- Volume 144, Issue 9, 1998
Volume 144, Issue 9, 1998
- Sgm Special Lecture
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Biochemistry of methanogenesis: a tribute to Marjory Stephenson:1998 Marjory Stephenson Prize Lecture
More LessMax-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Straße, D-35043 Marburg, and Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Karl-von-Frisch-Straße, D-35032 Marburg, Germany
In 1933, Stephenson & Stickland (1933a) published that they had isolated from river mud, by the single cell technique, a methanogenic organism capable of growth in an inorganic medium with formate as the sole carbon source.
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- Microbiology Comment
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- Biochemistry
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Altered expression and modification of proteases from an avirulent mutant of Porphyromonas gingivalis W50 (W50/BE1)
More LessProteases of Porphyromonas gingivalis are considered to be important factors in the virulence of this organism. A non-pigmenting mutant of P. gingivalis W50 (W50/BE1) has been shown to be less virulent in animal models and to produce significantly less Arg-specific protease activity than the parent strain. Three proteases are present in the culture supernatant of P. gingivalis W50: RI, RIA and RIB. All three proteases are derived from prpR1, which encodes a polypeptide of 1706 amino acids that is organized into distinct domains (pro, α, β and γ). The aim of the present investigation was to purify and characterize the Arg-specific proteases produced by the avirulent W50/BE1 strain. Significant differences were observed between the proteases of P. gingivalis W50 and W50/BE1. The levels of RI present in the culture supernatant of W50/BE1 were lower than those present in W50, and RIA and RIB were absent. RI from W50/BE1 was composed of three polypeptide chains, unlike the enzyme from W50, which is a heterodimer. The remainder of the Arg-specific protease activity in W50/BE1 was derived from a second gene, prR2, and was present in two fractions, RIIAs/BE (soluble) and RIIAv/BE (vesicle-bound). This activity contained two peptide chains: a ~ 54 kDa chain corresponding to the protease domain and a ~ 26 kDa chain, derived from the propeptide domain of the PrRII precursor. No enzyme with large glycan additions, equivalent to RIB in the vesicle fraction of the wild-type W50, was present. These data indicate that the reduced level of extracellular protease activity in W50/BE1 reflects reduced synthesis and/or export of prpR1 enzymes, which is only partially compensated by synthesis of prR2-derived enzymes, and that all of these proteases undergo altered post-translational modification compared to the parent strain.
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Isolation of a unique benzothiophene-desulphurizing bacterium, Gordona sp. strain 213E (NCIMB 40816), and characterization of the desulphurization pathway
More LessGordona sp. strain 213E (NCIMB 40816) grew in pure culture in a mineral salts medium containing fructose as a source of carbon and energy, and benzothiophene (BTH) as the sole source of sulphur. During growth a phenolic compound accumulated, as indicated by the production of a blue colour on addition of Gibb's reagent. Therefore this pathway is analogous to the dibenzothiophene (DBT) desulphurization pathway of Rhodococcus sp. strain IGTS8, in which 2-hydroxybiphenyl accumulates during growth with DBT as the sole sulphur source. Ethyl acetate extraction of the culture medium yielded the metabolites benzothiophene S-oxide (BTHO), benzothiophene S,S-dioxide (BTHO2), benzo[c][1,2]oxathiin 6-oxide (BcOTO), 2-(2'-hydroxyphenyl)ethan 1-al (HPEal) and benzofuran (BFU). The deduced pathway for BTH desulphurization is BTH ? BTHO ? BTHO2 ? HPESi- ? HPEal. HPESi- is (Z)-2-(2'-hydroxyphenyl)ethen 1-sulphinate, the stable aqueous-solution form of BcOTO. It was concluded that HPEal was the Gibb's-reagent-reactive phenolic compound which accumulated in the culture medium of strain 213E during growth, and that the presence of BFU was due to partial condensation of HPEal during the ethyl acetate extraction procedure. Gordona sp. strain 213E was unable to grow in a mineral salts medium containing fructose as a source of carbon and energy and DBT as the sole sulphur source. BTH-desulphurization-active cells (grown using BTH as sole sulphur source) were unable to desulphurize DBT. Likewise Rhodococcus sp. strain IGTS8 was unable to grow using BTH as the sole sulphur source, and DBT-desulphurization-active cells of strain IGTS8 (grown using DBT as sole sulphur source) were unable to desulphurize BTH. This absence of cross-reactivity is discussed in terms of fundamental differences in the chemistry of the DBT- and BTH-desulphurization reactions.
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Bacillus subtilis ORF yybQ encodes a manganese-dependent inorganic pyrophosphatase with distinctive properties: the first of a new class of soluble pyrophosphatase?
More LessThe N-terminal 15 amino acids of the major protein associated with inorganic pyrophosphatase activity in Bacillus subtilis WB600 are identical to those of B. subtilis ORF yybQ. This ORF was amplified from B. subtilis WB600 DNA by PCR and cloned into an overexpression vector in Escherichia coli. Induction of overexpression produced a soluble protein of 34000 Da by SDS-PAGE and by matrix-assisted laser desorption and ionization mass spectrometry. The overexpressed protein had a high specific activity for the hydrolysis of magnesium pyrophosphate, and was specifically and reversibly activated by Mn2+ ions. These properties are identical to those of inorganic pyrophosphatase purified from B. subtilis WB600. No significant similarity was found between the derived sequence of the B. subtilis yybQ-encoded protein and published sequences of identified inorganic pyrophosphatases of Eukarya, Bacteria or Archaea domains. However, there is significant similarity to three putative proteins of unknown function from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus, and from Streptococcus gordonii. The genomes of B. subtilis, M. jannaschii and A. fulgidus do not contain sequences similar to those of hitherto known soluble inorganic pyrophosphatases. The present findings, together with a survey of the properties of inorganic pyrophosphatases from 38 different sources, suggest that the B. subtilis yybQ-encoded protein is the first fully characterized member of a new class of inorganic pyrophosphatase.
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Biosynthesis of triacylglycerol in the filamentous fungus Mucor circinelloides
More LessLipid metabolism was studied in 2-d-old liquid cultures of Mucor circinelloides grown at 25 C. Under these conditions, oil accumulated to 0.5 g I-1 with a ?-linolenic acid content (?18:3) of 60 mg I-1. The major labelled lipids in cultures incubated with [14C]acetate were triacylglycerol (TAG), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The proportion of label declined in the phospholipids and increased in TAG with time. [14C]18:1 and [14C]18:2 rapidly appeared in PC and PE and later accumulated in [14C]?18:3. TAG-synthesizing capacity was greatest in the microsomal membrane fraction, which accumulated high levels of phosphatidic acid in the presence of glycerol 3-phosphate and acyl-CoA substrates at pH 7.0. Further metabolism of phosphatidic acid to diacylglycerol and TAG was achieved by increasing the pH to 8.0. Lysophosphatidic acid:acyl-CoA acyltransferase (LPAAT) activity was particularly high and may have accounted for the rapid accumulation of phosphatidic acid in the membranes. The glycerol-3-phosphate:acyl-CoA acyltransferase (GPAAT) and LPAAT were non-specific for a range of saturated and unsaturated species of acyl-CoA although the GPAAT showed a marked selectivity for palmitoyl-CoA and the LPAAT for oleoyl- and linoleoyl-CoA. ?-Linolenic acid was detected at all three positions of sn-TAG and was particularly enriched at the sn-3 position. The preparation of active in vitro systems (microsomal membranes) capable of the complete biosynthetic pathway for TAG assembly may be valuable in understanding the assembly of oils in future transgenic applications.
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- Bioenergetics And Transport
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Mechanism of isoniazid uptake in Mycobacterium tuberculosis
More LessInitial transport kinetics of isoniazid (INH) and its uptake at the plateau were studied in Mycobacterium tuberculosis H37Rv under various experimental conditions. The initial uptake velocity increased linearly with INH concentration from 2 x 10-6 M to 10-2 M. It was modified neither by addition of a protonophore that abolished proline transport, nor following ATP depletion by arsenate, which inhibited glycerol uptake, two transport processes taken as controls for secondary active transport and facilitated diffusion, respectively. Microaerobiosis or low temperature (4 °) were without effect on initial uptake. It is thus likely that INH transport in M. tuberculosis proceeds by a passive diffusion mechanism, and that catalase-peroxidase (KatG) is not involved in the actual transport. However, conditions inhibiting KatG activity (high INH concentration, microaerobiosis, low temperature) decrease cell radioactivity at the uptake plateau. It is proposed that INH transport occurs by passive diffusion. KatG is involved only in the intracellular accumulation of oxidized derivatives of INH, especially of isonicotinic acid, which is trapped inside cells in its ionized form. This model explains observed and previously known characteristics of the accumulation of radioactivity in the presence of [14C]INH for various species and strains of mycobacteria.
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- Biotechnology
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Genetic engineering of an industrial strain of Saccharopolyspora erythraea for stable expression of the Vitreoscilla haemoglobin gene (vhb)
More LessSeveral Actinomycetes/Streptomycetes expression vectors are described for expression of the Vitreoscilla haemoglobin gene (vhb) in an industrial erythromycin-producing strain of Saccharopolyspora erythraea. Cloning of vhb under the control of either the thiostrepton-inducible PtipA promoter or the constitutive PermE* promoter led to the production of chemically active haemoglobin (VHb) in Streptomyces lividans TK24 transformed with these constructs. However, the plasmids could not be transformed into Sac. erythraea. Transformants of Sac. erythraea and/or exconjugants were obtained using a novel Escherichia coli/Streptomyces shuttle vector comprised of vhb under the control of the PermE* promoter, the Streptomyces plasmid pIJ350 origin of replication, the thiostrepton-resistance gene (tsr) for selection, and the oriT region which is necessary for conjugal transfer. Increased plasmid stability in Sac. erythraea was obtained by construction of a vector for chromosomal integration. This vector contained the Streptomyces phage øC31 attachment site for chromosomal integration and vhb expressed under the PmerR promoter and was stably maintained in the chromosome of Sac. erythraea. Shake-flask cultivations of the transformed Sac. erythraea strain with the chromosomally integrated vhb gene show that vhb is expressed in an active form. The corresponding amount of erythromycin produced in the vhb-expressing strain was approximately 60% higher relative to the original VHb-negative strain.
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- Environmental Microbiology
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Bacteria in post-glacial freshwater sediments
More LessProkaryote communities in post-glacial profundal freshwater sediments of Windermere, representing 10-12000 years of deposition, were examined for culturability, viability and community structure. The potential for active geochemical cycles was inferred from the presence of specific groups of bacteria. Direct count procedures revealed 1012 cells (g dry wt sediment)-1 in the surface sediments, which declined to approximately 109 cells (g dry wt sediment)-1 at 6 m depth of core (representing approximately 10000 years of deposition). The majority of the cells in the upper sediments were metabolically active when challenged with viability probes and responded to the direct viable count method. Below 250 cm, viability shown by 5-cyano-2,3-diotyl tetrazolium chloride (CTC) dye was not significantly different from the direct count; however, counts obtained with 5-carboxyfluorescein diacetate (CFDA) and the direct viable count both declined significantly from the direct count below 250 cm and 1 m, respectively. Culture was achieved from samples throughout the core, although the numbers of culturable bacteria decreased significantly with depth, from 107 c.f.u. (g dry wt sediment)-1 to 101-102 c.f.u. (g dry wt sediment)-1 below 3 m depth. Among culturable isolates, Gram-positives and Gram-negatives were found at all levels of the core, and spore-forming heterotrophs dominated. Although sulphate-reducing bacteria were not detected below 20 cm, isolates demonstrating denitrifying activity were detected at all depths. PCR performed on samples taken below 3 m (deposited more than 7000 years ago) using eubacterial and archaeal primers revealed sequences similar to those found in deep sediments of the Pacific Ocean and the presence of methanogenic archaea. These observations indicate that bacteria and archaea are capable of long-term persistence and activity in deep, aged freshwater sediments.
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Phylogenetic diversity of mesophilic and thermophilic granular sludges determined by 16S rRNA gene analysis
The microbial diversity of two types of methanogenic granular sludge, mesophilic (35 °C) and thermophilic (55 °C), which had been treating sucrose/propionate/acetate-based artificial wastewater were compared. 16S rDNA clone libraries were constructed by PCR with a prokaryote-specific primer set, and partial sequencing of the clonal 16S rDNAs was conducted for phylogenetic analysis. Of 115 mesophilic granule and 110 thermophilic granule clones sequenced, 19 and 22%, respectively, were phylogenetically affiliated with the domain Archaea, and the remainder in each case were assigned to the domain Bacteria. Within the domain Archaea, the 16S rDNA clones in both libraries showed relatively close relationships with those of methanogens. Within the Bacteria, a major group represented in the mesophilic clone library was the delta subclass of the Proteobacteria (27%), in which high degrees of relatedness were observed between the clonal 16S rDNA sequences and those of previously identified syntrophic bacteria and sulfate-reducing bacteria. In contrast, in the thermophilic clone library, the Thermodesulfovibrio group (19%), the green non-sulfur bacteria (18%) and the low G+C subclass of the Gram-positive bacteria (18%) were predominant. A significant difference between the two libraries was that no clone affiliated with the Proteobacteria was detected in the thermophilic clone library, whereas the Proteobacteria was the most predominant group in the mesophilic clones. Thirty-six and 24 different sequences were found in the mesophilic and thermophilic clones, respectively, suggesting that the microbial diversity of the thermophilic granule was lower than that of the mesophilic granule.
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- Genetics And Molecular Biology
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A controllable gene-expression system for the pathogenic fungus Candida glabrata
A system for controlling gene expression was established in the pathogenic fungus Candida glabrata to elucidate the physiological functions of genes. To control the expression of the gene of interest, the C. glabrata cells were first transformed with the plasmid carrying the tetracycline repressor-transactivator fusion tetR::GAL4, then with the DNA fragment containing the controllable cassette, the tetracycline operator chimeric promoter (tetO::ScHOP1). The peptide elongation factor 3 (CgTEF3) and DNA topoisomerase II (CgTOP2) genes from C. glabrata were cloned and their expression assessed using this system. When the promoter of CgTEF3 or CgTOP2 was replaced with tetO::ScHOP1, doxycycline almost completely repressed the expression of both mRNAs, and impaired growth. Repression of the TOP2 or TEF3 gene by doxycycline also hampered the survival of C. glabrata cells in mice; in mouse kidneys the number of C. glabrata cells, in which the TOP2 or TEF3 promoter was replaced with the tetO::ScHOP1 controllable cassette, did not increase when the mice were given doxycycline. Thus, it appears that the gene repression mediated by doxycycline occurred not only in culture media but also in animals; therefore, this system can be used to elucidate the function of the gene in fungal infections and pathogenesis.
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Cloning and sequencing of the Candida albicans homologue of SRB1/PSA1/VIG9, the essential gene encoding GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae
More LessTwo genomic fragments have been isolated from Candida albicans which strongly hybridize to SRB1/PSA1/VIG9, an essential gene which encodes GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae. A common 2.5 kb Xbal-Pstl fragment has been identified, which Southern analysis suggests is most likely unique in the C. albicans genome. The fragment contains an ORF, which is 82% identical and 90% homologous to the Srb1p/Psa1p/Vig9p from S. cerevisiae, contains one additional amino acid at position 254 and is able to functionally complement the major phenotypic characteristics of S. cerevisiae srb1 null and conditional mutations. The authors therefore conclude that they have cloned and sequenced from C. albicans the bona fide homologue of SRB1/PSA1/VIG9, named hereafter CaSRB1. Northern analysis data indicate that the gene is expressed in C. albicans under conditions of growth in the yeast and hyphal form and suggest that its expression might be regulated.
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Structural, functional and mutational analysis of the pfr gene encoding a ferritin from Helicobacter pylori
The function of the pfr gene encoding the ferritin from Helicobacter pylori was investigated using the Fur titration assay (FURTA) in Escherichia coli, and by characterization of a pfr-deficient mutant strain of H. pylori. Nucleotide sequence analysis revealed that the pfr region is conserved among strains (>95% nucleotide identity). Two transcriptional start sites, at least one of them preceded by a s70-dependent promoter, were identified. Provision of the H. pylori pfr gene on a multicopy plasmid resulted in reversal of the Fur-mediated repression of the fhuF gene in E. coli, thus enabling the use of the FURTA for cloning of the ferritin gene. Inactivation of the pfr gene, either by insertion of a resistance cassette or by deletion of the up- and downstream segments, abolished this function. Immunoblot analysis with a Pfr-specific antiserum detected the Pfr protein in H. pylori and in E. coli carrying the pfr gene on a plasmid. Pfr-deficient mutants of H. pylori were generated by marker-exchange mutagenesis. These were more susceptible than the parental strain to killing by various metal ions including iron, copper and manganese, whereas conditions of oxidative stress or iron deprivation were not discriminative. Analysis by element-specific electron microscopy revealed that growth of H. pylori in the presence of iron induces the formation of two kinds of cytoplasmic aggregates: large vacuole-like bodies and smaller granules containing iron in association with oxygen or phosphorus. Neither of these structures was detected in the pfr-deficient mutant strain. Furthermore, the ferritin accumulated under iron overload and the pfr-deficient mutant strains lacked expression of a 12 kDa protein which was negatively regulated by iron in the parental strain. The results indicate that the nonhaem-iron ferritin is involved in the formation of iron-containing subcellular structures and contributes to metal resistance of H. pylori. Further evidence for an interaction of ferritin with iron-dependent regulation mechanisms is provided.
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Close proximity of the tdh, trh and ure genes on the chromosome of Vibrio parahaemolyticus
The distribution and location of the virulence-factor genes of Vibrio parahaemolyticus, tdh and trh, and the structural gene of urease, ureC, were examined on the genomic DNAs of 115 clinical isolates of V. parahaemolyticus. The majority of strains (81%) had two copies of tdh on the chromosome, and no copies of trh or ure. Southern hybridization with a tdh probe, after pulsed-field gel electrophoresis of Noti-digested genomic DNA of each strain revealed only single bands, suggesting that the two copies of tdh exist on single Notl fragments in each strain. Of the 115 strains, 7% had the tdh, trh and ure genes on chromosomal DNA. The three genes were also detected on single Notl fragments in these strains. More detailed analysis revealed that the three genes were localized within 40 kb. By long and accurate polymerase chain reactions (LA-PCR), the distance between trh and ure was shown to be less than 8.5 kb. These results reveal a close proximity of the tdh, trh and ure genes on the chromosome of pathogenic V. parahaemolyticus strains.
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An in silico evaluation of Tn916 as a tool for generalized mutagenesis in Haemophilus influenzae Rd
The transposon Tn916 was evaluated as a tool for generalized mutagenesis of the genome of Haemophilus influenzae. This was achieved in silico by searching the genome sequence of H. influenzae Rd for the published Tn916 target site consensus sequence 5′ TT/ATTTT(N)6AAAAAA/TA. This search identified 16 putative target sites. In subsequent experiments, integration of Tn916 did not occur at any of these sites. Using the nucleotide sequences of these observed integration sites, a new consensus sequence, 5′ TTTTT(N)xAAAAA (4x7), was derived. This sequence reflects the curve-twist-curve DNA topology which is a feature common to all Tn916 integration sites. A search of the H. influenzae Rd genome using the new consensus sequence identified 167 potential target sites, representing approximately 1% of the total genome. Only 80 of these sites were located within ORFs. The presence of such a limited number of target sites places severe constraints on the use of Tn916 as a tool for generalized mutagenesis of the genome of H. influenzae.
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Construction of a single-copy integration vector and its use to study gene expression in Bacillus licheniformis
More LessA versatile system consisting of an integrational vector and a bacitracin (Bt)-producing ß-galactosidase (ß-Gal)-negative (Lac−) Bacillus licheniformis TLH strain was constructed to quantify promoter activity and to study gene regulation in a single-copy set-up. The vector pTLH utilizes the promoterless Escherichia coli lacZ gene derived from pQF52 and contains the pBR322 origin of replication and a kanamycin-resistance gene for selection in both B. licheniformis and E. coli. The vector also contains an inner part of the first gene of the Bt synthetase (bts) operon which enables its integration into the bts of B. licheniformis by Campbell-type recombination. This recombination event can be easily tested on a Micrococcus flavus lawn where loss of Bt production, i.e. no clearing zone on the lawn, is indicative of the proper integration. The Lac− B. licheniformis TLH strain was developed by elimination of the natural ß-Gal activity of B. licheniformis strain ATCC 10716 UM12 using NTG mutagenesis.
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Intracellular chitinase gene from Rhizopus oligosporus: molecular cloning and characterization
More LessMultiple chitinases have been found in hyphae of filamentous fungi, which are presumed to have various functions during hyphal growth. Here it is reported, for the first time, the primary structure of one such intracellular chitinase, named chitinase III, from Rhizopus oligosporus, a zygomycete filamentous fungus. Chitinase III was purified to homogeneity from actively growing mycelia of R. oligosporus using three steps of column chromatography. Its molecular mass was 43.5 kDa and the pH optimum was 6.0 when p-nitrophenyl N,N’,N"-β-D-triacetylchitotrioside was used as a substrate. Chitinase III also hydrolysed chromogenic derivatives of chitobiose, but had no N-acetylglucosaminidase activity. The gene encoding chitinase III (chi3) was cloned using PCR with degenerate oligonucleotide primers from the partial amino acid sequence of the enzyme. The deduced amino acid sequence of chi3 was similar to that of bacterial chitinases and chitinases from mycoparasitic fungi, such as Aphanocladium album and Trichoderma harzianum, but it had no potential secretory signal sequence in its amino terminus. Northern blot analysis showed that chi3 was transcribed during hyphal growth. These results suggest that chitinase III may function during morphogenesis in R. oligosporus.
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- Pathogenicity And Medical Microbiology
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Fluorescence in situ hybridization shows spatial distribution of as yet uncultured treponemes in biopsies from digital dermatitis lesions
Fluorescence in situ hybridization (FISH) was performed on sections of plastic-embedded tissue using 16S rRNA-directed oligonucleotide probes to visualize uncultured treponemes in skin biopsies of cows with digital dermatitis. Plastic as embedding material allowed sectioning of hard and soft tissue with a defined thickness, avoiding the risk of dragging bacteria into the tissue while sectioning. Furthermore, it provided a good signal-to-noise ratio. Using this method the spatial distribution of three different bacterial phylotypes was visualized simultaneously within the tissue. Whereas debris covering the ulcers contained a mixture of different micro-organisms, a layering of certain treponemal phylotypes was observed deeper in the epidermis. Confocal laser scanning microscopy and subsequent three-dimensional reconstruction of series of optical sections confirmed that the treponemes migrated intercellularly around the cells, most of them directed towards the dermis. In situ hybridization on tissue embedded in plastic proved to be a useful method to study mixed bacterial infections since it combines excellent histological conservation of tissue with identification of bacterial species by simultaneous use of probes labelled with different fluorescent dyes. This technique may have implications for in situ detection, identification and localization of microorganisms in veterinary as well as in human medicine.
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The role of environmental factors in the regulation of virulence-determinant expression in Staphylococcus aureus 8325-4
More LessStaphylococcus aureus is a major human pathogen, which produces a variety of virulence determinants. To study environmental regulation of virulence-determinant production, several transcriptional reporter gene fusions were constructed. Chromosomal fusions were made with the staphylococcal accessory regulator (sarA), α-haemolysin (hla), surface protein A (spa) and toxic-shock syndrome toxin-1 (tst) genes. The effect of many different environmental conditions on the expression of the fusions was examined. Expression of hla, tst and spa was strongly repressed in the presence of sodium chloride (1 M) or sucrose (20 mM), but sarA was relatively unaffected. The global regulator of expression of virulence-determinant genes, agr (accessory gene regulator) was not involved in the salt or sucrose repression. Novobiocin, a DNA gyrase inhibitor, did not significantly increase the expression of tst in wild-type or agr backgrounds and failed to relieve the salt suppression. Expression of tst was strongly stimulated in several low-metal environments, independently of agr, whilst spa levels were significantly reduced by EGTA. The complex, interactive role of environmental factors in the control of expression of the virulence determinants is discussed.
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