Showing papers on "Escherichia coli published in 1994"

Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes

Abstract: In Pseudomonas aeruginosa the LasR protein is required for activation of lasB and several other virulence genes. A diffusible signal molecule, the P. aeruginosa autoinducer (PAI), produced by the bacterial cell and released into the growth medium, is required for activity of LasR. By cloning a lasB::lacZ fusion and a lasR gene under control of the lac promoter in Escherichia coli, we have developed a quantitative bioassay for PAI. We have used this assay to follow the purification of PAI from cell-free culture supernatant fluids in which P. aeruginosa or E. coli containing the P. aeruginosa gene required for autoinducer synthesis, lasI, had been grown. Chemical analyses indicated the purified material was 3-oxo-N-(tetrahydro-2-oxo-3-furanyl)dodecanamide. To confirm this assignment, the compound was synthesized and the synthetic compound was shown to have chemical and biological properties identical to those of PAI purified from culture supernatant fluids. The elucidation of the PAI structure suggests therapeutic approaches toward control of P. aeruginosa infections.

Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA.

Abstract: In bacteria, genetic recombination is catalysed by RecA protein, the product of the recA gene. A human gene that shares homology with Escherichia coli recA (and its yeast homologue RAD51) has been cloned from a testis cDNA library, and its 37 kDa product (hRad51) purified to homogeneity. The human Rad51 protein binds to single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using a topological assay, we demonstrate that hRad51 underwinds duplex DNA, in a reaction dependent upon the presence of ATP or its non-hydrolysable analogue ATP gamma S. Complexes formed with single- and double-stranded DNA have been observed by electron microscopy following negative staining. With nicked duplex DNA, hRad51 forms helical nucleoprotein filaments which exhibit the striated appearance characteristic of RecA or yeast Rad51 filaments. Contour length measurements indicate that the DNA is underwound and extended within the nucleoprotein complex. In contrast to yeast Rad51 protein, human Rad51 forms filaments with single-stranded DNA in the presence of ATP/ATP gamma S. These resemble the inactive form of the RecA filament which is observed in the absence of a nucleotide cofactor.

The Cold-Shock Response--A Hot Topic

Abstract: The cold-shock response of Escherichia coli describes a specific pattern of gene expression in response to abrupt shifts to lower temperatures. This pattern includes the induction of cold-shock proteins, synthesis of proteins involved in transcription and translation, and repression of heat-shock proteins. The identified cold-shock proteins are involved in various cellular functions from supercoiling of DNA to initiation of translation. The major cold-shock protein, CspA, has high sequence similarity with three other E. coli proteins--CspB, CspC, and CspD. Using translational lacZ fusions, cspB was found to be cold-shock inducible at the level of transcription like cspA, while cspC and cspD were not. The Csp proteins, which share sequence similarity with other prokaryotic proteins and with the 'cold-shock domain' of eukaryotic Y-box proteins, may have a function in activating transcription or unwinding or masking RNA molecules. Because the cold-shock response can also be induced by the addition of certain inhibitors of translation, it has been proposed that the state of the ribosome is the physiological sensor for the induction. In addition to E. coli, cold-shock proteins have also been found in other prokaryotic and eukaryotic organisms.

Molecular characterization of an enterobacterial metallo beta-lactamase found in a clinical isolate of Serratia marcescens that shows imipenem resistance.

Abstract: A clinical isolate of Serratia marcescens (TN9106) produced a metallo beta-lactamase (IMP-1) which conferred resistance to imipenem and broad-spectrum beta-lactams. The blaIMP gene providing imipenem resistance was cloned and expressed in Escherichia coli HB101. The IMP-1 was purified from E. coli HB101 that harbors pSMBNU24 carrying blaIMP, and its apparent molecular mass was calculated to be about 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic studies of IMP-1 against various beta-lactams revealed that this enzyme hydrolyzes not only various broad-spectrum beta-lactams but also carbapenems. However, aztreonam was relatively stable against IMP-1. Although clavulanate or cloxacillin failed to inhibit IMP-1, Hg2+, Fe2+, or Cu2+ blocked the enzyme's activity. Moreover, the presence of EDTA in the reaction buffer resulted in a decrease in the enzyme's activity. Carbapenem resistance was not transferred from S. marcescens TN9106 to E. coli CSH2 by conjugation. A hybridization study confirmed that blaIMP was encoded on the chromosome of S. marcescens TN9106. By nucleotide sequencing analysis, blaIMP was found to encode a protein of 246 amino acid residues and was shown to have considerable homology to the metallo beta-lactamase genes of Bacillus cereus, Bacteroides fragilis, and Aeromonas hydrophila. The G+C content of blaIMP was 39.4%. Four consensus amino acid residues, His-95, His-97, Cys-176, and His-215, which form putative zinc ligands, were conserved in the deduced amino acid sequence of IMP-1. By determination of the amino acid sequence at the N terminus of purified mature IMP-1, 18 amino acid residues were found to be processed from the N terminus of the premature enzyme as a signal peptide. These results clearly show that IMP-1 is an enterobacterial metallo beta-lactamase, of which the primary structure has been completely determined, that confers resistance to carbapenems and other broad-spectrum beta-lactams.

Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH.

Abstract: Escherichia coli K-12 strains and Shigella flexneri grown to stationary phase can survive several hours at pH 2 to 3, which is considerably lower than the acid limit for growth (about pH 4.5). A 1.3-kb fragment cloned from S. flexneri conferred acid resistance on acid-sensitive E. coli HB101; sequence data identified the fragment as a homolog of rpoS, the growth phase-dependent sigma factor sigma 38. The clone also conferred acid resistance on S. flexneri rpoS::Tn10 but not on Salmonella typhimurium. E. coli and S. flexneri strains containing wild-type rpoS maintained greater internal pH in the face of a low external pH than strains lacking functional rpoS, but the ability to survive at low pH did not require maintenance of a high transmembrane pH difference. Aerobic stationary-phase cultures of E. coli MC4100 and S. flexneri 3136, grown initially at an external pH range of 5 to 8, were 100% acid resistant (surviving 2 h at pH 2.5). Aerobic log-phase cultures grown at pH 5.0 were acid resistant; survival decreased 10- to 100-fold as the pH of growth was increased to pH 8.0. Extended growth in log phase also decreased acid resistance substantially. Strains containing rpoS::Tn10 showed partial acid resistance when grown at pH 5 to stationary phase; log-phase cultures showed < 0.01% acid resistance. When grown anaerobically at low pH, however, the rpoS::Tn10 strains were acid resistant. E. coli MC4100 also showed resistance at alkaline pH outside the growth range (base resistance). Significant base resistance was observed up to pH 10.2. Base resistance was diminished by rpoS::Tn10 and by the presence of Na+. Base resistance was increased by an order of magnitude for stationary-phase cultures grown in moderate base (pH 8) compared with those grown in moderate acid (pH 5). Anaerobic growth partly restored base resistance in cultures grown at pH 5 but not in those grown at pH 8. Thus, both acid resistance and base resistance show dependence on growth pH and are regulated by rpoS under certain conditions. For acid resistance, and in part for base resistance, the rpoS requirement can be overcome by anaerobic growth in moderate acid.

A role for exopolysaccharides in the protection of microorganisms from desiccation.

Abstract: Mucoid strains of Escherichia coli, Acinetobacter calcoaceticus, and Erwinia stewartii were significantly more resistant to desiccation than corresponding isogenic nonmucoid mutants (survival rates of up to 35% in mucoid strains and between 0.7 and 5% in nonmucoid variants), even in colonies containing both cell types. Desiccation was found to bring about an induction of beta-galactosidase in Lon strains of E. coli K-12 carrying transcriptional lac fusions in the capsule biosynthetic (cps) regulon. This induction was dependent on the transcriptional activators RcsA and RcsB. Induction was lower in cells carrying mutations in the membrane sensor protein RcsC.

Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway

Abstract: Genes coding for poly-beta-hydroxybutyrate were removed from Alcaligenes eutrophus H16 and cloned into Escherichia coli. Some of the clones produced PHB to 90% of the cell weight.

Excision of large DNA regions termed pathogenicity islands from tRNA-specific loci in the chromosome of an Escherichia coli wild-type pathogen.

Abstract: Uropathogenic Escherichia coli 536 (O6:K15:H31) carries two unstable DNA regions, which were shown to be responsible for virulence. These regions, on which the genes for hemolysin production (hly) and P-related fimbriae (prf) are located, are termed pathogenicity islands (PAI) I and II, and were mapped to positions 82 and 97, respectively, on the E. coli K-12 linkage map. Sequence analysis of the PAI region junction sites revealed sequences of the leuX and selC loci specific for leucine and selenocysteine tRNAs. The tRNA loci function as the targets for excision events. Northern (RNA) blot analysis revealed that the sites of excision are transcriptionally active in the wild-type strain and that no tRNA-specific transcripts were found in the deletion mutant. The analysis of deletion mutants revealed that the excision of these regions is specific and involves direct repeats of 16 and 18 nucleotides, respectively, on both sides of the deletions. By using DNA long-range mapping techniques, the size of PAI I, located at position 82, was calculated to be 70 kb, while PAI II, mapped at position 97, comprises 190 kb. The excision events described here reflect the dynamics of the E. coli chromosome.

Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones

Abstract: A 4.6 kb Staphylococcus aureus DNA fragment containing DNA gyrase-like genes (grlA and grlB) was cloned and sequenced. The proteins GrlA and GrlB exhibit more than 30% identity with E. coli DNA topoisomerase IV subunits and with the gyrase subunits from S. aureus and Escherichia coli. The combined E. coli cell extracts of GrlA and GrlB overproducing strains catalysed ATP-dependent relaxation and decatenation specific to DNA topoisomerase IV. The temperature-sensitive phenotype of Salmonella typhimurium parC and parE mutants was complemented by the S. aureus grlA and grlB genes, when the two genes were co-expressed. These results show that GrlA and GrlB are the subunits of S. aureus DNA topoisomerase IV. The GyrA subunit of DNA gyrase has been previously defined as a primary target of quinolones based on genetic and biochemical experiments essentially carried out in E. coli. Single-point mutations occurring in the 'quinolone resistance-determining region' (QRDR) of GyrA were found in bacteria exhibiting quinolone resistance, the most common mutation being a substitution of Ser-83 on the E. coli GyrA sequence. We analysed eight S. aureus fluoroquinolone-resistant clinical isolates and observed that mutations in the QRDR of GyrA are not present in the low-quinolone-resistant isolates. In contrast, Ser-80 of GrlA, which corresponds to Ser-83 of E. coli GyrA, is substituted to Phe or Tyr in both high- and low-quinolone-resistant isolates. We propose that DNA topoisomerase IV is a primary target of fluoroquinolones in S. aureus.

Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments.

Abstract: Plasmids harboring multiple antimicrobial-resistance determinants (R plasmids) were transferred in simulated natural microenvironments from various bacterial pathogens of human, animal, or fish origin to susceptible strains isolated from a different ecological niche. R plasmids in a strain of the human pathogen Vibrio cholerae O1 E1 Tor and a bovine Escherichia coli strain were conjugated to a susceptible strain of the fish pathogenic bacterium Aeromonas salmonicida subsp. salmonicida in marine water. Conjugations of R plasmids between a resistant bovine pathogenic E. coli strain and a susceptible E. coli strain of human origin were performed on a hand towel contaminated with milk from a cow with mastitis. A similar conjugation event between a resistant porcine pathogenic E. coli strain of human origin was studied in minced meat on a cutting board. Conjugation of R plasmids between a resistant strain of the fish pathogenic bacterium A. salmonicida subsp. salmonicida and a susceptible E. coli strain of human origin was performed in raw salmon on a cutting board. R plasmids in a strain of A. salmonicida subsp. salmonicida and a human pathogenic E. coli strain were conjugated to a susceptible porcine E. coli strain in porcine feces. Transfer of the different R plasmids was confirmed by plasmid profile analyses and determination of the resistance pattern of the transconjugants. The different R plasmids were transferred equally well under simulated natural conditions and under controlled laboratory conditions, with median conjugation frequencies ranging from 3 x 10(-6) to 8 x 10(-3). The present study demonstrates that conjugation and transfer of R plasmids is a phenomenon that belongs to the environment and can occur between bacterial strains of human, animal, and fish origins that are unrelated either evolutionarily or ecologically even in the absence of antibiotics. Consequently, the contamination of the environment with bacterial pathogens resistant to antimicrobial agents is a real threat not only as a source of disease but also as a source from which R plasmids can easily spread to other pathogens of diverse origins.

A Swimming-Associated Outbreak of Hemorrhagic Colitis Caused by Escherichia coli O157:H7 and Shigella Sonnei

Abstract: Background In the summer of 1991, simultaneous outbreaks of bloody diarrhea and hemolytic-uremic syndrome caused by Escherichia coli O157:H7 and of bloody diarrhea caused by Shigella sonnei were traced to a lakeside park near Portland, Oregon Methods We identified cases primarily from routine surveillance reports In case-control studies, the activities of persons with park-associated E coli O157:H7 or S sonnei infections were compared independently with those of three sets of controls We also evaluated environmental conditions at the park and subtyped the bacterial isolates Results We identified 21 persons with park-associated E coli O157:H7 infections (all of them children; median age, six years) and 38 persons with S sonnei infections (most of them children) These 59 people had visited the park over a 24-day period Their illnesses were not associated with food or beverage consumption All the case patients reported swimming, however, and in case-control studies swimming was strongly associated

The FlhD/FlhC complex, a transcriptional activator of the Escherichia coli flagellar class II operons.

Abstract: The Escherichia coli flhD operon encodes two genes, flhD and flhC. Both gene products were overproduced and purified. The purified proteins formed a complex consisting of two FlhD and two FlhC molecules. Mobility shift assays showed that the FlhD/FlhC complex had a DNA-binding activity and bound to the upstream regions of fliA, flhB, and fliL operons (class II), which are under direct control of the flhD operon. DNase I footprinting analyses of FlhD/FlhC binding to the three class II promoter regions revealed protection of a 48-bp region of the fliA operon between positions -41 to -88, a 50-bp region of the flhB operon between positions -28 to -77, and a 48-bp region of the fliL operon between positions -29 to -76. In vitro transcription experiments demonstrated that the FlhD/FlhC complex is a transcriptional activator required for the transcription of the three class II operons examined in vitro.

Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster

Abstract: Escherichia coli K-12 has long been known not to produce an O antigen. We recently identified two independent mutations in different lineages of K-12 which had led to loss of O antigen synthesis (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994) and constructed a strain with all rfb (O antigen) genes intact which synthesized a variant of O antigen O16, giving cross-reaction with anti-O17 antibody. We determined the structure of this O antigen to be -->2)-beta-D-Galf-(1-->6)-alpha-D-Glcp- (1-->3)-alpha-L-Rhap-(1-->3)-alpha-D-GlcpNAc-(1-->, with an O-acetyl group on C-2 of the rhamnose and a side chain alpha-D-Glcp on C-6 of GlcNAc. O antigen synthesis is rfe dependent, and D-GlcpNAc is the first sugar of the biological repeat unit. We sequenced the rfb (O antigen) gene cluster and found 11 open reading frames. Four rhamnose pathway genes are identified by similarity to those of other strains, the rhamnose transferase gene is identified by assay of its product, and the identities of other genes are predicted with various degrees of confidence. We interpret earlier observations on interaction between the rfb region of Escherichia coli K-12 and those of E. coli O4 and E. coli Flexneri. All K-12 rfb genes were of low G+C content for E. coli. The rhamnose pathway genes were similar in sequence to those of (Shigella) Dysenteriae 1 and Flexneri, but the other genes showed distant or no similarity. We suggest that the K-12 gene cluster is a member of a family of rfb gene clusters, including those of Dysenteriae 1 and Flexneri, which evolved outside E. coli and was acquired by lateral gene transfer.

Escherichia coli in Domestic Animals and Humans

Abstract: This book presents an authoritative and comprehensive review of disease processes and diseases caused by the bacterium Escherichia coli. It is divided into four parts covering: characteristics of E. coli; diseases caused by E. coli; virulence factors of E. coli; and diagnosis and prevention of diseases caused by E. coli. It represents a reference work for veterinary and medical microbiologists.

Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.

Abstract: A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced. This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and zeta-carotene. The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic beta-carotene, an essential component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition. The enzyme also converts neurosporene to the monocyclic beta-zeacarotene but does not cyclize zeta-carotene, indicating that desaturation of the 7-8 or 79-89 carbon-carbon bond is required for cyclization. The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions. A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL. The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 411 amino acids with a molecular weight of 46,125 and a pI of 6.0. An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide. DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942. Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria. Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and algal genes encoding this enzyme.

A comparison of immunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli 0157 from bovine faeces

Abstract: Summary Enrichment culture (EC) in modified buffered peptone water followed by immunomagnetic separation (IMS) with magnetic beads coated with an antibody against Escherichia coli 0157 (Dynabeads anti-E. coli 0157; Dynal, Oslo) was compared with direct culture on cefixime rhamnose sorbitol MacConkey agar (CR-SMAC) and cefixime tellurite sorbitol MacConkey agar (CT-SMAC) for the isolation of E. coli 0157 from bovine faeces. When used to examine bovine faecal suspensions inoculated with 12 different strains of E. coli 0157, EC-IMS was c. 100-fold more sensitive for detection of the organism than direct culture on either medium. During monitoring of a dairy herd, E. coli 0157 was isolated from 84 (8.2 %) of 1024 rectal swabs taken from cattle over a 4-month period; 23 (27.4% of the 84 strains were isolated by both direct culture and IMS (15 of the 23 were isolated on both media, five on CT-SMAC only and three on CR-SMAC only), whereas 61 (72.6%) strains were isolated by IMS only. IMS is a sensitive and simple technique for the isolation of E. coli 0157 from bovine faecal samples and should prove useful in elucidating further the epidemiology of this organism.

Neisseria gonorrhoeae acquires mutations in analogous regions of gyrA and parC in fluoroquinolone‐resistant isolates

Abstract: Neisseria gonorrhoeae homologues of gyrA and parC have been identified using hybridization probes generated from conserved regions of diverse gyrA genes. These genes have been tentatively identified as gyrA and parC, based on predicted amino acid sequence homologies to known GyrA homologues from numerous bacterial species and to ParC from Escherichia coli and Salmonella typhimurium. The gyrA gene maps to a physical location distant from the gyrB locus on the gonococcal chromosome, which is similar to the situation found in E. coli. The parC gene is not closely linked (i.e. greater than 9 kb) to an identifiable parE gene in N. gonorrhoeae. The gonococcal GyrA is slightly larger than its E. coli homologue and contains several small insertions near the C-terminus of the predicted open reading frame. A series of ciprofloxacin-resistant mutants were selected by passage of N. gonorrhoeae on increasing concentrations of the antibiotic. Sequential passage resulted in the selection of isolates with minimum inhibitory concentrations approximately 10,000-fold higher than the parental strain. Mutations within gyrA resulted in low to moderate levels of resistance, while strains with high-level resistance acquired analogous mutations in both gyrA and parC. Resistance mutations were readily transferred between N. gonorrhoeae strains by transformation. The frequencies of transformation, resulting in different levels of ciprofloxacin resistance, further support the notion that both gyrA and parC genes are involved in the establishment of extreme levels of ciprofloxacin resistance.

Evolutionary conservation of components of the protein translocation complex

Abstract: PROTEIN translocation into the mammalian endoplasmic reticulum requires the Sec61p complex, which consists of three membrane proteins1. The α-subunit, the homologue of Sec61p of yeast2–4, shows some similarity to SecYp5, a key component of the protein export apparatus of bacteria6,7. In Escherichia coli, SecYp is also associated with two other proteins (SecEp and band-1 protein)8,9. We have now determined the sequences of the β- and γ-subunits of the mammalian Sec61p complex. Sec61-γ is homologous to SSSlp, a suppressor of sec61 mutants in Saccharomyces cerevisiae, and can functionally replace it in yeast cells. Moreover, Sec61-γ and SSSlp are structurally related to SecEp of E. coli and to putative homologues in various other bacteria. At least two sub-units of the Sec61/SecYp complex therefore seem to be key components of the protein translocation apparatus in all classes of organisms.

Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases.

Abstract: The deamination of adenine residues in DNA generates hypoxanthine, which is mutagenic since it gives rise to an A.T to G.C transition. Hypoxanthine is removed by hypoxanthine DNA glycosylase activity present in Escherichia coli and mammalian cells. Using polydeoxyribonucleotides or double-stranded synthetic oligonucleotides that contain dIMP residues, we show that this activity in E. coli is associated with the 3-methyladenine DNA glycosylase II coded for by the alkA gene. This conclusion is based on the following facts: (i) the two enzymatic activities have the same chromatographic behavior on various supports and they have the same molecular weight, (ii) both are induced during the adaptive response, (iii) a multicopy plasmid bearing the alkA gene overproduces both activities, (iv) homogeneous preparation of AlkA has both enzymatic activities, (v) the E. coli alkA- mutant does not show any detectable hypoxanthine DNA glycosylase activity. Under the same experimental conditions, but using different substrates, the same amount of AlkA protein liberates 1 pmol of 3-methyladenine from alkylated DNA and 1.2 fmol of hypoxanthine from dIMP-containing DNA. The Km for the latter substrate is 420 x 10(-9) M as compared to 5 x 10(-9) M for alkylated DNA. Hypoxanthine is released as a free base during the reaction. Duplex oligodeoxynucleotides containing hypoxanthine positioned opposite T, G, C, and A were cleaved efficiently. ANPG protein, APDG protein, and MAG protein--the 3-methyladenine DNA glycosylases of human, rat, and yeast origin, respectively--were also able to release hypoxanthine from various DNA substrates containing dIMP residues. The mammalian enzyme is by far the most efficient hypoxanthine DNA glycosylase of all the enzymes tested.

Expression of G-protein alpha subunits in Escherichia coli.

Abstract: Publisher Summary This chapter describes a general method for expressing several G-protein α subunits in Escherichia coli (E. coli) at levels 10-100 times higher than achieved previously. G proteins are a family of guanine nucleotide-binding regulatory proteins that link a large number of cell surface receptors to regulation of several intracellular effectors. The chapter describes a method for purification of the recombinant proteins by affinity chromatography on a resin containing chelated Ni2+ after addition of an amino-terminal hexahistidine tag to the recombinant protein. Such purification is rapid and results in the isolation of highly purified protein in a single step. Furthermore, the introduction of a tobacco etch virus (TEV) polyprotein cleavage site between the hexahistidine tag and the G-protein α subunit permits the efficient removal of the tag by recombinant TEV protease.

Characterization of the desulfurization genes from Rhodococcus sp. strain IGTS8.

Abstract: Rhodococcus sp. strain IGTS8 possesses an enzymatic pathway that can remove covalently bound sulfur from dibenzothiophene (DBT) without breaking carbon-carbon bonds. The DNA sequence of a 4.0-kb BstBI-BsiWI fragment that carries the genes for this pathway was determined. Frameshift and deletion mutations established that three open reading frames were required for DBT desulfurization, and the genes were designated soxABC (for sulfur oxidation). Each sox gene was subcloned independently and expressed in Escherichia coli MZ1 under control of the inducible lambda pL promoter with a lambda cII ribosomal binding site. SoxC is an approximately 45-kDa protein that oxidizes DBT to DBT-5,5'-dioxide. SoxA is an approximately 50-kDa protein responsible for metabolizing DBT-5,5'-dioxide to an unidentified intermediate. SoxB is an approximately 40-kDa protein that, together with the SoxA protein, completes the desulfurization of DBT-5,5'-dioxide to 2-hydroxybiphenyl. Protein sequence comparisons revealed that the predicted SoxC protein is similar to members of the acyl coenzyme A dehydrogenase family but that the SoxA and SoxB proteins have no significant identities to other known proteins. The sox genes are plasmidborne and appear to be expressed as an operon in Rhodococcus sp. strain IGTS8 and in E. coli.

The rpoB gene of Mycobacterium tuberculosis.

Abstract: A portion of the Mycobacterium tuberculosis gene encoding the beta subunit of RNA polymerase (rpoB) was amplified by PCR using degenerate oligonucleotides and used as a hybridization probe to isolate plasmid clones carrying the entire rpoB gene of M. tuberculosis H37Rv, a virulent, rifampin-susceptible strain. Sequence analysis of a 5,084-bp SacI genomic DNA fragment revealed a 3,534-bp open reading frame encoding an 1,178-amino-acid protein with 57% identity with the Escherichia coli beta subunit. This SacI fragment also carried a portion of the rpoC gene located 43 bp downstream from the 3' end of the rpoB open reading frame; this organization is similar to that of the rpoBC operon of E. coli. The M. tuberculosis rpoB gene was cloned into the shuttle plasmid pMV261 and electroporated into the LR223 strain of Mycobacterium smegmatis, which is highly resistant to rifampin (MIC > 200 micrograms/ml). The resulting transformants were relatively rifampin susceptible (MIC = 50 micrograms/ml). Using PCR mutagenesis techniques, we introduced a specific rpoB point mutation (associated with clinical strains of rifampin-resistant M. tuberculosis) into the cloned M. tuberculosis rpoB gene and expressed this altered gene in the LR222 strain of M. smegmatis, which is susceptible to rifampin (MIC = 25 micrograms/ml). The resulting transformants were rifampin resistant (MIC = 200 micrograms/ml). The mutagenesis and expression strategy of the cloned M. tuberculosis rpoB gene that we have employed in this study will allow us to determine the rpoB mutations that are responsible for rifampin resistance in M. tuberculosis.

Cloning and sequencing of the genes encoding Escherichia coli cytolethal distending toxin.

Abstract: Escherichia coli strains expressing cytolethal distending toxin (CDT) cause elongation of CHO cells at 24 h, followed by progressive cellular distention and death for up to 120 h. Similar distention and cytotoxicity are seen in HeLa, HEp-2, and, to a lesser extent, Vero cells. The initial elongation in CHO cells is indistinguishable from that caused by E. coli heat-labile toxin (LT). In contrast to those from LT strains, supernatants from these strains have no effect on Y-1 adrenal cells. TnphoA was introduced into CDT-positive E. coli E6468/62 (O86:H34), isolated from a child with diarrhea, and 13 CDT-negative transconjugants were identified. DNA probes constructed from DNA flanking the TnphoA insertion sites of CDT-negative mutants were used to identify a CDT-positive clone from an E6468/62 genomic library with a 5.5-kb insert. Exonuclease deletions were created and assayed in CHO cells. In this manner, a 2.3-kb CDT-active region was defined, and the nucleotide sequence was determined. Sequence analysis identified three open reading frames (ORFs), designated cdtA, cdtB, and cdtC. These contain 711, 819, and 570 bp, respectively, and encode polypeptides with predicted molecular masses of 25.5, 29.8, and 20.3 kDa, respectively. Each ORF has a putative signal sequence, and there are 4-bp overlaps between cdtA and cdtB and between cdtB and cdtC. The nucleotide and predicted amino acid sequences have no significant homology with those of any previously reported genes or proteins. By in vitro transcription-translation and an anti-alkaline phosphatase immunoblot, native proteins and/or fusion proteins corresponding to each ORF were identified.

Isolation and characterization of a gene involved in hemagglutination by an avian pathogenic Escherichia coli strain.

Abstract: In this article, we report the isolation and characterization of a gene that may be important in the adherence of avian pathogenic Escherichia coli to the avian respiratory tract. The E. coli strain HB101, which is unable to agglutinate chicken erythrocytes, was transduced with cosmid libraries from the avian pathogenic E. coli strain chi 7122. Enrichment of transductants that could agglutinate chicken erythrocytes yielded 19 colonies. These isolates contained cosmids that encompassed four nonoverlapping regions of the E. coli chromosome. Only one group of cosmids, represented by pYA3104, would cause E. coli CC118 to agglutinate chicken erythrocytes. A 10-kb fragment of this cosmid was subcloned in pACYC184. Transposon mutagenesis of this fragment with Tn5seq1 indicated that a contiguous 4.4-kb region of cloned DNA was required for hemagglutination. In vitro transcription/translation assays indicated that this 4.4-kb region of DNA encoded one protein of approximately 140 kDa. The nucleotide sequence of this region was determined and found to encode one open reading frame of 4,134 nucleotides that would encode a protein of 1,377 amino acids with a deduced molecular weight of 148,226. This gene confers on E. coli K-12 a temperature-sensitive hemagglutination phenotype that is best expressed when cells are grown at 26 degrees C, and we have designated this gene tsh and the deduced gene product Tsh. Insertional mutagenesis of the chromosomal tsh gene in chi 7122 had no effect on hemagglutination titers. The deduced protein was found to contain significant homology to the Haemophilus influenzae and Neisseria gonorrhoeae immunoglobulin A1 proteases. These data indicate that (i) a single gene isolated from the avian pathogenic E. coli strain chi 7122 will confer on E. coli K-12 a hemagglutination-positive phenotype, (ii) chi 7122 contains at least two distinct mechanisms to allow hemagglutination to occur, and (iii) the hemagglutinin Tsh has homology with a class of proteins previously not known to exist in E. coli.

Vero cytotoxin‐producing Escherichia coli O157 in beefburgers linked to an outbreak of diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome in Britain

Abstract: Vero cytotoxin (VT)-producing Escherichia coli O157 (0157 VTEC) were isolated from a raw beefburger obtained from a retail source linked to a small community outbreak of 0157 VTEC infection in Wales. Strains from the meat and from seven of eight patients belonged to phage type 49 and were indistinguishable by their VT-type, plasmid content and hybridization with DNA of a VT-encoding phage from an 0157 VTEC strain. This first report of the isolation of 0157 VTEC from a beef product in Britain supports the view that there is a bovine reservoir for this organism.