often to about pH 2. Modelling of Fe(II) speciation in the presence of oxalate has revealed that Fe(II)-oxalate complexes are important at pH 4-5, but at pH 2 almost all Fe(II) is in an uncomplexed state which reacts very slowly with dioxygen. Diffusion of Fe(II) away from the hyphae will promote conversion to Fe(II)-oxalate and autoxidation with H2O2 as product. Thus the critical Fe(II)/H2O2 combination will be generated at a distance, enabling hydroxyl radicals to be formed without damage to the hyphae.
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- Bioenergetics And Transport
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Expulsion of Uracil and Thymine from the Yeast Saccharomyces Cerevisiae: Contrasting Responses to Changes in the Proton Electrochemical Gradient
More LessThe outflow of uracil from the yeast Saccharomyces cerevisiae is known to be relatively fast in certain circumstances, to be retarded by proton conductors and to occur in strains lacking a uracil proton symport. In the present work, it was shown that uracil exit from washed yeast cells is an active process, creating a uracil gradient of the order of -80 mV relative to the surrounding medium. Glucose accelerated uracil exit, while retarding its entry. DNP or sodium azide each lowered the gradient to about -30 mV, simultaneously increasing the rate of uracil entry. They also lowered cellular ATP content. Manipulation of the external ionic conditions governing Δμ;H+ at the plasma membrane had no detectable effect on uracil transport in yeast preparations thoroughly depleted of ATP. It was concluded that uracil exit is probably not driven by the proton gradient but may utilize ATP directly. It is known that thymine is not normally absorbed by yeast. However, thymine expulsion was here observed during deamination of the substrate 5-methylcytosine in the presence of glucose. In the absence of glucose, or following ATP depletion, thymine uptake from the medium only occurred when Δμ;H+ was dissipated, either by DNP or azide, or by manipulation of the external ionic environment. The yeast expelled absorbed thymine when Δμ;H+ was restored to the physiological range. The properties of the system corresponded to those of an H+/thymine antiport that is distinct from the mechanism expelling uracil.
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Expression and Content of Terminal Oxidases in Azotobacter Vinelandii Grown with Excess NH4+ are Modulated by O2 Supply
More LessThe influence of the rate of O2 supply to batch cultures on the contents of cytochromes bd and ‘o’ in NH4 +-grown Azotobacter vinelandii has been investigated. Difference spectra at room temperature (reduced + CO minus reduced) were recorded for whole cells of a wild-type strain and mutants which either lacked or over-produced the cytochrome bd-type terminal oxidase encoded by cydAB. A Tn5-B20 insertion in cydB in the former mutant also provided a means of monitoring cydAB gene expression from measurements of β-galactosidase activity. The content of cytochrome d in the wild-type, and the expression of cydAB-lacZ, in the mutant, increased as the O2 supply was raised, suggesting that O2 regulates cydAB expression even in the absence of diazotrophy. In a strain carrying a mutation in cydR, a regulatory gene upstream of cydAB, and which over-produces cytochrome bd, the responses to O2 supply during growth at different O2 supply rates were reversed. Changes in the content of a haemoprotein detectable in low temperature photodissociation spectra, and attributed to cytochrome b 595 -the high-spin cytochrome b component of the cytochrome bd complex - followed the changes in cytochrome d levels. CO difference spectra of both the wild-type strain and the cytochrome bd-deficient mutant revealed a haemoprotein with spectral characteristics similar to cytochrome o, the levels of which increased as the O2 supply was raised. These results are discussed with reference to previous reports of cytochrome changes in cells grown under N2-fixing conditions.
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- Genetics And Molecular Biology
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The Lipid a Biosynthesis Deficiency of the Escherichia Coli Antibiotic-Supersensitive Mutant LH530 is Suppressed by a Novel Locus, ORF195
More LessA new mutant of Escherichia coli K-12 supersensitive to both hydrophobic and large hydrophilic antibiotics was isolated and characterized. The mutant grew well at 28 °C, poorly at 37 °C, and did not grow at 42 °C. The rate of its lipid A biosynthesis was reduced as compared to that of the parent strain. This deficiency was rescued by a novel locus, ORF195, the function of which has not been elucidated. ORF195 is located in the 76 min region in the E. coli chromosome and encodes a hypothetical 21.8 kDa protein with no signal sequence. ORF195 isolated from the mutant strain had an identical sequence to the wild-type allele, indicating a suppressor function of the gene product.
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Thioredoxin is Essential for Rhodobacter Sphaeroides Growth by Aerobic and Anaerobic Respiration
More LessTo investigate the biological role of thioredoxin in the facultative photosynthetic bacterium Rhodobacter sphaeroides, attempts were made to construct a thioredoxin-deficient mutant by site-specific mutagenesis, using the Tn903 kanamycin resistance gene for selection. In situ and Southern hybridization analyses have demonstrated that the TrxA-mutation is lethal for R. sphaeroides growth under anaerobic conditions with DMSO as terminal electron acceptor and under aerobic conditions. In addition, the DNA region upstream of the trxA initiation codon is essential for aerobic growth of R. sphaeroides. An ORF of unknown function was identified in this region and is suggested to encode a product essential for aerobic metabolism of R. sphaeroides. The mechanism of thioredoxin action was also analysed by using the procedure for gene replacement to introduce a Cys33 to Ser mutation into the trxA chromosomal copy. The strain carrying this mutation produced a thioredoxin impaired in its protein-disulfide reductase activity and was also not viable. These data suggest that the physiological function of R. sphaeroides thioredoxin is redox-dependent. Thioredoxin purified from R. sphaeroides was shown to have a glutathione-disulfide oxidoreductase activity typical of glutaredoxins. This unexpected finding suggests that R. sphaeroides thioredoxin, in contrast to Escherichia coli thioredoxin, has the potential to act in GSH-dependent processes. Thus, the fundamental role of R. sphaeroides thioredoxin in cell growth probably originates from the multiple functions it can serve in vivo.
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Plasmid-Encoded Genes Specifying Aniline Oxidation from Acinetobacter sp. Strain YAA
More LessAcinetobacter sp. strain YAA is able to use aniline and o-toluidine as the sole carbon and energy source. This strain has several different plasmids and acridine orange curing suggested that aniline utilization in strain YAA was plasmid-encoded. The gene cluster involved in aniline oxidation was cloned in Escherichia coli JM109 from the total plasmid DNA of strain YAA. A recombinant E. coli containing an 18.5 kb insert fragment showed yellow colouration on aniline-containing plates, indicating the formation of 2-hydroxymuconic semialdehyde from aniline. In addition, subcloning of a 9.0 kb Sall fragment from the insert in E. coli resulted in the accumulation of catechol. Southern hybridization studies indicated that the aniline oxygenase gene (atdA) was present on one of the plasmids, pYA1. These results suggest that in strain YAA aniline is degraded via catechol through a pathway involving meta-cleavage of the benzene-ring by plasmid-encoded genes including atdA.
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xyIUW, Two Genes at the Start of the upper Pathway Operon of TOL Plasmid Pwwo, Appear to Play no Essential Part in Determining its Catabolic Phenotype
More LessThe upper pathway operon of the toluene catabolic pathway of TOL plasmid pWW0 was shown to carry two open reading frames between the start of transcription and xylC (encoding benzaldehyde dehydrogenase), the first previously reported gene of the operon. These were designated xyIUW: xyIU encoded a protein of 131 amino acid residues (Mr 14244) which bore no relationship with any protein in the databases, and xyIW encoded a protein of 348 residues (Mr 36992) which was strongly homologous to other long-chain Zn-containing alcohol dehydrogenases. Extracts of Escherichia coli carrying xyIUW in expression vector pTrc99A contained a novel protein corresponding to XyIW, but no NAD+ -dependent dehydrogenase activity against benzyl alcohol, mandelate or benzylamine. A mini-Tn5 transposon carrying the meta pathway operon was constructed and from it two strains of Pseudomonas putida were constructed with the normally plasmid-encoded catabolic operons integrated into the chromosome. Three derivatives of plasmid pKNG101 containing modified xyIUW genes were constructed, two of which had frameshifts in xyIU and xyIW, respectively, and a third with a deletion from the 3′ end of xyIU into the 5′ end of xyIW. The wild-type genes of the two Pseudomonas strains were substituted by the mutant alleles by reverse genetics. The ability of the constructed mutant strains to utilize the aromatic substrates of the TOL pathway was not significantly affected.
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The Plasmid-Located Haloalkane Dehalogenase Gene from Rhodococcus Rhodochrous NCIMB 13064
More LessThe haloalkane dehalogenase (dhaA) gene from Rhodococcus rhodochrous NCIMB 13064 was cloned and sequenced. Its comparison with the previously studied dhIA gene from Xanthobacter autotrophicus GJ10 did not show homology. However, the amino acid sequences of the products of these genes showed approximately 30% identity and several of the catalytic amino acid residues were conserved in the NCIMB 13064 dehalogenase. A high level of dhaA expression was demonstrated in Escherichia coli cells and this gene was shown to encode a dehalogenase with the activity against chloroalkanes of chain length C3-C10. Also, some dehalogenase activity against 1,2-dichloroethane encoded by the cloned dhaA gene was detected. The analysis of NCIMB 13064 derivatives lacking dehalogenase activity showed that the dhaA gene was located on the 100 kbp pRTL1 plasmid. It was also found that reversible rearrangements of DNA in the dhaA region may be responsible for the control of expression of haloalkane dehalogenase in R. rhodochrous NCIMB 13064. A number of repeated and inverted sequences which may cause genetic instability at the locus were found in the haloalkane dehalogenase gene region.
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ScCypB is a Novel Second Cytosolic Cyclophilin from Streptomyces Chrysomallus which is Phylogenetically Distant from ScCypA
More LessA novel second streptomycete cyclophilin gene - designated sccypB - was isolated from a cosmid gene library of Streptomyces chrysomallus by using as gene probe a fragment of the previously isolated cyclophilin gene sccypA of the same organism. From its sequence the gene sccypB should encode a protein of Mr 18868. Expression of sccypB in Escherichia coli as a hexaHis-tagged fusion protein (H6ScCypB) and enzymic characterization of the purified protein showed that, like ScCypA, ScCypB is a peptidyl-prolyl cis-trans isomerase (PPIase). The specific activity and substrate specificity of the enzyme were comparable to that of ScCypA, but it was threefold less sensitive to inhibition by cyclosporin A (CsA). In contrast to ScCypA, which is abundant and exists in free and liganded form, ScCypB was 50- to 100-fold less abundant in cytosol-derived protein fractions of S. chrysomallus or Streptomyces lividans, as revealed by Western blot analyses, suggesting a specialized function for this enzyme in the streptomycete cell. Both sccypB and sccypA were found to be present as single copies in the genome of S. chrysomallus and hybridized to a single band in chromosomal DNAs of other streptomycetes. High-level expression of sccypB as well as of sccypA cloned into the expression vector pIJ702 did not produce detectable changes in growth and morphology of S. chrysomallus and S. lividans. Calculations of similarities to known cyclophilin sequences and construction of phylogenetic trees indicated that ScCypB and ScCypA are phylogenetically distant from each other. While ScCypA is clearly related to the eukaryotic cyclophilins, the analyses show the sequence of ScCypB to be the most divergent of all cyclophilin sequences, indicating that it possibly constitutes a cluster by itself.
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High Affinity Iron Acquisition in Rhizobium Leguminosarum Requires the cycHJKL Operon and the feuPQ Gene Products, which belong to the Family of Two-Component Transcriptional Regulators
More LessThe cycHJKL operon of Rhizobium leguminosarum has previously been shown to be involved in the maturation of cytochrome c, possibly by its involvement in the covalent attachment of haem to the apoprotein. Mutations in the cycHJKL genes abolish symbiotic nitrogen fixation. Here, we show that cyc mutants are pleiotropically defective. They have lost a high affinity iron acquisition system due to their failure to make or to export siderophores. They also accumulate protoporphyrin IX, the immediate precursor of haem. A model to account for these phenotypes is presented. Immediately upstream of cycH is a gene, lipA, which is predicted to encode an outer-membrane lipoprotein. Further upstream of lipA, there are two other genes, whose products are similar in sequence to the widespread family of two-component transcriptional regulators. These two genes, feuP and feuQ, did not affect the transcription of lipA, or of the cycHJKL operon. However, a mutation in feuQ also led to the loss of the high affinity iron uptake system, although siderophores were still produced.
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An iron-Regulated Outer-Membrane Protein Specific to Bordetella Bronchiseptica and Homologous to Ferric Siderophore Receptors
More LessThe bfrA (Bordetella bronchiseptica ferric iron repressed outer-membrane protein) gene was cloned from Bordetella bronchiseptica by screening a library of TnphoA insertion mutants for iron-repressed fusions to phoA. The bfrA gene encoded an 80 kDa outer-membrane protein with a high level of amino acid sequence identity to several bacterial proteins belonging to the family of Ton B-dependent outer-membrane receptors. BfrA was especially homologous to Cir of Escherichia coli, IrgA of Vibrio cholerae and to three previously characterized ferric enterobactin receptors. DNA hybridization results indicated that bfrA was not present in other Bordetella species. Expression of the bfrA gene was induced by low iron availability from a promoter overlapped by a sequence resembling a consensus Fur-binding sequence, and bfrA expression was derepressed in a B. bronchiseptica fur mutant. Utilization of the Bordetella siderophore alcaligin and the exogenous siderophore enterobactin was unaffected in bfrA mutants. Upon attempting to find the specificity of BfrA, 2,3-dihydroxybenzoylserine (DHBS) was shown to be utilized in a bfeA (Bordetella ferric enterobactin receptor gene)-dependent manner by B. bronchiseptica and B. pertussis. In addition, the hydroxamate siderophores ferrichrome and desferrioxamine B, and the iron source haemin were shown to be utilized independently of bfeA and bfrA in B. bronchiseptica and B. pertussis.
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bofC Encodes a Putative Forespore Regulator of the Bacillus Subtilis σk Checkpoint
More LessA mutation, bofC1, that restores σK activation in Bacillus subtilis strains unable to produce active σG has been identified. This mutation defines a new sporulation gene, bofC, that has been cloned and sequenced and encodes a 19 kDa protein. bofC is transcribed in the forespore by RNA polymerase associated with the transcription factors σF (EσF) and σG (EσG). BofC acts negatively on SpolVB and the results described suggest that BofC regulates SpolVB activity and its intercompartmental signalling role in the σK checkpoint
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Expression of CEL2 and CEL4, Two Proteins from Agaricus Bisporus with Similarity to Fungal Cellobiohydrolase I and -mannanase, Respectively, is Regulated by the Carbon Source
More LessTwo new cellulose-growth specific (cel) cDNAs, cel2 and cel4, have been isolated from an Agaricus bisporus cDNA expression library by immunoscreening with an A. bisporus anti-endoglucanase antibody. The deduced amino acid sequences showed that both CEL2 and CEL4 proteins have a modular structure consisting of a fungal-type cellulose-binding domain (CBD) and a catalytic domain separated by a linker region rich in Pro, Ser and Thr. The CEL2 and CEL4 catalytic domains were homologous to fungal cellobiohydrolases (CBH) in family 7 and to fungal mannanases in family 5 of the glycosyl hydrolases, respectively. A previously isolated cDNA derived from a constitutive gene was also sequenced. The deduced amino acid sequence corresponded to 5-aminolaevulinic acid synthase (ALA), the first enzyme in the haem biosynthetic pathway, and was most similar to other fungal ALAs. RNA analysis showed that the expression of cel2 and cel4 genes was induced by cellulose and repressed by glucose, fructose and lactose. The soluble cellulose derivative CM-cellulose induced mRNA accumulation for cel1 but not cel2, cel3 or cel4. Mannitol, maltose, sorbitol and glycerol decreased cel2 and cel4 mRNA levels to different extents, cel1, cel2, cel3 and cel4 mRNAs all disappeared after the addition of glucose with apparent half-lives of less than 20 min. Whether cel mRNAs have short half-lives or glucose affects the stability of cel transcripts remains to be investigated.
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The Phytase Subfamily of Histidine Acid Phosphatases: Isolation of Genes for Two Novel Phytases from the Fungi Aspergillus Terreus and Myceliophthora Thermophila
More LessPhytases catalyse the hydrolysis of phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate. In this study genes encoding novel phytases from two different filamentous fungi, Aspergillus terreus strain 9A-1 and Myceliophthora thermophila were isolated. The encoded PhyA phytase proteins show 60% (A. terreus) and 48% (M. thermophila) identity, respectively, to the PhyA of Aspergillus niger and have 21-29% identity compared to other histidine acid phosphatases. All three PhyA proteins, in contrast to the A. niger pH 2.5-optimum acid phosphatase, prefer phytic acid as substrate and show enzyme activity at a broad range of acidic pH values. Based on their enzyme characteristics and protein sequence homology, the phytases form a novel subclass of the histidine acid phosphatase family.
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- Pathogenicity And Medical Microbiology
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Colonial Opacity Variations among the Choleragenic Vibrios
Cultures of Vibrio cholerae O1, biotype El Tor, from the current epidemic of cholera in the Western Hemisphere, and of the new V. cholerae serogroup O139, from the current outbreak in India and Bangladesh, revealed marked colonial heterogeneity when received by the authors. By comparison with reference colony types, using a stereoscope and transmitted oblique illumination, colonies of approximately 10 different degrees of opacity could be distinguished. In contrast, strains freshly isolated from patients and rapidly and carefully preserved were more homogeneous although still differentiable by this technique. These (and older) observations prompted the questions: (1) why is a V. cholerae colony opaque or translucent? and (2) what benefit is it to the vibrios to vary their colonial appearance? The observed changes in colonial opacity, which are reversible, are sometimes (rarely) accompanied by changes in virulence for infant rabbits and, more frequently, by other phenotypic variations including the ability to produce poly-β-hydroxybutyrate inclusion bodies on glycerol-containing medium, the degree of encapsulation in O139, changes in outer-membrane proteins, alteration in lipopolysaccharide structure, changes in expression of glycolytic pathways, and differences in ability to survive under adverse conditions. Colonial variations in choleragenic vibrios are phenotypically multifactorial. The genetic mechanisms(s) underlying the observed phenotypic changes remain to be defined.
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Use of Siderophores to Type Pseudomonads: The Three Pseudomonas Aeruginosa Pyoverdine Systems
Eighty-eight Pseudomonas aeruginosa isolates, most of them from the Collection of Bacterial Strains of the Institut Pasteur, Paris, were analysed for their pyoverdine-mediated iron incorporation system by different methods, including pyoverdine isoelectrofocusing analysis, pyoverdine-mediated growth stimulation, immunoblot detection of (ferri)pyoverdine outer-membrane receptor and pyoverdine-facilitated iron uptake. The same grouping of the strains was reached by each of these methods, resulting in the classification of the P. aeruginosa isolates, even those which were devoid of pyoverdine production, into three different siderophore types. Forty-two percent of the strains were identified with the type-strain P. aeruginosa ATCC 15692 (group I). 42% were identical with the second type-strain P. aeruginosa ATCC 27853 (group II) and 16% reacted identically with the clinical isolate P. aeruginosa Pa6, whose pyoverdine was recognized in this study to be identical in structure to the pyoverdine produced by a natural isolate, P. aeruginosa strain R. No new pyoverdine species was detected among these strains.
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TNF-a, IL-1a, IL-6 and ICAM-1 Expression in Human Keratinocytes Stimulated in Vitro with Escherichia Coli Heat-Shock Proteins
Bacterial heat-shock proteins (HSPs) from Escherichia coli (GroES, GroEL and DnaK) were studied for their ability to induce by themselves the expression and release of tumour necrosis factor-a (TNF-a), interleukin-6 (IL-1a), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) by cultured human keratinocytes. The surface expression of ICAM-1 was also investigated. In the supernatants of untreated cells none or a minimal amount of these molecules was found. After 48 h of stimulation with GroEL significant amounts of TNF-a, IL-1a, IL-6 and soluble ICAM-1 were detected, reaching maximum concentrations at 1 g ml-1. The same effect was elicited by DnaK but to a lesser extent. Treatment of keratinocytes with GroEL and DnaK also increased TNF-a, IL-1a, IL-6 and ICAM-1 mRNA levels. GroES showed significant activity only on the expression and release of IL-6. GroEL and DnaK were also able to up-regulate the surface expression of ICAM-1 on keratinocytes. The effects on ICAM-1 expression seemed to be directly due to HSPs and not mediated via cytokines. Furthermore, these effects were due to the properties of HSPs because they were inhibited by specific monoclonal antibodies. These findings support the potential role of HSPs in modulating cell interactions during immunological and inflammatory responses in the skin.
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Monoclonal Antibodies Against Streptococcus Pneumoniae Detect Epitopes on Eubacterial Ribosomal Proteins L7/L12 and on Streptococcal Elongation Factor Ts
More LessTwo monoclonal antibodies (mAbs) designated 144,H-3 (lgG2a) and 218,C-5 (lgM) were produced after immunization of mice with two different heattreated and sonicated pneumococcal strains. Western blotting, with solubilized proteins from different bacterial genera and from mammalian lymphocytes, showed that both mAbs reacted with a protein of approximately 12 kDa in all 66 strains of eubacteria examined, representing 27 different species. The 12 kDa protein was isolated by immunoaffinity chromatography. Subsequent preparative Western blotting enabled N-terminal amino acid sequence analysis by microsequencing. A high degree of amino acid sequence similarity with eubacterial ribosomal proteins L7/L12 was demonstrated. One of the mAbs (144,H-3) also cross-reacted in Western blotting with a 43 kDa protein, but only from streptococci. The 43 kDa protein carrying the common streptococcal epitope was isolated and sequenced in the N-terminal region. A high degree of amino acid sequence identity was found to elongation factor Ts from Escherichia coli.
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Endotoxic Properties of Free Lipid a from Porphyromonas Gingivalis
More LessThe relationship between chemical structure and biological activity of the lipid A from Porphyromonas gingivalis, which we recently isolated and whose complete chemical structure was determined [Kumada et al. (1995). J Bacteriol 177, 2098--2106], was studied. The lipid A exhibited endotoxic activity in all the assay systems tested: Limulus gelation activity, lethal toxicity in galactosamine-sensitized mice, mitogenicity in mouse spleen cells and induction of nitric oxide (NO) and tumour necrosis factor alpha (TNF) release from both mouse peritoneal macrophages and the J774-1 mouse macrophage-like cell line. The activity was, however, about 100-fold less than that of Salmonella minnesota LPS used as a control. The moderate activity of the lipid A may be partially explained by its unique fatty acid composition and the lack of a phosphate group in position 4. In contrast, the lipid A as well as whole LPS of P. gingivalis unexpectedly exhibited an even stronger induction of TNF from the human monocytic THP-1 cell line than control LPS when measured by the minimum stimulatory dose. The difference in sensitivity of human and mouse cells to P. gingivalis lipid A suggests that the recognition mechanism, including that for the receptor for endotoxin, may be regulated in different ways in the two cells.
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- Physiology And Growth
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Bacteriophage T4 Development Depends on the Physiology of its Host Escherichia Coli
More LessSeveral parameters of phage T4 adsorption to and growth in Escherichia coli B/r were determined. All changed monotonously with the bacterial growth rate (μ), which was modified by nutritional conditions. Adsorption rate was faster at higher μ values, positively correlated to cell size, and increased by pretreatment with low penicillin (Pn) concentrations; it was directly proportional to total cellular surface area, indicating a constant density of T4 receptors on cell envelopes irrespective of growth conditions. Parameters of phage development and cell lysis were μ-dependent. The rate of phage release and burst size increased, while the eclipse and latent periods decreased with increasing μ. Differentiation between the contribution of several physiological parameters to the development of T4 was performed by manipulating the host cells. A competitive inhibitor of glucose uptake, methyl α-D-glucoside, was exploited to reduce the growth rate in the same effective carbon source. Synchronous cells were obtained by the ‘baby-machine’ and large cells were obtained by pretreatment with low Pn concentrations. Lysis was delayed by superinfection, and DNA content and concentration were modified by growing a thy mutant in limiting thymine concentrations. The results indicate that burst size is not limited by cell size or DNA composition, nor directly by the rate of metabolism, but rather by the rates of synthesis and assembly of phage components and by lysis time. The rates of synthesis and assembly of phage components seem to depend on the content of the protein-synthesizing system and lysis time seems to depend on cellular dimensions.
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The IdhA Gene Encoding the Fermentative Lactate Dehydrogenase of Escherichia Coli
More LessUnder anaerobic conditions, especially at low pH, Escherichia coli converts pyruvate to D-lactate by means of an NADH-linked lactate dehydrogenase (LDH). This LDH is present in substantial basal levels under all conditions but increases approximately 10-fold at low pH. The IdhA gene, encoding the fermentative lactate dehydrogenase of E. coli, was cloned using λ10E6 of the Kohara collection as the source of DNA. The IdhA gene was subcloned on a 2.8 kb MIuI-MIuI fragment into a multicopy vector and the region encompassing the gene was sequenced. The IdhA gene of E. coli was highly homologous to genes for other D-lactate-specific dehydrogenases but unrelated to those for the L-lactate-specific enzymes. We constructed a disrupted derivative of the IdhA gene by inserting a kanamycin resistance cassette into the unique KpnI site within the coding region. When transferred to the chromosome, the IdhA::Kan construct abolished the synthesis of the D-LDH completely. When present in high copy number, the IdhA gene was greatly overexpressed, suggesting escape from negative regulation. Cells expressing high levels of the D-LDH grew very poorly, especially in minimal medium. This poor growth was largely counteracted by supplementation with high alanine or pyruvate concentrations, suggesting that excess LDH converts the pyruvate pool to lactate, thus creating a shortage of 3-carbon metabolic intermediates. Using an IdhA-cat gene fusion construct we isolated mutants which no longer showed pH-dependent regulation of the IdhA gene. Some of these appeared to be in the pta gene, which encodes phosphotransacetylase, suggesting the possible involvement of acetyl phosphate in IdhA regulation.
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Use of Morphology Index Histograms to Quantify Populations of the Fungal Pathogen Paracoccidioides Brasiliensis
More LessTo quantify the dimorphic process in wild and mutant strains of Paracoccidioides brasiliensis, we defined a morphology index (Mi) in terms of the maximum cell length (I), maximum cell diameter (d), and septal diameter (s), according to the equation Mi = 2.13 + 1.13 log10(Is/d 2), whose intercept and slope were such that Mi was around 1 for yeast (spherical) cells or 4 for hyphal (elongated) cells. This discriminatory power was used to quantify morphological population mixtures through Mi histograms. During the temperature-induced dimorphic transition (either way), mean Mi (Mi) varied linearly with time, suggesting a continuity in the process. Also, in wild strains and mutants thereof we found an inverse relationship between Mi and content of both cell wall chitin and 1,3-a-glucan.
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Flux Distributions in Anaerobic, Glucose-Limited Continuous Cultures of Saccharomyces Cerevisiae
More LessA stoichiometric model describing the anaerobic metabolism of Saccharomyces cerevisiae during growth on a defined medium was derived. The model was used to calculate intracellular fluxes based on measurements of the uptake of substrates from the medium, the secretion of products from the cells, and of the rate of biomass formation. Furthermore, measurements of the biomass composition and of the activity of key enzymes were used in the calculations. The stoichiometric network consists of 37 pathway reactions involving 43 compounds of which 13 were measured (acetate, CO2, ethanol, glucose, glycerol, NH+ 4, pyruvate, succinate, carbohydrates, DNA, lipids, proteins and RNA). The model was used to calculate the production rates of malate and fumarate and the ethanol measurement was used to validate the model. All rate measurements were performed on glucose-limited continuous cultures in a high-performance bioreactor. Carbon balances closed within 98%. The calculations comprised flux distributions at specific growth rates of 0.10 and 0.30 h−1. The fluxes through reactions located around important branch points of the metabolism were compared, i.e. the split between the pentose phosphate and the Embden-Meyerhoff-Parnas pathways. Also the model was used to show the probable existence of a redox shunt across the inner mitochondrial membrane consisting of the reactions catalysed by the mitochondrial and the cytosolic alcohol dehydrogenase. Finally it was concluded that cytosolic isocitrate dehydrogenase is probably not present during growth on glucose. The importance of basing the flux analysis on accurate measurements was demonstrated through a sensitivity analysis. It was found that the accuracy of the measurements of CO2, ethanol, glucose, glycerol and protein was critical for the correct calculation of the flux distribution.
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- Plant-Microbe Interactions
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Quantitative Assessment of in Planta Distribution of Metabolic Activity and Gene Expression of an Endophytic Fungus
More LessUsing perennial ryegrass infected with an Acremonium transformant carrying the Escherichia coli β-D-glucuronidase gene (gusA) (GUS system) under control of a constitutive promoter, we have developed methods for the quantitative extraction of endophyte-associated GUS activity from plant material. Fluorometric assays of these extracts allow quantitative assessment of the distribution of endophyte-associated GUS activity within single plants (tillers) with high resolution. Fluorescence microscopy with the dye Imagene Green can in addition visualize individual GUS-expressing hyphae. Since the transformant expresses the GUS gene constitutively, GUS activity can be used as an indicator of in planta endophyte metabolic activity. Using this approach we found that (i) the concentration of endophyte metabolic activity in plant tissue decreases with increasing plant size, (ii) approximately 70% of endophyte metabolic activity present in a plant is located in the leaf sheaths, (iii) basal-apical gradients and lateral (younger to older tissue) gradients of endophyte metabolic activity exist and (iv) basal-apical gradients are established early in leaf development. Our data suggest that the concentration of endophyte in each part of the plant is regulated so that a predetermined threshold of total endophyte activity per plant is not exceeded and a consistent distribution pattern is maintained.
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- Genome Analysis
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A Bacillus Cereus Member of the SNF2 Family
More LessThe complete sequence of a Bacillus cereus member of the SNF2 family of putative helicases showed conservation of all seven motifs typical of this family. Bcsnf predicted a protein of 1064 aa where the conserved SNF2 domain was located at the carboxy terminus, whereas the 633 amino-terminal aa showed no homology to any protein in the databases. A putative transcriptional start was identified by primer extension, indicating that Bcsnf is not a part of a larger operon. No phenotypical changes were observed after insertional inactivation of Bcsnf. The completely sequenced genomes of Mycoplasma genitalium and Haemophilus influenzae contain one ORF each with similarity to the SNF2 family: MG018 and HI0616, respectively. A phylogenetic tree of the SNF2 family showed that BcSNF and MG018 were most closely related, and appeared closer to the eukaryotic members of the SNF2 family than to the two other bacterial members of the family, HepA from Escherichia coli and HI0616.
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A 10.3 kbp Segment from nprB to argJ at the 102 Region of the Bacillus Subtilis Chromosome
More LessThe approximately 10 kbp region encompassing nprB and argJ at 102 on the Bacillus subtilis chromosome was sequenced, revealing 12 ORFs, four known genes (argJ, argC, ipi and nprB) and two genes, yitY and yitS, whose products respectively display significant homology with L-gulono-?-lactone oxidase of rat and dihydrofolate reductase of Staphylococcus aureus. The data also indicated that nprB mapped to a different position than previously published.
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- Guidelines For Authors
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