摘要:

The study of pathogenic is often limited toex vivoassays and cell‐culture correlates. A greater understanding of infectious diseases would be facilitated byin vivoanalyses. Therefore, we have developed a method for detecting bacterial pathogens in a living host and used this method to evaluate disease processes for strains ofSalmonella typhimuriumthat differ in their virulence for mice. Three strains ofSalmonellawere marked with bioluminescence through transformation with a plasmid conferring constitutive expression of bacterial luciferase. Detection of photons transmitted through tissues of animals infected with bioluminescentSalmonellaallowed localization of the bacteria to specific tissues. In this manner progressive infections were distinguished from those that were persistent or abortive. We observed patterns of bio‐luminescence that suggested the caecum may play a pivotal role inSalmonellapathogenesis.In vivo efficacy of an antibiotic was monitored using this optical method. This study demonstrates that the real time non‐invasive analyses of pathogenic events and pharmacological monitoring can be performed in

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18040593.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

The upstream activation sequence (UAS) in theSaccharomyces cerevisiaeactin gene promoter contains three different motifs, specifically two AT‐rich tracts, two binding sites for the yeast protein REB1, and anMlul site. Synthetic UAS elements containing individual motifs, or combinations of them, were inserted in place of the natural UAS, and assayed using alacZreporter gene. The REB1 binding sites were found to be essential for, and sufficient to restore partial, UAS activity. AT‐rich tracts alone were inactive. Multimerization of a REB1 binding site created a UAS that in galactose is more active, but in glucose less active, than a UAS having a single REB1 site with one AT‐rich tract. In general, transcription during growth in galactose or glycerol/lactate responds more to multimerization of motifs. The results suggest that the natural actin promoter UAS retains activity on these alternative carbon sources because of reiteration of sequence elements within it; the additional elements appear to be redundant when cells are grown on glucose. Themlul site, which is present upstream of a number of yeast genes involved in DNA synthesis and confers cell cycle periodicity to those genes, contributes to the activity of the synthetic UAS elements, but not in a cell‐cycle‐depende

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18040605.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Expression of the lactose operon upon induction by IPTG was studied withEscherichia coliB/r and K‐12 strains as a function of exposure to ultraviolet light. Patterns of expression inactivation were compared in cells with wild‐type UvrABC nucleotide excision repair, with transcription‐coupled excision repair (TCR) specifically defective because of a defect atmfd, or with excision repair (ER) and TCR eliminated by defects atuvrAoruvrC.Sets of inactivation patterns were also determined for cells expressing the lactose operon via the ‘UV5’ promoter, an alternative to the wild‐type promoter that eliminates dependence of expression on negative DNA supercoiling. The results demonstrated a major contribution by TCR to successful gene expression. Gene expression was more sensitive to u.v. inactivation when TCR was defective and similarly more sensitive when both ER and TCR were defective. Thus, TCR may be the only means of repairing transcription‐blocking damage at active genes. Contrasting results with wild‐type and UV5 promoters suggested that relaxed supercoiling might accompany repair and reduce expression even though a template lesion is removed. A test of mismatch repair defects on ultraviolet inactivation of gene expression found only limited interference with TCR as it benefits

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18040615.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Diphtheria toxin enters toxin‐sensitive mammalian cells by receptor‐mediated endocytosis employing the heparin‐binding EGF‐like growth factor precursor as its receptor. We reported previously (Almond and Eidels, 1994) that cytoplasmic domain mutants of the toxin receptor and cells expressing wild‐type receptor internalize toxin slowly, the rate being approximately that of normal turnover of the plasma membrane. To determine whether it was possible to increase toxin sensitivity by increasing the rate of toxin internalization, we constructed diphtheria toxin cytoplasmic domain mutant cell lines containing rapid‐internalization signals from either the low density lipoprotein receptor or from the lysosomal acid phosphatase precursor. Although cells transfected with mutant receptor genes internalized toxin at a faster rate than those expressing the wild‐type receptor, they showed a decrease in toxin sensitivity. This decreased sensitivity may be accounted for by an observed decrease in the number of toxin‐binding sites and by an increased rate of toxin internalization and degradation. These results suggest that the rate of toxin internalization may not be the rate‐limiting step in the

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18040623.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

In this study, we show that bacteriocin production inLactobacillus plantarumC11 is an inducible process triggered by a secreted protein factor produced by the bacteriocin producer itself. The induction factor was identified to be plantaricin A, a bacteriocin‐like peptide whose gene (plnA) is located in the same operon as a two‐component regulatory system (plnBCD). WhenL. plantarumC11 cultures were depleted for plantaricin A, either by growing individual colonies on agar plates or by starting a new culture with a highly diluted inoculum, no bacteriocin was produced during the following growth. When chemically synthesized plantaricin A or purified bacterially produced plantaricin A was added to non‐producing cultures, bacteriocin production was induced. Only 1 ng ml−1plantaricin A is sufficient to induce the bacteriocin production in non‐producingL. plantarumC11, and bacteriocin activity appears in the growth medium approximately 150 min after induction. Northern analyses, using aplnA‐specific probe, demonstrated that plantaricin A is able to induce its own synthesis by transcription of theplnABCDoperon, and this is observed approximately 15 min after adding plantaricin A. Furthermore, heterologous expression of theplnABCDoperon in aLactobacillus sakestrain showed that the conditioned growth medium contained the active induction factor. Neither synthetic nor expressed plantaricin A from the heterologous system possesses any bacteriocin activity, suggesting that plantaricin A is primarily an induction factor and not a bacteriocin as clai

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18040631.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Rhodobacter sphaeroidesis chemotactic to glutamate and most other amino acids. InEscherichia coli, chemotaxis involves a membrane‐bound sensor that either binds the amino acid directly or interacts with the binding protein loaded with the amino acid. InR. sphaeroides, chemotaxis is thought to require both the uptake and the metabolism of the amino acid. Glutamate is accumulated by the cells via a binding protein‐dependent system. To determine the role of the binding protein and transport in glutamate taxis, mutants were created by Tn5insertion mutagenesis and selected for growth in the presence of the toxic glutamine analogue γ‐glutamyl‐hydrazide. One of the mutants,R. sphaeroidesMJ7, was defective in glutamate uptake but showed wild‐type levels of binding protein. The mutant showed no chemotactic response to glutamate. Both glutamate uptake and chemotaxis were recovered when thegltPgene, coding for the H+‐linked glutamate carrier ofE. coli, was expressed inR. sphaeroidesMJ7. It is concluded that the chemotactic response to glutamate strictly requires uptake of glutamate, supporting the view that intracellular metabolism is needed for chemotaxis inR.

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18040641.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Mycoplasma hyorhiniscontains clusteredvlpgenes encodingvariablelipoproteins (Vlps), the major coat proteins and surface antigens of this wall‐less prokaryotic pathogen.vlpgenes are subject to discrete, high frequency mutations independently affecting the size or the expression of variant Vlp products. Change in Vlp size occurs by mutations altering the number of tandem intragenic repeats at the 3′ end of each single‐copyvlpgene. In this report, phase‐variant Vlp expression is shown to result from alteredvlpgene transcription.vlpA, vlpBandvlpCtranscripts were monitored in a clonal lineage selected to display various Vlp phenotypes. Eachvlpgene was expressed as a distinct transcript, which was subject to drastic ON/OFF switches associated with random insertion/ deletion mutations in a homopolymeric tract of adenine residues in the promoter region of allvlpgenes. Unexpectedly, the level ofvlptranscripts appeared to depend on the length of the corresponding genes in the ON configuration. Higher proportional levels of shortervlptranscripts were shown to reflect a greater abundance of short Vlp lipoproteins present inl‐[35S]‐cysteine‐labelled membrane protein preparations. Thevlpcluster provides a heritable, and highly mutable, locus for the generation of surface diversity through random promoter mutations affecting the expression of genes, whose products also vary in length and abundance, by virtue of separate mutations in structural regions

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18040649.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Two divergently transcribed operons inEscherichia colirequired for the expression of fibronectin‐ and Congo red‐binding curli polymers were identified and characterized by transposon mutagenesis, sequencing and transcriptional analyses, as well as for their ability to produce the curli subunit protein. ThecsgBAoperon encodes CsgA, the major subunit protein of the fibre, and CsgB, a protein with sequence homology to CsgA. A non‐polarcsgBmutant is unaffected in its production of CsgA, but the subunit protein is not assembled into insoluble fibre polymers. A third open reading frame,orfC, positioned downstream ofcsgAmay affect some functional property of curli since an insertion in this putative gene abolishes the autoagglutinating ability typical of curliated cells without affecting the production of the fibre. The promoter for the oppositely transcribedcsgDEFGoperon was identified by primer extension and shown, like thecsgBApromoter, to be dependent upon the alternate stationary phase‐specific sigma factor σsin wild‐type cells, but not in mutants lacking the nucleoid associated protein H‐NS. Insertions incsgDabolish completely trancription from thecsgBApromoter. Therefore, any regulatory effect on thecsgBApromoter might be secondary to events controlling thecsgDEFGpromoter and/or activation of CsgD. Insertions incsgE, csgFandcsgGabolish curli formation but allow CsgA expression suggesting that one or more of these gene products are involved in secretion/assembly of the CsgA subunit protein. No amino acid sequence homologies were found between the CsgE, CsgF and CsgG proteins and secretion/assembly proteins for other known bacterial fibres, suggesting that the formation of curli follows a n

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18040661.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

A complete understanding of host‐parasite interactions must necessarily include the identification and characterization of gene products expressed by both parties during the infectious process. We have developed a new screen to identify bacterial genes that are transcriptionally induced during infection of a host animal. The method is based on pre‐selection of strains carryingtnpRoperon fusions (encoding resolvase, a site‐specific DNA recombinase) which are not expressedin vitro, followed by screening for a subset of these strains that subsequently express resolvase within the host environment. The latter subset was recognized as recombinants that had deleted a resolvase‐specific reporter construct. Thirteen transcription units ofVibrio choleraewere identified that were induced during infection in an infant mouse model of cholera. Five of these were predicted to encode polypeptides with diverse functions in metabolism, biosynthesis and motility; one encoded a secreted lipase; two appear to be antisense to genes involved in motility; and five are predicted to encode polypeptides of unknown function. Three of the transcripts were shown to be required for full virulence in infant mice, as assessed by competition expe

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18040671.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Temperate bacteriophage K139 was isolated from aVibrio choleraeO139 isolate and characterized in this study. The phage genome consists of a 35 kbp, double‐stranded, linear DNA molecule that circularizes and integrates into the chromosome in a site‐specific manner. DNA sequences that cross‐hybridize with K139 phage DNA are present in all strains ofV. choleraeserogroup O1 of the classical biotype examined and in some strains of the El Tor biotype. Phage K139 produces plaques on El Tor O1 strains that do not carry the K139‐related sequences but does not plaque on O139 strains that lack detectable phage DNA. This results suggests that O139 strains arose in part by horizontal gene transfer of the O139 antigen genes into an El Tor O1 strain that harboured a K139 prophage. Consistent with this interpretation, the morphology of K139 phage particles is identical to that displayed by the widely distributed family of O1 phages referred to as ‘kappa’. In order to test whether K139 phage is involved in lysogenic conversion ofV. cholerae, we constructed a novel mini‐transposon, Tn10d‐bla, which was designed to produce β‐lactamase fusions to phage‐encoded, exported proteins. All Tn10d‐blainsertions obtained were closely linked to one location on the K139 phage genome. DNA sequence determination of the fusion joints revealed an open reading frame (ORF1), encoding a gene product of 137 amino acids with a typical N‐terminal hydrophobic signal sequence. ORF1 was designated theglogene (Gprotein‐likeORF) because its amino acid sequence shows similarity to eukaryotic Gs(α) protein (34.5% identity over an 81‐amino‐acid overlap) and its C‐terminus displays the consensus motif (CAAX) which is found in many small eukaryotic GTP‐binding proteins. LD50assays with isogenic Glo+and Glo−K139 lysogens suggest thatgloencodes a secret

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18040685.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY