Protein engineering of cytochrome p450(cam) (CYP101) for the oxidation of polycyclic aromatic hydrocarbons.

Mutations of the active site residues F87 and Y96 greatly enhanced the activity of cytochrome P450(cam) (CYP101) from Pseudomonas putida for the oxidation of the polycyclic aromatic hydrocarbons phenanthrene, fluoranthene, pyrene and benzo[a]pyrene. Wild-type P450(cam) had low (<0.01 min(-1)) activity with these substrates. Phenanthrene was oxidized to 1-, 2-, 3- and 4-phenanthrol, while fluoranthene gave mainly 3-fluoranthol. Pyrene was oxidized to 1-pyrenol and then to 1,6- and 1,8-pyrenequinone, with small amounts of 2-pyrenol also formed with the Y96A mutant. Benzo[a]pyrene gave 3-hydroxybenzo[a]pyrene as the major product. The NADH oxidation rate of the mutants with phenanthrene was as high as 374 min(-1), which was 31% of the camphor oxidation rate by wild-type P450(cam), and with fluoranthene the fastest rate was 144 min(-1). The oxidation of phenanthrene and fluoranthene were highly uncoupled, with highest couplings of 1.3 and 3.1%, respectively. The highest coupling efficiency for pyrene oxidation was a reasonable 23%, but the NADH turnover rate was slow. The product distributions varied significantly between mutants, suggesting that substrate binding orientations can be manipulated by protein engineering, and that genetic variants of P450(cam) may be useful for studying the oxidation of polycyclic aromatic hydrocarbons by P450 enzymes.

The oxidation of naphthalene and pyrene by cytochrome P450cam.

Mutants of the heme monooxygenase cytochrome P450cam in which Y96 had been replaced with hydrophobic residues, have been shown to oxidise naphthalene and pyrene with rates one to two orders of magnitude faster than the wild-type. Naphthalene was oxidised to 1- and 2-naphthol, probably via the 1,2-oxide intermediate. In the case of the Y96F mutant, naphthalene was oxidised at a rate comparable to camphor. Pyrene oxidation gave 1,6- and 1,8-pyrenequinone with no evidence for attack at the K-region, in contrast to mammalian enzymes. The results show that the Y96 residue plays a key role in controlling the substrate range of P450cam.

Copper-inducible transcriptional regulation at two promoters in the Escherichia coli copper resistance determinant pco.

The pco determinant of Escherichia coli plasmid pRI1004 encodes inducible resistance to the trace element copper. The identification of two copper-dependent transcriptional initiation regions within pco that each contain a similar upstream hyphenated dyad motif is described. Deletion constructs showed that this 'copper box' motif was essential for copper-inducible activity at both pco promoters, PpcoA and PpcoE. The placement of the motif differs in the two promoters, and PpcoA contains an extended -10 nonamer typical of promoters for which RNA polymerase does not bind specifically to -35 sequences. PpcoE does not contain this motif and is the more strongly expressed promoter. The transcript from PpcoA contains the pcoABCDRS genes, while PpcoE expresses only pcoE. The induction profiles for PpcoA- and PpcoE-IacZ fusions were flattened sigmoidal curves with a gradual response to increasing copper concentration. On high-copy-number plasmids, zinc was found also to induce transcription from both promoters in vivo. Both promoters showed inducible activity in the absence of pcoRS, the plasmid-borne two-component regulatory system, indicating that a second trans-acting regulatory system is present on the chromosome. The pcoR product showed repressor action in the absence of pcoS, while still allowing induction, suggesting the chromosome encoded a similar two-component system to pco. TnphoA insertion mutagenesis identified chromosomal genes which affected promoter expression, including ptsH, ptsI (sugar phosphotransferase system) and cya (adenylate cyclase). The results support that idea that pco-encoded copper resistance is an auxiliary mechanism for handling copper, the regulation of which is integrated with the chromosomal regulation of cellular copper metabolism.

Molecular genetics and transport analysis of the copper-resistance determinant (pco) from Escherichia coli plasmid pRJ1004.

The copper-resistance determinant (pco) of Escherichia coli plasmid pRJ1004 was cloned and sequenced. Tn1000 transposon mutagenesis identified four complementation groups, mutations in any of which eliminated copper resistance. DNA sequence analysis showed that the four complementation groups contained six open reading frames, designated pco-ABCDRS. The protein product sequences derived from the nucleotide sequence show close homology between this copper-resistance system and the cop system of a plasmid pPT23D of Pseudomonas syringae pv. tomato. The PcoR and PcoS protein sequences show homology to the family of two-component sensor/responder phosphokinase regulatory systems. A seventh reading frame (pcoE) was identified from DNA sequence data, and lies downstream of a copper-regulated promoter. Transport assays with 64Cu(II) showed that the resistant cells containing the plasmid had reduced copper accumulation during the log phase of growth, while increased accumulation had previously been observed during stationary phase. Chromosomal mutants defective in cellular copper management were obtained and characterized. In two of these mutants pco resistance was rendered totally inactive, whilst in another two mutants pco complemented the defective genes. These data indicate that plasmid-borne copper resistance in E. coli is linked with chromosomal systems for copper management.

Understanding cellular responses to toxic agents: a model for mechanism-choice in bacterial metal resistance.

Bacterial resistances to metals are heterogeneous in both their genetic and biochemical bases. Metal resistance may be chromosomally-, plasmid- or transposon-encoded, and one or more genes may be involved: at the biochemical level at least six different mechanisms are responsible for resistance. Various types of resistance mechanisms can occur singly or in combination and for a particular metal different mechanisms of resistance can occur in the same species. To understand better the diverse responses of bacteria to metal ion challenge we have constructed a qualitative model for the selection of metal resistance in bacteria. How a bacterium becomes resistant to a particular metal depends on the number and location of cellular components sensitive to the specific metal ion. Other important selective factors include the nature of the uptake systems for the metal, the role and interactions of the metal in the normal metabolism of the cell and the availability of plasmid (or transposon) encoded resistance mechanisms. The selection model presented is based on the interaction of these factors and allows predictions to be made about the evolution of metal resistance in bacterial populations. It also allows prediction of the genetic basis and of mechanisms of resistance which are in substantial agreement with those in well-documented populations. The interaction of, and selection for resistance to, toxic substances in addition to metals, such as antibiotics and toxic analogues, involve similar principles to those concerning metals. Potentially, models for selection of resistance to any substance can be derived using this approach.

New techniques for liver transplantation: reduced-size, split-liver, living-related and auxiliary liver transplantation.

Mortality among patients on a waiting list for orthotopic liver transplantation continues to be 10-15%; this is of particular concern in the pediatric population and may become more problematic in adult patients as longer waiting lists for cadaveric transplantation accrue. The longer cold ischemia times afforded by use of University of Wisconsin (UW) solution and improved hepatic surgery techniques have allowed the development of reduced-size liver transplantation (RSLT), split-liver transplantation (SLT), and living-related liver transplantation (LRLT). These new surgical techniques have been predominantly employed in children, up to 40% of whom may be candidates for one of these modified procedures. With the exception of SLT, these approaches have been associated with comparable rates of biliary tract and vascular complications, rejection episodes and graft and patient survival when compared to whole organ transplantation. Right hepatic lobe graft recipients have approximately 15% decreased graft survival rates, limiting the acceptance of SLT as a standard approach to decrease waiting list times. Application of LRLT to the adult population, where 5-10% of recipients are potential candidates, is expected to increase. Over 100 LRLTs have been performed worldwide and while recipient survival with LRLT is excellent, concerns about donor morbidity and mortality, psychosocial factors and reimbursement issues remain obstacles. Living-unrelated liver transplantation and auxiliary orthotopic partial liver transplantation are developing approaches to be considered only in highly selected cases.

Phosphatase-mediated heavy metal accumulation by a Citrobacter sp. and related enterobacteria.

A Citrobacter sp. was reported previously to accumulate heavy metals as cell-bound heavy metal phosphates. Metal uptake is mediated by the activity of a periplasmic acid-type phosphatase that liberates inorganic phosphate to provide the precipitant ligand for heavy metals presented to the cells. Amino acid sequencing of peptide fragments of the purified enzyme revealed significant homology to the phoN product (acid phosphatase) of some other enterobacteria. These organisms, together with Klebsiella pneumoniae, previously reported to produce acid phosphatase, were tested for their ability to remove uranium and lanthanum from challenge solutions supplemented with phosphatase substrate. The coupling of phosphate liberation to metal bioaccumulation was limited to the metal accumulating Citrobacter sp.; therefore the participation of species-specific additional factors in metal bioaccumulation was suggested.

Accumulation and intracellular fate of tellurite in tellurite-resistant Escherichia coli: a model for the mechanism of resistance.

The tellurite accumulation properties of three Escherichia coli strains containing different tellurium-resistance determinants of Gram-negative origin, from plasmids pMER610, pHH1508a and RK2, were compared. In all three cases membrane-associated tellurium crystallization was observed, and neither reduced uptake nor increased export contributed to the resistance. Specific membrane-proximal reduction is proposed as the mechanism of resistance to tellurite coded by all three determinants, despite their lack of sequence homology.

Successful allogeneic bone marrow transplantation in an adult with aplastic anemia following orthotopic liver transplantation for non-A, non-B, non-C hepatitis.

A 29-year-old male patient presented with acute liver failure from non-A, non-B and non-C hepatitis, necessitating orthotopic liver transplantation. After operation he developed progressive pancytopenia on the basis of aplastic anemia, which was probably hepatitis associated. After therapy with GM-CSF had failed, he underwent allogeneic BMT from his HLA genotypically identical brother following a conditioning regimen of CY 50 mg/kg x 4 and 500 cGy total lymphoid irradiation. He engrafted promptly but transfusion dependency did not resolve until CMV viremia was treated with ganciclovir. The patient is alive and well 2 years after BMT.

Copper-resistant enteric bacteria from United Kingdom and Australian piggeries.

Thirty-three enteric isolates from Australian (Escherichia coli only) and United Kingdom (U.K.) (Salmonella sp., Citrobacter spp., and E. coli) piggeries were characterized with respect to their copper resistance. The copper resistance phenotypes of four new Australian E. coli isolates were comparable with that of the previously studied E. coli K-12 strain ED8739(pRJ1004), in that the resistance level in rich media was high (up to 18 mM CuSO4) and resistance was inducible. Copper resistance was transferable by conjugation from the new Australian isolates to E. coli K-12 recipients. DNA similarity between the new Australian isolates and the pco copper resistance determinant located on plasmid pRJ1004 was strong as measured by DNA-DNA hybridization; however, the copper resistance plasmids were nonidentical as indicated by the presence of restriction fragment length polymorphisms between the plasmids. DNA-DNA hybridization and polymerase chain reaction analysis demonstrated DNA homology between the pco determinant and DNA from the U.K.E. coli, Salmonella sp., and Citrobacter freundii isolates. However, the copper resistance level and inducibility were variable among the U.K. strains. Of the U.K. E. coli isolates, 1 demonstrated a high level of copper resistance, 4 exhibited intermediate resistance, and 16 showed a low level of copper resistance; all of these resistances were expressed constitutively. A single U.K. C. freundii isolate, had a high level of copper resistance, inducible by subtoxic levels of copper. Transconjugants from one E. coli and one C. freundii donor, with E. coli K-12 strain UB1637 as a recipient, showed copper resistance levels and inducibility of resistance which differed from that expressed from plasmid pRJ1004.(ABSTRACT TRUNCATED AT 250 WORDS)

The Chlamydia trachomatis Mip-like protein is a lipoprotein.

The Mip-like protein of Chlamydia trachomatis is similar to the Mip protein of Legionella pneumophila and may be equally important for the initiation of intracellular infection. This article presents data which identify the chlamydial Mip-like protein as a lipoprotein. The amino acid sequence of the Mip-like protein contains a signal peptidase II recognition sequence, as is seen in procaryotic lipoproteins. Palmitic acid was incorporated into the recombinant chlamydial Mip-like protein. Globomycin, known to inhibit signal peptidase II, inhibited processing of the recombinant Mip-like protein. Labelling of chlamydial organisms with palmitic acid revealed incorporation into the native Mip-like protein.

Intraoperative ultrasound of hepatic tumors.

Intraoperative ultrasound (IOUS) scanning of liver masses was compared with those of preoperative computed tomography (CT) and intraoperative palpation. Between March 1989 and May 1991, 24 patients underwent 25 IOUS procedures during laparotomy. Intraoperative ultrasound provided more information than the other modalities in 10 patients (40%) and affected operative management in eight patients (32%). It was concluded that IOUS, when used in conjunction with CT and palpation, is an important technique in the surgical management of patients with hepatic neoplasms.

Mercuric reductase in environmental gram-positive bacteria sensitive to mercury.

According to existing data, mercury resistance operons (mer operons) are in general thought to be rare in bacteria, other than those from mercury-contaminated sites. We have found that a high proportion of strains in environmental isolates of Gram-positive bacteria express mercuric reductase (MerA protein): the majority of these strains are apparently sensitive to mercury. The expression of MerA was also inducible in all cases. These results imply the presence of phenotypically cryptic mer resistance operons, with both the merA (mercuric reductase) and merR (regulatory) genes still present, but the possible absence of the transport function required to complete the resistance mechanism. This indicates that mer operons or parts thereof are more widely spread in nature than is suggested by the frequency of mercury-resistant bacteria.

Chlamydia trachomatis Mip-like protein.

A 27 kDa Chlamydia trachomatis Mip-like protein with homology of a 175-amino-acid C-terminal fragment to the surface-exposed Legionella pneumophila mip-gene product has previously been described. In this paper the entire chlamydia Mip-like sequence of C. trachomatis serovar L2 (lymphogranuloma venereum (LGV) biovar) is presented. The sequence shows high similarity to the legionella Mip protein and its C-terminal region, like that of the legionella Mip, has high amino acid similarity to eukaryotic and prokaryotic FK506-binding proteins. The chlamydial mip-like gene was detected by polymerase chain reaction (PCR) in other C. trachomatis serovars and by sequencing of the mip-like genes of serovars B and E (trachoma biovar) was shown to be highly conserved within the two major biovars of C. trachomatis. Monoclonal and polyclonal antibodies raised against the recombinant Mip-like protein failed to demonstrate surface-exposed epitopes on infectious elementary bodies or reproductive reticulate body forms either by immunofluorescence or immuno-gold electron microscopy. However, a complement-dependent inhibition of up to 91% of infectivity for cell cultures was observed with antibodies to the N-terminal fragment of the Mip-like protein suggesting that antibody-accessible epitopes are present on infectious EBs.

Membrane transport proteins: implications of sequence comparisons.

Analyses of the sequences and structures of many transport proteins that differ in substrate specificity, direction of transport and mechanism of transport suggest that they form a family of related proteins. Their sequence similarities imply a common mechanism of action. This hypothesis provides an objective basis for examining their mechanisms of action and relationships to other transporters.

Copper resistance determinants in bacteria.

Copper is an essential trace element that is utilized in a number of oxygenases and electron transport proteins, but it is also a highly toxic heavy metal, against which all organisms must protect themselves. Known bacterial determinants of copper resistance are plasmid-encoded. The mechanisms which confer resistance must be integrated with the normal metabolism of copper. Different bacteria have adopted diverse strategies for copper resistance, and this review outlines what is known about bacterial copper resistance mechanisms and their genetic regulation.

Bacterial resistances to mercury and copper.

Heavy metals are toxic to living organisms. Some have no known beneficial biological function, while others have essential roles in physiological reactions. Mechanisms which deal with heavy metal stress must protect against the deleterious effects of heavy metals, yet avoid depleting the cell of a heavy metal which is also an essential nutrient. We describe the mechanisms of resistance in Escherichia coli to two different heavy metals, mercury and copper. Resistance of E. coli to mercury is reasonably well understood and is known to occur by transport of mercuric ions into the cytoplasmic compartment of the bacterial cell and subsequent reductive detoxification of mercuric ions. Recent mutational analysis has started to uncover the mechanistic detail of the mercuric ion transport processes, and has shown the essential nature of cysteine residues in transport of Hg(II). Resistance to copper is much less well understood, but is known to involve the increased export of copper from the bacterial cell and modification of the copper; the details of the process are still being elucidated. Expression of both metal resistance determinants is regulated by the corresponding cation. In each case the response enables the maintenance of cellular homeostasis for the metal. The conclusions drawn allow us to make testable predictions about the regulation of expression of resistance to other heavy metals.

Molecular characterization of the gor gene encoding glutathione reductase from Pseudomonas aeruginosa: determinants of substrate specificity among pyridine nucleotide-disulphide oxidoreductases.

In order to investigate the basis of functional diversity among the pyridine nucleotide-oxidoreductases the gor gene from Pseudomonas aeruginosa PAO, which encodes glutathione reductase, was analysed. The P. aeruginosa gor gene was identified by hybridization with a short DNA sequence from the gene encoding mercuric reductase in transposon Tn501. The gene was cloned, sequenced and overexpressed in Escherichia coli. Expression of the gene enabled rescue of an E. coli gor- mutant, confirming the identity of the cloned gene. The predicted sequence of the gene product showed homology with other members of the pyridine nucleotide-disulphide oxidoreductase family, and allowed determination of positions that may be involved in substrate specificity. These predictions provided information on the relationship of sequence to function, independently of structural data used in previous studies.

Molecular characterization of the gor gene encoding glutathione reductase from Pseudomonas aeruginosa: determinants of substrate specificity among pyridine nucleotide-disulphide oxidoreductases.

In order to investigate the basis of functional diversity among the pyridine nucleotide-oxidoreductases the gor gene from Pseudomonas aeruginosa PAO, which encodes glutathione reductase, was analysed. The P. aeruginosa gor gene was identified by hybridization with a short DNA sequence from the gene encoding mercuric reductase in transposon Tn501. The gene was cloned, sequenced and overexpressed in Escherichia coli. Expression of the gene enabled rescue of an E. coli gor- mutant, confirming the identity of the cloned gene. The predicted sequence of the gene product showed homology with other members of the pyridine nucleotide-disulphide oxidoreductase family, and allowed determination of positions that may be involved in substrate specificity. These predictions provided information on the relationship of sequence to function, independently of structural data used in previous studies.