Cloning and expression of certain herpes simplex virus type 1 genes in Escherichia coli

Complete genes IE12, IE63, IE68, IE175, UL19, UL29 of herpes simplex virus type I or their fragments have been cloned in Escherichia coli cells. The peptides expressed were shown to be fused with cro-beta-galactosidase proteins. The recombinant proteins containing amino acid sequences of ICP4, ICP27 and ICP47, major capsid protein, and major DNA-binding protein react in immunoblotting with the anti-HSVI serum from hyperimmune rabbit. The recombinant proteins can be used for creation of diagnosticums and other scientific and practical purposes. The immunological properties of the recombinant proteins are being investigated.     

Large plasmids of avian Escherichia coli isolates

The plasmid DNA of 30 Escherichia coli isolates from chickens was extracted and examined using techniques designed to isolate large plasmids. This plasmid DNA was examined for the presence of certain known virulence-related genes including cvaC, traT, and some aerobactin-related sequences. Seventeen of the 30 isolates contained from one to four plasmids greater than 50 kb in size. Eleven of these 17 strains possessed plasmids greater than 100 kb in size. Therefore, E. coli isolates of chickens frequently contain large plasmids, and many of these plasmids are likely to contain virulence-related sequences.     

Small cryptic plasmids of multiplasmid, clinical Escherichia coli

Clinical isolates of Escherichia coli were found to host a multiplicity of plasmids. These were resolved from plasmid gel profiles, from the properties of various transconjugants and transformants of E. coli DH1, by the topoisomerase I relaxation of covalently closed circle plasmid DNA, by electron microscopy, and by the determination of their compatibilities. The majority of these were unusually small, cryptic plasmids (SCPs). From one strain, KL4, 13 electrophoretic bands were resolved to five plasmids, three of which were SCPs. SCPs were phenotypically barren, and the smallest of these, pKL1, contained barely enough information for self-replication. A derivative of pKL1, pKL1Km, in which the transposon was restricted to a small 350-bp region, was stably maintained in Shigella, Salmonella, Serratia, and Citrobacter species and its replication was polA independent. pKL1 encoded only a single protein, RepA (Mr 17960), which specifically bound to pKL1 DNA. No apparent homologies with other RepA protein sequences could be detected. Thus the SCP, pKL1, is a novel minimal plasmid replicon encoding only enough information to ensure perpetuation. A hypothesis is presented describing SCPs as a class of selfish DNA that persists simply due to its ability to replicate and to its stability based on high copy number.     

Purification of Escherichia coli chromosomal segments without cloning

Pairs of genomic insertions made with elements carrying any one of several frequently used rare restriction sites allow physical purification of insertion delimited genes. However, native rare restriction sites can, either by causing (i) fragmentation of targeted intervals or (ii) generation of additional fragments that overlap electrophoretically with targeted ones, place severe limitations on this approach. We present a series of Escherichia coli mini-Tn10 insertions containing the rare-cutting polylinker 2 (RCP2) of rare restriction sites, which includes the 18-base-pair I-SceI site (absent from native E. coli sequences). Pulsed-field gel purification from RCP2 double insertion mutants of both an I-SceI fragment from strain K-12 (containing approximately 90-95 min) and an allelic I-SceI fragment from a pathogenic strain is demonstrated. The complete series of RCP2 insertions, containing different antibiotic resistances at intervals of approximately 35 kb in prototype K-12 strain MG1655, allows rapid purification of the genes from any E. coli chromosomal interval as an isolated I-SceI fragment.     

Transfer of plasmids between strains of Escherichia coli under rumen conditions

Strains of Escherichia coli originally isolated from the rumen of sheep were shown to be capable of exchanging a 60kb plasmid, conferring resistance to tetracycline and ampicillin, at low frequencies (below 10(-6) per recipient) under anaerobic conditions in the presence of (a) autoclaved and clarified rumen fluid, (b) raw clarified rumen fluid, or (c) whole rumen fluid. Under anaerobic conditions the two rumen strains showed no inhibition of growth rate when 50 mmol l-1 volatile fatty acids were added to LB medium at pH 7, although significant inhibition resulted with 100 mmol l-1 VFA. The two rumen strains, and four strains from the pig gut, showed less inhibition of anaerobic growth by volatile fatty acids than did three laboratory strains examined for comparison. These findings indicate that plasmid transfer between certain E. coli strains can occur under conditions that closely simulate an anaerobic but environment.     

Protein expression in response to folate stress in Escherichia coli

Interruption of folate metabolism by trimethoprim results in the elevated expression of folate stress proteins in Escherichia coli. E. coli grown in culture medium supplemented with the folate-dependent metabolites glycine, methionine, and the purine nucleoside inosine shows reduced expression of folate stress proteins. The folate stress proteins include the universal stress protein, the ferric uptake regulatory repressor, and possibly, lipoamide dehydrogenase, the L protein component of the glycine cleavage enzyme complex.     

Genetic control of the resistance to phage C1 of Escherichia coli K-12

Escherichia coli K-12 lytic phage C1 was earlier isolated in our laboratory. Its adsorption is controlled by at least three bacterial genes: dcrA, dcrB, and btuB. Our results provide evidence that the dcrA gene located at 60 min on the E. coli genetic map is identical to the sdaC gene. This gene product is an inner membrane protein recently identified as a putative specific serine transporter. The dcrB gene, located at 76.5 min, encodes a 20-kDa processed periplasmic protein, as determined by maxicell analysis, and corresponds to a recently determined open reading frame with a previously unknown function. The btuB gene product is known to be an outer membrane receptor protein responsible for adsorption of BF23 phage and vitamin B12 uptake. According to our data the DcrA and DcrB proteins are not involved in these processes. However, the DcrA protein probably participates in some cell division steps.     

Escherichia coli genes expressed preferentially in an aquatic environment

We isolated genomic clones that contain the 5'-flanking region of the mouse activin beta A subunit gene. The nucleotide sequence determination of the 5'-flanking region of the gene and the comparison of that with the reported mouse cDNA structure identified the putative 5' regulatory region, a novel first exon and a part of the first intron of the gene within this region. The putative 5' regulatory region of the mouse activin beta A subunit gene directed the expression of CAT gene in transfected HT1080 cells. Successive deletions of this region demonstrated a 400-bp region that exerts a strong positive effect on promoter activity of the mouse activin beta A subunit gene.     

A rare 920-kilobase chromosomal inversion mediated by IS1 transposition causes constitutive expression of the yiaK-S operon for carbohydrate utilization in Escherichia coli

The regulator of the yiaK-S operon, currently assigned a carbohydrate utilization function in Escherichia coli, is inactivated by a genome rearrangement that leads to the constitutive expression of the operon. The yiaK-S constitutive cells acquire the ability to utilize the rare pentose L-lyxose. Restriction analysis and sequencing of the regulator gene indicate that it is disrupted by foreign DNA. The insert consists of a large inverted fragment of DNA of 920 kilobases flanked by two IS1 elements with opposite polarity. One corresponds to that found naturally at min 0.4 of the bacterial chromosome and the other to a new copy transposed into the regulator gene located at min 80.6. This insertion-inversion could be the result of the intramolecular transposition mechanism itself, a gene rearrangement rarely originated by IS1. Alternatively, it could be attributed to the homologous recombination between the IS1 at min 0.4 and the IS1 transposed intermolecularly into the yiaK-S regulator gene. The participation of a rare IS1-mediated inversion in the evolution of a stable phenotype is thus identified.     

Small genes/gene-products in Escherichia coli K-12

OBJECTIVE: We tested the hypothesis that right heart failure during endotoxin shock may result from altered ventriculovascular coupling responsible for impeding power transfer to the pulmonary circulation. METHODS: The changes in vascular pulmonary input impedance and right ventricular contractility produced by low-dose endotoxin infusion were studied in 6 intact anesthetized dogs. RESULTS: Endotoxin insult resulted in pulmonary hypertension (from 22 +/- 2 to 33 +/- 3 mmHg) associated with significant decreases in stroke volume (from 26.9 +/- 4 to 20.2 +/- 3 ml) and right ventricular ejection fraction (from 41 +/- 3 to 32 +/- 2%). The first minimum of input impedance spectrum and zero phase were shifted towards higher frequencies. Input resistance and characteristic resistance were dramatically increased. The latter change contributed to a significant increase in the pulsatile component of total right ventricular power output from 13 to 21%, indicating a reduction in the hydraulic right ventricle power output delivered into the main pulmonary artery. Overall changes in input pulmonary impedance were indicative of increased afterload facing the right ventricle leading to depressed performance. In contrast, right ventricular systolic elastance was simultaneously increased from 0.56 to 0.93 mmHg/ml indicating an increase in right heart contractility. CONCLUSION: These data suggest that pulmonary hypertension in the setting of experimental endotoxin shock is accompanied by deleterious changes in the pulmonary impedance spectrum, which are responsible for a mismatch of increased contractile state of the right ventricle to the varying hydraulic load ultimately leading to ventricular-vascular uncoupling.     

Expression of mutant horse cytochrome c genes in Escherichia coli

Here we describe genetically engineered constructs for the expression in Escherichia coli of genes for horse cytochrome c mutants. These constructs allow the expression of the cytochrome c genes together with hemeligase, an enzyme which covalently links heme to cytochrome. Careful selection of producer strains and the adjustment of the conditions of expression provided for expression levels of 10-15 mg of protein per liter of culture. This is by an order of magnitude greater than the expression previously achieved in yeast. A series of horse cytochrome c mutants were obtained in this way.     

Isolation and characterization of the heat-responsive genes in Escherichia coli

Isocitrate lyase from Escherichia coli has been expressed in transformed E. coli JE10 cells lacking the isocitrate lyase (icl) gene. After directed mutagenesis of icl by the restriction-site elimination method, partially purified isocitrate lyase mutants in which His 356 has been converted to Lys, Arg, Gln, Asp, or Leu have been characterized after induction of transformed, induced JE10 cells. Values of kcat compared to those for wild-type (wt) enzyme (100) at 37 degrees C, pH 7.3, are 18, 1, <1, 0, and 0 for H356K, H356R, H356E, H356Q, and H356L mutant enzymes, respectively. Km values for the 1:1 Mg-isocitrate complex (in millimolar units) are: 0.13, wt; 0.11, H356K; and 0.63, H356R. Further chromatographic purification of isocitrate lyase yields highly purified wt, H356K, and H356R enzymes. The pH profile of the stability of isocitrate lyase, which has never been reported, showed that the H356R enzyme was unstable in the pH range investigated; the wt and H356R variant differed but each was sufficiently stable to study the pH dependence of catalysis. The log kcat/pH profiles for highly purified wt and H356K enzymes are roughly bell-shaped and have pKa and pKb values for dissociation of an ionizable group on the enzyme-substrate complex of <6.3 and 8.4 for wt and 5.9 and 7.9 for H356K enzymes. Plots of pKm vs pH were different for the wt and H356K variant. Values of pKa and pKb (derived from log kcat/Km plots vs pH) for the dissociation of an activity-related ionizable group on the variant were 5.3 and 7.6, whereas the analogous pKb value for the wt enzyme was 8.4. The data suggest that His 356 is an important functional residue in isocitrate lyase, perhaps in deprotonating isocitrate during catalytic cleavage.     

Cell division genes ftsQAZ in Escherichia coli require distant cis-acting signals upstream of ddlB for full expression

The incidence of septic shock caused by gram-positive bacteria has risen markedly in the last few years. It is largely unclear how gram-positive bacteria (which do not contain endotoxin) cause shock and multiple organ failure. We have discovered recently that two cell wall fragments of the pathogenic gram-positive bacterium Staphylococcus aureus, lipoteichoic acid (LTA) and peptidoglycan (PepG), synergize to cause the induction of nitric oxide (NO) formation, shock, and organ injury in the rat. We report here that a specific fragment of PepG, N-acetylglucosamine-beta-[1--> 4]-N-acetylmuramyl-L-alanine-D-isoglutamine, is the moiety within the PepG polymer responsible for the synergism with LTA (or the cytokine interferon gamma) to induce NO formation in the murine macrophage cell line J774.2. However, this moiety is also present in the PepG of the nonpathogenic bacterium Bacillus subtilis. We have discovered subsequently that S. aureus LTA synergizes with PepG from either bacterium to cause enhanced NO formation, shock, and organ injury in the rat, whereas the LTA from B. subtilis does not synergize with PepG of either bacterium. Thus, we propose that the structure of LTA determines the ability of a particular bacterium to cause shock and multiple organ failure (pathogenicity), while PepG acts to amplify any response induced by LTA.     

Nosocomial spread of cephem-resistant Escherichia coli strains carrying multiple Toho-1-like beta-lactamase genes

Escherichia coli HKY56, which demonstrated resistance to various beta-lactams except carbapenems, was isolated from the throat swab of an inpatient in 1994. Conjugal transfer of cephem resistance from HKY56 to E. coli CSH2 was not successful. Three cefotaxime-resistant E. coli clones harboring plasmid pMRE001, pMRE002, or pMRE003, each of which carried a 3.4-, 5.8-, or 6.2-kb EcoRI fragment insert, respectively, were obtained from HKY56. Although restriction analysis suggested their different origins, these clones showed similar profiles of resistance to various beta-lactams. The sequence of 10 amino acid residues at the N terminus of beta-lactamase purified from E. coli HB101(pMRE001) was identical to that of Toho-1. This Toho-1-like beta-lactamase-1 (TLB-1) was able to hydrolyze cefoperazone and cefotaxime efficiently, but it failed to hydrolyze cephamycins. A Toho-1-specific DNA probe was hybridized with three distinct EcoRI fragments derived from the chromosomal DNA of strain HKY56, and these fragments corresponded to DNA inserts carried by pMRE001, pMRE002, and pMRE003, respectively. PCR and Southern hybridization analysis suggested that all six cephem-resistant E. coli strains, strains HKY273, HKY285, HKY288, HKY305, HKY316, and HKY335, which were isolated in 1996 at the same hospital where strain HKY56 had been isolated, also possessed multiple Toho-1-like beta-lactamase (TLB) genes, and the hybridization patterns obtained with the Toho-1-specific probe were quite similar among these six isolates. The DNA fingerprinting patterns observed by pulsed-field gel electrophoresis revealed that among the E. coli isolates tested, all isolates except HKY56 possessed a similar genetic background. These findings suggested that E. coli strains that carry chromosomally multiplied TLB genes may have been proliferating and transmitted among patients in the same hospital.     

Combinatorial selection of a small RNA that induces amplification of IncFII plasmids in Escherichia coli

Patients with arteriovenous malformation (AVM)-associated aneurysms are a well-recognized subset of the cerebrovascular disease population. The origin of these dual lesions is likely multifactorial, with hemodynamic stresses having a dominant influence. In most patients who present with hemorrhage, the aneurysm is the usual source. Since aneurysm rupture continues to carry a more significant morbidity and mortality when compared to AVM hemorrhage, the authors recommend that the aneurysm be treated first or simultaneously with the AVM in the majority of cases. Treatment of the AVM first is primarily reserved for patients who have suffered bleeds from resectable AVMs.     

Effect of expression of human spermidine/spermine N1-acetyltransferase in Escherichia coli

A plasmid expression vector, pINSAT2, was constructed in order to express spermidine/spermine N1-acetyltransferase (SSAT) in Escherichia coli. Cells transfected with this vector produced large amounts of SSAT, amounting to up to 2% of the soluble protein when isopropyl beta-D-thiogalactopyranoside (IPTG) was added and 0.3% of the soluble protein in the absence of inducer. The growth rate of cells expressing SSAT was reduced, and all of the cellular spermidine was converted to N1-acetylspermidine, much of which was excreted. Putrescine and 1-methylspermidine, which is not a substrate for SSAT, could reverse the effects of SSAT expression on growth, but spermidine was only effective when the amount of SSAT expression was limited by omitting the IPTG inducer. The lack of stimulation of growth by spermidine correlated with its complete conversion to N1-acetylspermidine. These results show that N1-acetylspermine is not able to substitute for the unmodified polyamines in supporting growth and suggest that acetylation is a physiological response to convert excess polyamines to a physiologically inert form which is readily excreted. Cells expressing large amounts of SSAT were much more sensitive to the growth inhibitory action of the antitumor agent N1,N12-bis(ethyl)spermine, supporting the hypothesis that the ability of such bis(ethyl) polyamines to induce SSAT contributes to their antiproliferative actions. SSAT was readily purified to homogeneity from extracts of DH5 alpha cells containing pINSAT2. The purified enzyme had a similar specific activity and Km values for spermine and spermidine as the enzyme purified from human colon cancer cells, suggesting that posttranslational modifications specific to eukaryotes are not needed for enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Positive selection of recombinant plasmids based on the EcoK restriction activity of Escherichia coli K-12

We have constructed a pTZ19R-derived vector which allows efficient positive selection of recombinant plasmids. The system uses the EcoK restriction activity of Escherichia coli K-12 to select against non-recombinant plasmids. The vector contains an EcoK site which, if deleted or disrupted by ligating a DNA fragment, yields recombinant plasmids that are no longer susceptible to EcoK restriction when transformed into a restriction-proficient E. coli host.