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.

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.

IS257 from Staphylococcus aureus: member of an insertion sequence superfamily prevalent among gram-positive and gram-negative bacteria.

The nucleotide sequences for the IS257 family of insertion sequences from Staphylococcus aureus were compared with those of the ISS1 family from Streptococcus lactis and the IS15 family which is widespread amongst Gram-negative bacteria. These elements have a striking degree of similarity in both their putative transposase polypeptide sequences and their nucleotide sequences (40 to 64% between pairs), including 12 out of 14 bp conservation in their terminal inverted repeats. The evolutionary distance between the IS15 family and the IS257 and ISS1 families of Gram-positive origin is approximately twice that between the IS257 and ISS1 families. Analysis of base substitutions in the three sequences has provided insights into the effect of selection for the G + C content of immigrant genes to conform to that of their hosts, and into the evolution of biases in overall amino acid composition of cellular proteins in prokaryotes and eukaryotes. The IS257, ISS1, IS15 families form a superfamily of insertion sequences that has been involved in the spread of a number of antimicrobial resistance determinants in Gram-positive and Gram-negative pathogens.

Nucleotide sequence analysis of IS256 from the Staphylococcus aureus gentamicin-tobramycin-kanamycin-resistance transposon Tn4001.

Resistance to the aminoglycosides gentamicin, tobramycin and kanamycin (GmTmKmR) in Australian clinical strains of Staphylococcus aureus is commonly carried on the composite transposon Tn4001. The resistance gene aacA-aphD of Tn4001, which encodes a bifunctional AAC(6')-APH(2") modifying enzyme, is flanked by two 1324-bp inverted repeats, IS256L and IS256R, that are identical in sequence. Analysis of the IS256 sequence revealed structural features characteristic of IS elements including 26-bp imperfect terminal inverted repeats and a single open reading frame with coding capacity for a 45.6 kDa protein. The nucleotide sequence of IS256 described here, together with the sequence of the aacA-aphD gene reported previously [Rouch et al., J. Gen. Microbiol. 133 (1987) 3039-3052], completes the entire sequence of Tn4001, which totals 4566 bp.

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.

Liver transplantation with reduced-size donor organs.

Orthotopic liver transplantation (OLT) of the pediatric patient is often limited by the availability of a size-matched donor organ. Use of reduced liver transplantation (RLT) can increase the proportion of candidates transplanted and may reduce overall mortality. We report herein the initial clinical application of RLT in the United States. Indications for RLT included fulminant hepatic failure (n = 2), acute hepatic artery thrombosis (n = 3), and chronic liver disease unresponsive to inpatient support and more than 30 days on transplant list (n = 4). Donor hepatectomy was performed using standard techniques. Formal hepatic resection was performed ex-vivo to create a size-matched graft, from the larger donor organ, which was implanted in the orthotopic position. Between 11/84 and 4/87, 70 pediatric patients were evaluated for OLT, and 33 of these were transplanted. During this period only 5 patients (7%) died awaiting OLT. Of 33 patients treated at the University of Chicago, 5 received RLT. Donor: recipient weight ratios ranged from 2:1 to 8.1:1. For RLT median operative blood loss was 1.7 blood volumes (range 0.5-11.7) with an operative time of 9.3 + 3.5 hr. Acceptable early graft function was observed in five patients, all of whom were discharged from the hospital. Four of these five patients are alive between 2 and 48 months after transplantation. Marginal graft function with cholestasis and coagulopathy was associated with acute intracranial hemorrhage and neurologic death in one case. One patient died intraoperatively with non-function caused by the use of a liver from a donor with steatosis and a poor size match. Another patient died on day 5 with primary nonfunction and persistent hemorrhage. Systemic cytomegalovirus infection was the cause of death in the other two cases. RLT can provide life-sustaining liver function in urgent clinical settings. The graft can serve as a temporary or permanent liver replacement. With evolution of the technique RLT could eventually be offered to more elective candidates and increase the utilization of available donors by reducing size limitations in OLT.

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.

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.

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.

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.

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.

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.

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.

A family of ATPases involved in active partitioning of diverse bacterial plasmids.

Low copy-number bacterial plasmids F (the classical Escherichia coli sex factor) and prophage P1 encode partitioning functions which may provide fundamental insights into the active processes which ensure that bacterial genomes are segregated to both daughter cells prior to cell division. These partitioning systems involve two proteins: ParA and ParB. We report that incC from the broad host-range plasmid RK2 is a member of the family of ParA partitioning proteins and that these proteins (as well as related proteins encoded by plasmids from Agrobacterium tumefaciens and Chlamydia trachomatis) contain type I nucleotide-binding motifs. Also, we show that the cell division inhibitor MinD is homologous to members of the ParA family. Sequence comparisons of ParB proteins suggest that they may contain sites for phosphorylation. We propose that ATP hydrolysis by the ParA protein may result in phosphorylation of the ParB protein, thereby causing a conformational shift necessary to separate paired plasmid molecules at the cell division plane.

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.

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.

Efflux-mediated antiseptic resistance gene qacA from Staphylococcus aureus: common ancestry with tetracycline- and sugar-transport proteins.

Resistance to intercalating dyes (ethidium, acriflavine) and other organic cations, such as quaternary ammonium-type antiseptic compounds, mediated by the Staphylococcus aureus plasmid pSK1 is specified by an energy-dependent export mechanism encoded by the qacA gene. From nucleotide sequence analysis, qacA is predicted to encode a protein of Mr 55017 containing 514 amino acids. The gene is likely to initiate with a CUG codon, and a 36 bp palindrome immediately preceding qacA, along with an upstream reading frame with homology to the TetR repressors, may be components of a regulatory circuit. The putative polypeptide specified by qacA has properties typical of a cytoplasmic membrane protein, and is indicated to be a member of a transport protein family that includes proteins responsible for export-mediated resistance to tetracycline and methylenomycin, and uptake of sugars and quinate. The analysis suggests that N- and C-terminal regions of these proteins are involved in energy coupling (proton translocation) and substrate transport, respectively. The last common ancestor of the qacA and related tet (tetracycline resistance) lineages is inferred to have been repressor controlled, as occurs for modern tet determinants from Gram-negative, but not those from Gram-positive, 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.