Vet Microbiol, 1996 Apr, 49(3-4), 209 - 17 Serogroups, toxins and antibiotic resistance of Escherichia coli strains isolated from diarrhoeic lambs in Spain; Blanco J et al.; One hundred and forty-four Escherichia coli strains isolated from 144 diarrhoeic lambs (5 to 21 days old) from 38 flocks in Spain were serotyped and investigated for production of enterotoxins (LT and STa), verotoxins (VT1 and VT2), cytotoxic necrotizing factors (CNF1 and CNF2), alpha-haemolysin (Hly) and enterohaemolysin (EntHly), for necrotic and lethal activities and for antibiotic resistance . The strains belonged to 39 different serogroups; however, 58% were of one of 13 serogroups (O4, O6, O7, O8, O9, O11, O23, O26, O77, O80, O101, O103 and O161) and only four of them (O8, O9, O11 and O77) accounted for 34% of strains . In total 10 (7%) toxigenic strains were detected: two LT+, two VT1+ EntHly+, four VT1+ EntHly-, one CNF1+ Hly+ and one CNF2+ . The highest percentages of antibiotic resistance were reached in the group of antibiotics (tetracycline, streptomycin, sulphadiazine, ampicillin, kanamycin, neomycin, chloramphenicol, trimethoprim and cotrimoxazole) that are most generally used by Spanish veterinary clinics . We conclude that E . coli strains isolated from diarrhoeic lambs are not generally toxigenic and belong to a large number of serogroups. J Bacteriol, 1996 Apr, 178(8), 2334 - 42 Directed mutagenesis of the Rhodobacter capsulatus puhA gene and orf 214: pleiotropic effects on photosynthetic reaction center and light-harvesting 1 complexes; Wong DK et al.; Rhodobacter capsulatus puhA mutant strains containing either a nonpolar, translationally in-frame deletion or a polar insertion of an antibiotic resistance cartridge were constructed and evaluated for their photosynthetic growth properties, absorption spectroscopy profiles, and chromatophore protein compositions . Both types of mutants were found to be incapable of photosynthetic growth and deficient in the reaction center (RC) and light-harvesting 1 (LH1) complexes . The translationally in-frame puhA deletion strains were restored to the parental strain phenotypes by complementation with a plasmid containing the puhA gene, whereas the polar puhA mutants were not . Analogous nonpolar and polar disruptions of orf 214 (located immediately 3' of the puhA gene) were made, and the resultant mutant strains were evaluated as described above . The strain containing the nonpolar deletion of orf 214 exhibited severely impaired photosynthetic growth properties and had greatly reduced levels of the RC and LH1 complexes . Complementation of this strain with a plasmid that expressed orf 214 from the nifHDK promoter restored photosynthetic growth capability, as well as the RC and LH1 complexes . The polar disruption of orf 214 yielded cells that were incapable of photosynthetic growth and had even lower levels of the RC and LH1 complexes, and complementation in trans with orf 214 only marginally improved these deficiencies . These results indicate that orf 214 and at least one additional gene located 3' of orf 214 are required to obtain the RC and LH1 complexes, and transcription read-through from the puhA superoperon is necessary for optimal expression of these new photosynthesis genes. J Bacteriol, 1996 Apr, 178(8), 2216 - 23 Autoactivation of the marRAB multiple antibiotic resistance operon by the MarA transcriptional activator in Escherichia coli; Martin RG et al.; Transcriptional activation of the promoters of the mar/soxRS regulons by the sequence-related but independently inducible MarA and SoxS proteins renders Escherichia coli resistant to a broad spectrum of antibiotics and superoxide generators . Here, the effects of MarA and SoxS on transcription of the marRAB promoter itself were assayed in vitro by using a minimal transcription system and in vivo by assaying beta-galactosidase synthesized from marR::lacZ fusions . Purified MarA and MalE-SoxS proteins stimulated mar transcription about 6- and 15-fold, respectively, when the RNA polymerase/DNA ratio was 1 . Purified MarA bound as a monomer to a 16-bp "marbox" located 69 to 54 nucleotides upstream of a putative RNA initiation site . Deletion of the marbox reduced MarA-mar binding 100-fold, abolished the stimulatory effects of MarA and SoxS on transcription in vitro, and reduced marR::lacZ synthesis about 4-fold in vivo . Deletion of upstream DNA adjoining the marbox reduced MarA binding efficiency 30-fold and transcriptional activation 2- to 3-fold, providing evidence for an accessory marbox . Although MarA and the mar operon repressor, MarR, bound to independent sites, they competed for promoter DNA in band shift experiments . Assays of marR::lacZ transcriptional fusions in marRAB deletion or soxRS deletion strains showed that the superoxide generator paraquat stimulates mar transcription via soxRS and that salicylate stimulates mar transcription both by antagonizing MarR and by a MarR-independent mechanism . Thus, transcription of the marRAB operon is autorepressed by MarR and autoactivated by MarA at a site that also can be activated by SoxS. J Fam Pract, 1996 Apr, 42(4), 357 - 61 Antibiotics and upper respiratory infection: do some folks think there is a cure for the common cold; Mainous AG 3rd et al.; BACKGROUND: Symptomatic treatment is the only recommended therapy for the uncomplicated "common cold." The purpose of this study was to examine the use of antibiotics and other prescription medications for the common cold in a Medicaid population seen in ambulatory care settings . METHODS: A cross-sectional sample of Kentucky Medicaid claims from July 1, 1993, through June 30, 1994, was analyzed . Subjects were patients seen in an ambulatory setting for the common cold, defined as acute nasopharyngitis . A total of 1439 individuals were seen for 2171 separate outpatient and emergency department encounters for the common cold . Outpatient visits accounted for 99% (2144) of the encounters . RESULTS: Patients in 35% (752) of the encounters did not fill a prescription for medication, 6% (129) filled a prescription for an antihistamine or other symptomatic medication, and 60% (1290) filled a prescription for an antibiotic for the common cold . Nineteen different antibiotics, 54% of which were amoxicillin, were prescribed for the common cold . Less than 2% of the encounters had a secondary diagnosis of either acute sinusitis or otitis media . These encounters were not more likely than the total sample to receive antibiotics . Adults were more likely than children to receive an antibiotic (P<.001), and urban physicians were more likely than rural physicians to prescribe antibiotics (P=.02) . A conservative estimate of the annual cost of antibiotic prescribing for the common cold in the United States was $37.5 million . CONCLUSIONS: A majority of persons receiving medical care for the common cold are given prescriptions for an unnecessary antibiotic . Unchecked, this practice may lead to greater antibiotic resistance and unnecessary use of health care resources . Future research should focus on the ability to institute behavioral changes for treatment of the common cold in both closed systems (eg, managed care) and open systems (eg, general community of physicians). J Clin Microbiol, 1996 Mar, 34(3), 628 - 33 Determination of stability of Brucella abortus RB51 by use of genomic fingerprint, oxidative metabolism, and colonial morphology and differentiation of strain RB51 from B . abortus isolates from bison and elk; Jensen AE et al.; Brucella abortus RB51 and isolates from cattle, bison, and elk were characterized by pulsed-field gel electrophoresis and standard techniques for biotyping Brucella species, which included biochemical, morphological, and antigenic techniques, phage susceptibility, and antibiotic resistance . The objectives were to ascertain the stability of RB51 and to differentiate RB51 from other brucellae . Genomic restriction endonuclease patterns produced by pulsed-field gel electrophoresis demonstrated a unique fingerprint for RB51 relative to other brucellae . Comparisons of the oxidative metabolic profiles of RB51 after time in vivo (14 weeks) and in vitro (75 passages) showed no change in characteristic patterns of oxygen uptake on selected amino acid and carbohydrate substrates . Strain RB51 was biotyped as a typical rough B . abortus biovar 1 (not strain 19) after animal passage or a high number of passages in vitro and remained resistant to rifampin or penicillin and susceptible to tetracycline . No reactions with A or M antiserum or with a monoclonal antibody to the O antigen of Brucella lipopolysaccharides were detected; however, RB51 agglutinated with R antiserum . The results indicate that the genomic fingerprint and rough colonial morphology of RB51 are stable characteristics and can be used to differentiate this vaccine strain from Brucella isolates from cattle, bison, and elk. RNA, 1996 Mar, 2(3), 254 - 63 A translational fidelity mutation in the universally conserved sarcin/ricin domain of 25S yeast ribosomal RNA; Liu R et al.; Recent evidence suggests that ribosomal RNAs have functional roles in translation . We describe here a new ribosomal RNA mutation that causes translational suppression and antibiotic resistance in eukaryotic cells . Using random mutagenesis of the cloned ribosomal RNA gene and in vivo selection, we isolated a C --> U mutation in the universally conserved sarcin/ricin domain in Saccharomyces cerevisiae 25S ribosomal RNA . This mutation changes the putative CG pair, which closes the GAGA tetraloop in the sarcin/ricin domain, into a weaker UG pair without eliminating ribosomal sensitivity to ricin . We show that suppression of several UGA, UAG, and frameshift mutations is evident when a portion of the cellular ribosomal RNA contains the C --> U mutation . Cells that contain essentially all mutant ribosomal RNA grow only 10% slower than the wild-type, but show increased suppression as well as resistance to paramomycin, G418, and hygromycin, and sensitivity to cycloheximide . Our results provide genetic evidence for the participation of the sarcin/ricin loop in maintaining translational accuracy and are discussed in terms of a hypothesis that this ribosomal RNA region normally undergoes a conformational change during translation. Behav Brain Res, 1996, 73(1-2), 51 - 8 A molecular analysis of vesicular amine transport; Liu Y et al.; To package classical neurotransmitters into vesicles so that their release can be regulated by activity, neuronal cells express a set of specific vesicular transport proteins . We have used selection in MPP+ to clone the cDNAs encoding two vesicular monoamine transporters, the first members of this novel gene family that now also includes the vesicular transporter for acetylcholine . The sequences show similarity to several bacterial antibiotic resistance proteins, further supporting a role in detoxification and possibly Parkinson's disease . The two vesicular amine transporters show differences in their affinity for substrates, their turnover number and their pharmacology . In particular, the proteins differ in their interactions with the potent inhibitor tetrabenazine and with amphetamines, accounting for several classic pharmacological observations . Since the subcellular localization of the transport proteins determines the site of monoamine storage and the site of monoamine storage appears to differ from other classical transmitters, we have also raised polyclonal antibodies to the transporters and used these to demonstrate localization in dense core vesicles rather than synaptic vesicles . In addition to the implications for monoamine release, these observations also indicate a vesicular amine transporter as the first integral membrane protein restricted to the regulated secretory pathway. Scand J Gastroenterol Suppl, 1996, 215, 82 - 9 Eradication of Helicobacter pylori: omeprazole in combination with antibiotics; Axon AT et al.; Until recently, the mainstay of treatment for Helicobacter pylori infection was either dual therapy, using omeprazole with amoxycillin or clarithromycin, or traditional triple therapy comprising bismuth and two antibiotics . Success with these treatment strategies has, however, varied widely between centres . Furthermore, the side-effects reported for bismuth triple therapy and the 2-week treatment period recommended have limited its popularity . These drawbacks have thus stimulated research aimed at identifying better drug combinations, with a simpler dosage for a shorter period, fewer side-effects, and greater and more consistent efficacy . A number of studies have now been undertaken using an acid inhibitor in combination with two antibiotics . Omeprazole, a highly effective acid pump inhibitor, has been investigated most extensively in this context, and is markedly effective in eradicating H . pylori when combined with any two of clarithromycin, a nitroimidazole and amoxycillin . These omeprazole triple therapy combinations provide eradication rates that are usually in the range of 85-95%, when assessed on a per protocol basis . Side-effects are minor and rarely interfere with compliance . Increasingly, these combinations are being given in a twice-daily dosage, making them more acceptable for the patient, and the dosage of antibiotics, in some cases, can be reduced . Furthermore, 1 week of treatment has been shown to be effective . In a few patients, however, even these highly effective eradication regimens fails, and anecdotal reports suggest that, once this has happened, other treatments are often similarly ineffective . Failure is not simply a matter of antibiotic resistance because patients with resistant organisms are often cured . In some patients, poor compliance, antibiotic resistance, coccoid bacterial forms, or the presence of sanctuary sites may be the cause of failure, in others, it has been suggested that pretreatment with an acid inhibitor may be the explanation . Research into these particular areas will be required, unless a new and universally effective drug combination can be identified. S Afr Med J, 1996 Jan, 86(1), 45 - 9 Unexpectedly high strain diversity of Mycobacterium tuberculosis in a high-incidence community; Warren R et al.; OBJECTIVE: To characterise Mycobacterium tuberculosis strains present in a community experiencing an epidemic, in order to establish whether a high rate of transmission results in low strain diversity . DESIGN: Sputum specimens collected for 18 months; IS6110-based DNA fingerprinting . SETTING: The communities of Ravensmead and Uitsig, Cape Town, South Africa . PARTICIPANTS: Three hundred and thirty-four pulmonary tuberculosis patients attending the Local Authority Health Care Clinic . MAIN OUTCOME MEASURE: DNA fingerprinting . RESULTS: A total of 334 M . tuberculosis isolates were characterised by IS6110-based DNA fingerprinting; 209 strains were identified, 199 having 5 or more insertions . Forty of these strains were present in 2 or more patients (clustering--126 patients in total), which indicates a recent transmission rate of 30% . The 163 unique strains suggest reactivation of latent infections . Computer analysis showed a high degree of strain diversity, and a common progenitor could only be linked to 33% of the strains . Clustering was shown in 50% of drug-resistant isolates . CONCLUSIONS: The low rate of transmission (30%) and the high degree of strain diversity (209 strains) was unexpected and unexplained, given the high burden of disease in this community . The clustering of drug-resistant strains suggests that transmission, rather than lack of compliance, drives the spread of antibiotic resistance in this community . Preliminary indications are that BCG vaccination, while having little effect on the incidence of tuberculosis in this community, may have altered the strain dynamics. Cleve Clin J Med, 1996 Jan-Feb, 63(1), 16 - 30 Community-acquired pneumonia: an update; Meeker DP et al.; Despite the discovery of new pathogens and the evolving problem of antibiotic resistance, the basic trends in community-acquired pneumonia remain remarkably constant . This article reviews the common pathogens, new pathogens, their clinical presentations, the diagnostic workup, the decision to hospitalize, antibiotic resistance, and antibiotic choices. J Bacteriol, 1996 Jan, 178(1), 306 - 8 AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants; Okusu H et al.; Multiple-antibiotic-resistance (Mar) mutants of Escherichia coli are resistant to a wide variety of antibiotics, and increased active efflux is known to be responsible for the resistance to some drugs . The identity of the efflux system, however, has remained unknown . By constructing an isogenic set of E . coli K-12 strains, we showed that the marR1 mutation was incapable of increasing the resistance level in the absence of the AcrAB efflux system . This experiment identified the AcrAB system as the major pump responsible for making the Mar mutants resistant to many agents, including tetracycline, chloramphenicol, ampicillin, nalidixic acid, and rifampin. Gene, 1995 Dec 29, 167(1-2), 333 - 4 Plasmids with a kanamycin-resistance gene for site-directed mutagenesis using the oligodeoxyribonucleotide-directed dual amber method; Hashimoto-Gotoh T et al.; Plasmid vectors carrying lacZ' and kanamycin-resistance (KmR) genes were constructed for site-directed mutagenesis (SDM) using the oligodeoxyribonucleotide (oligo)-directed dual amber (ODA) method {Hashimoto-Gotoh et al., Gene 152 (1995) 271-276} . The plasmids, designated pKF16k, pKF17k, pKF18k and pKF19k, correspond to the previously reported chloramphenicol resistant (CmR) ODA plasmids, pKF16c, pKF17c, pKF18c and pKF19c, respectively, but contain dual amber (am) codons in KmR instead of the CmR gene . The SDM procedure using the KmR ODA plasmids is essentially the same as that with CmR ODA plasmids, which utilizes two oligo primers for in vitro DNA synthesis, one (selection primer) for dual am reversions and the other (mutagenic primer) for the target site . The KmR ODA plasmids yield 5-10-times more DNA per culture volume as compared to the CmR ODA plasmids, and one can prepare selection agar medium simply by spreading Km solution on dried agar plate at a final concentration of 50-100 micrograms/ml; due to the broad range of selecting antibiotic resistance. Mol Gen Genet, 1995 Dec 20, 249(6), 622 - 8 Directed inactivation of the psbI gene does not affect photosystem II in the cyanobacterium Synechocystis sp . PCC 6803; Ikeuchi M et al.; PsbI is a small, integral membrane protein component of photosystem II (PSII), a pigment-protein complex in cyanobacteria, algae and higher plants . To understand the function of this protein, we have isolated the psbI gene from the unicellular cyanobacterium Synechocystis sp . PCC 6803 and determined its nucleotide sequence . Using an antibiotic-resistance cartridge to disrupt and replace the psbI gene, we have created mutants of Synechocystis 6803 that lack the PsbI protein . Analysis of these mutants revealed that absence of the PsbI protein results in a 25-30% loss of PSII activity . However, other PSII polypeptides are present in near wild-type amounts, indicating that no significant destabilization of the PSII complex has occurred . These results contrast with recently reported data indicating that PsbI-deficient mutants of the eukaryotic alga Chlamydomonas reinhardtii are highly light-sensitive and have a significantly lower (80-90%) titer of the PSII complex . In Synechocystis 6803, PsbI-deficient cells appear to be slightly more photosensitive than wild-type cells, suggesting that this protein, while not essential for PSII biogenesis or function, plays a role in the optimization of PSII activity. Mol Gen Genet, 1995 Dec 10, 249(4), 375 - 90 Gene silencing in transgenic tobacco hybrids: frequency of the event and visualization of somatic inactivation pattern; Schmulling T et al.; We have investigated the stability of the expression of different T-DNA-borne genes in hybrid tobacco lines . These lines were constructed to rescue rolC-induced male sterility in kanamycin-resistant P35s-rolC transgenic tobacco plants by expression of rolC antisense genes . Using five different tester lines, a total of 158 hybrids was obtained . We observed inactivation of transgene expression in 20% of the F1 progeny and in 35% of the backcrossed F2 progeny, as indicated by the loss of kanamycin resistance . In 3% of all crosses complete loss of antibiotic resistance was noted, while in most affected hybrid progeny only part of the population became kanamycin sensitive . Single genes could be selectively inactivated on T-DNAs harboring several genes . Gene inactivation was not restricted to one of the two T-DNAs examined . Somatic silencing, visualized by a cell-specific 35SGUSINT marker gene, occurred in a random fashion or exhibited an inherited specific pattern . The type of somatic silencing pattern observed indicated developmental control of the process . Two phenotypic classes could be distinguished with respect to frequency and timing of the inactivation process . Rapid gene inactivation, occurring within a few weeks after germination of hybrid seedlings, was characterized by complete methylation of restriction sites in the promoter of the silenced gene, resetting of gene expression during meiosis, heredity of the developmentally controlled program of gene silencing in subsequent generations, and rapid reactivation of gene expression after genetic separation of the different T-DNAs . In contrast, a slow type of gene inactivation was of a more stochastic nature and was recognized only in hybrids of the backcrossed F2 generation . In this case the degree of promoter methylation, which could extend beyond the T-DNA borders, was not correlated with the reduction in steady-state poly(A)+ mRNA levels, the silenced state was transmitted through meiosis and reactivation lasted several generations . The implications of the observations for our understanding of the gene inactivation process are discussed. Gene, 1995 Dec 1, 166(1), 175 - 6 Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes; Kovach ME et al.; Four new antibiotic-resistant derivatives of the broad-host-range (bhr) cloning vector pBBR1MCS have been constructed . These new plasmids have several advantages over many of the currently available bhr vectors in that: (i) they are relatively small (< 5.3 kb), (ii) they possess an extended multiple cloning site (MCS), (iii) they allow direct selection of recombinant plasmid molecules in Escherichia coli via disruption of the LacZ alpha peptide, (iv) they are mobilizable when the RK2 transfer functions are provided in trans and (v) they are compatible with IncP, IncQ and IncW group plasmids, as well as with ColE1- and P15a-based replicons. J Bacteriol, 1995 Dec, 177(23), 6798 - 803 Subunit interactions and protein stability in the cyanobacterial light-harvesting proteins; Plank T et al.; Strain 4R is a phycocyanin-minus mutant of the unicellular cyanobacterium Synechocystis sp . strain 6803 . Although it lacks the light-harvesting protein phycocyanin, 4R has normal levels of phycocyanin (cpc) transcripts . Sequence analysis of the cpcB gene encoding the phycocyanin beta subunit shows an insertion mutation in 4R that causes early termination of translation . Other work has shown that the phycocyanin alpha subunit and the linker proteins encoded on the cpc transcripts are all functional in 4R, yet the defective phycocyanin beta subunit results in the complete absence of the alpha subunit and the linkers . Phycocyanin-minus mutants were constructed in a wild-type background by interruption of cpcB and cpcA with an antibiotic resistance gene and were compared with the 4R strain . Immunoblot analysis of the mutants demonstrated that interruption of one subunit was accompanied by a complete absence of the unassembled partner subunit . Phycocyanin assembly begins with the formation of the alpha beta heterodimer (the monomer) and continues through higher-order trimeric and hexameric aggregates that associate with linker proteins to form the phycobilisome rods . The results in this paper indicate that monomer formation is a critical stage in the biliprotein assembly pathway and that unassembled subunits are subject to stringent controls that prevent their appearance in vivo. Nucleic Acids Res, 1995 Nov 11, 23(21), 4234 - 8 Nonsense suppressor and antisuppressor mutations at the 1409-1491 base pair in the decoding region of Escherichia coli 16S rRNA; Gregory ST et al.; Using a genetic selection for suppressors of a UGA nonsense mutation in trpA, we have isolated a G to A transition mutation at position 1491 in the decoding region of 16S rRNA . This suppressor displayed no codon specificity, suppressing UGA, UAG and UAA nonsense mutations and +1 and -1 frameshift mutations in lacZ . Subsequent examination of a series of mutations at G1491 and its base-pairing partner C1409 revealed various effects on nonsense suppression and frameshifting . Mutations that prevented Watson-Crick base pairing between these residues were observed to increase misreading and frameshifting . However, double mutations that retained pairing potential produced an antisuppressor or hyperaccurate phenotype . Previous studies of antibiotic resistance mutations and antibiotic and tRNA footprints have placed G1491 and C1409 near the site of codon-anticodon pairing . The results of this study demonstrate that the nature of the interaction of these two residues influences the fidelity of tRNA selection. Gene, 1995 Nov 7, 165(1), 141 - 2 New plasmids carrying antibiotic-resistance cassettes; Reece KS et al.; A series of new plasmid vectors is described that carry gene cassettes imparting resistance to the antibiotics chloramphenicol (CmR), kanamycin (KmR), tetracycline (TcR) and spectinomycin/streptomycin (Sp/SmR) . The gene cassettes are symmetrically flanked by several restriction sites . In addition, several restriction sites that are normally found internal to the gene cassettes have been eliminated, thereby expanding the number of restriction enzymes available to excise an intact antibiotic-resistance gene . The gene cassettes are carried by high-copy-number plasmids that confer ampicillin resistance (ApR). Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 1167 - 77 Antibiotic resistance mechanisms of mutant EF-Tu species in Escherichia coli; Kraal B et al.; Analysis of antibiotic-resistant EF-Tu mutants has revealed a connection between resistance and structural elements that participate in the GTPase switching mechanism . Both random and site-directed mutagenesis methods have yielded sets of purified mutant EF-Tu resistant to kirromycin (kirT) or pulvomycin (pulT) . All kirT mutations cluster in the interface of domain 1 and 3 of EF-Tu in its GTP-bound conformation, not in that of EF-Tu.GDP . Other evidence also suggests that kirromycin binds to the interface of wild-type EF-Tu, thereby jamming the GTPase switch . Various functional studies reveal two subsequent resistance mechanisms . The first hinders kirromycin binding to EF-Tu.GTP and the second occurs after GTP hydrolysis by rejection of bound kirromycin . All pulT mutations cluster in the three-domain junction interface of EF-Tu . GTP (which is an open hole in EF-Tu.GDP) and destabilize a salt-bridge network . Pulvomycin may bind nearby and overlap with tRNA binding . Mutations show that a D99-R230 salt bridge is not essential for the transduction of the GTPase switch signal from domain 1 . In vivo and in vitro studies reveal that pulvomycin sensitivity is dominant over resistance . This demands a revision of the current view of the mechanism of pulvomycin inhibition of protein synthesis and may support a translation model with two EF-Tus on the ribosome . Several mutant EF-Tu species display altered behaviour towards aminoacyl-tRNA with interesting effects on translational accuracy . KirT EF-Tu(A375T) is able to reverse the streptomycin-dependent phenotype of a ribosomal protein S12 mutant strain to streptomycin sensitivity. J Infect, 1995 Nov, 31(3), 225 - 7 Isolation of Arcobacter butzleri from a neonate with bacteraemia; On SL et al.; We describe the case of a neonate with bacteraemia from whom the recently described organism Arcobacter butzleri was isolated . This appears to be the first report of the organism causing neonatal infection . Clinical details suggest that the infection was contracted in utero, although the mother showed no evidence of disease before delivery . Treatment of the preterm infant was ultimately successful in resolving the infection but the organism proved resistant to a wide range of antibiotics . Similar patterns of antibiotic resistance were also observed in 39 reference and field strains of the genus Arcobacter . These findings, combined with available data on the distribution of Arcobacter species, suggest that these organisms may be important human pathogens . Optimized methods for their isolation and identification are therefore required so as to ascertain their role in human disease. J Bacteriol, 1995 Nov, 177(22), 6440 - 8 The hglK gene is required for localization of heterocyst-specific glycolipids in the cyanobacterium Anabaena sp . strain PCC 7120; Black K et al.; Mutant strain 543 of the cyanobacterium Anabaena sp . strain PCC 7120 was originally isolated as a Fox- mutant following chemical mutagenesis . Ultrastructural analysis shows that in nitrogen-replete media the vegetative cells of the mutant are more cylindrical and have thicker septa than those of the wild type, while in nitrogen-free media the mutant heterocysts lack the normal glycolipid layer external to the cell wall . Although this layer is absent, strain 543 heterocysts nevertheless contain heterocyst-specific glycolipids, as determined by thin-layer chromatography . The mutation in strain 543 is in a gene we have named hglK, encoding a protein of 727 amino acids . The wild-type HglK protein appears to contain four membrane-spanning regions followed by 36 repeats of a degenerate pentapeptide sequence, AXLXX . The mutation in strain 543 introduces a termination codon immediately upstream of the pentapeptide repeat region . A mutant constructed by insertion of an antibiotic resistance cassette near the beginning of the hglK gene has the same phenotype as strain 543 . We propose that hglK encodes a protein necessary for the localization of heterocyst glycolipids and that this function requires the pentapeptide repeats of the HglK protein. J Biol Chem, 1995 Oct 27, 270(43), 25798 - 804 Identification of residues involved in substrate recognition by a vesicular monoamine transporter; Merickel A et al.; To identify the residues involved in substrate recognition by recently cloned vesicular monoamine transporters (VMAT1 and VMAT2), we have mutagenized the conserved residues in a cytoplasmic loop between transmembrane domains two and three of VMAT2 . Although studies of related bacterial antibiotic resistance proteins indicate an important functional role for this region, we found no effect of these mutations on VMAT2 activity . However, replacement of aspartate 33 in the first predicted transmembrane domain with an asparagine (D33N) eliminates transport . D33N shows normal levels of expression and normal binding at equilibrium to the potent inhibitor reserpine . However, in contrast to wild-type VMAT2, serotonin inhibits reserpine binding to D33N very poorly, indicating a specific defect in substrate recognition . Replacement of three serine residues in transmembrane domain three with alanine (Stmd3A) shows a similarly selective but even more profound defect in substrate recognition . The results suggest that by analogy to receptors and plasma membrane transporters for monoamines, the cationic amino group of the ligand interacts with an asparte in the first transmembrane domain of VMAT2 and hydroxyl groups on the catechol or indole ring interact with a group of serines in the third transmembrane domain . Importantly, D33N and Stmd3A retain coupling to the proton electrochemical gradient as measured by the delta microH(+)-induced acceleration of reserpine binding . This indicates that substrate recognition can be separated from coupling to the driving force. Nucleic Acids Res, 1995 Oct 25, 23(20), 4073 - 80 The expression of biologically active human p53 in Leishmania cells: a novel eukaryotic system to produce recombinant proteins; Zhang WW et al.; We have investigated the use of Leishmania cells as a novel eukaryotic expression system for the production of recombinant protein . These cells are easy to maintain, requiring no CO2 incubator or shaker, and can be grown in standard tissue culture media . Leishmania cells can be readily transfected with plasmid DNA by electroporation and transformants selected with antibiotic resistance . Recent studies have shown that it is possible to express foreign genes in Leishmania for the purpose of understanding the biology of this protozoan cell . In the present study we report the use of this system as a means of producing a biologically functional human p53 protein . The conformation of the p53 protein is critical for its ability to bind specific DNA sequences . It is demonstrated that Leishmania-synthesized human p53 is phosphorylated and can bind specifically to its enhancer DNA sequence . These data demonstrate that Leishmania may represent a simple eukaryotic expression system for the production of biologically active recombinant proteins. J Assoc Physicians India, 1995 Oct, 43(10), 679 - 84 Reassessment of frequency of occurrence of typhoid fever and cost efficacy analysis of antibiotic therapy; Sridhar CB et al.; Typhoid fever has assumed importance due to the increased incidence of drug resistance in India . The exact magnitude of the problem is not accurately known . The objective of this study was to collect retrospectively the data on the incidence and frequency of typhoid fever among hospital admissions at St . Johns Medical College Hospital (SJMCH), Bangalore during the year 1987 to 1992 and also to study the sensitivity pattern and the use of antibiotics . The study was also aimed at comparison of immunogenicity and tolerance of conventional vaccine to the newer polysaccharide vaccine . It was found that the incidence of typhoid fever showed change from epidemic to endemic situation with outbreaks of epidemics . Sensitivity pattern also showed change during the same period and antibiotic resistance was increasingly demonstrated from 1989 . Cost per patient and total cost to the hospital due to increased admissions also showed progressive increase . The polysaccharide vaccine (recently made available in India) had very low adverse reaction profile with higher immunogenicity as compared to conventional vaccine . Its single dose effect with long lasting immunity indicates it probable usefulness in the eradication of disease. Arch Biochem Biophys, 1995 Sep 10, 322(1), 291 - 4 A superoxide dismutase mimic protects sodA sodB Escherichia coli against aerobic heating and stationary-phase death; Benov L et al.; Superoxide appears to be a major cause of stationary-phase death and heat kill . In support of this conclusion are the following observations: (a) Stationary-phase death was apparent in the sodA sodB, but not in the superoxide dismutase (SOD)-competent parental strain; (b) Stationary phase death in the sodA sodB strain was dioxygen-dependent; (c) A manganic porphyrin, which catalyzes the dismutation of superoxide, protected the sodA sodB strain against stationary-phase death; (d) Heating the sodA sodB strain to 42 degrees C caused a loss of viability not seen with the SOD-competent parental strain and preventable by the manganic porphyrin . Exposure to aerobic conditions induced antibiotic resistance in the sodA sodB, but not in the parental strain and the manganic porphyrin prevented that induction . This again indicates its ability to substitute for SOD in Escherichia coli. Salud Publica Mex, 1995 Sep-Oct, 37(5), 408 - 16 {Risk factors for antitubercular drug resistance in Chiapas, Mexico}; Alvarez-Gordillo GC et al.; OBJECTIVES . To determine risk factors for antibiotic resistance in patients with pulmonary tuberculosis in four Health Jurisdictions of the state of Chiapas . MATERIAL AND METHODS . A case-control study was carried out in patients diagnosed by acid fast smear during 1992 . A questionnaire was applied which included variables related to the diagnosis, treatment and follow-up of the patients . Sputum samples were collected for culture and sensitivity tests . A case of drug-resistant pulmonary tuberculosis was defined as the presence of culture colonies showing resistance to one or more drugs . The control group was patients with negative smears and cultures or positive cultures for M . tuberculosis sensitive to the specific drugs . RESULTS . Of the total of 18 individuals reported to have positive cultures, 13 (72.2%) were resistant to one or more drugs . Resistance to two or more drugs was found in 10 of them of which three were resistant to five antituberculosis drugs . The most frequent resistance was to isoniazid (77%) . Risk factors for resistance encountered in this patient population were monotherapy (OR = 34.2), abandonment of treatment (OR = 6.86), a prolonged period of illness (OR = 6.40), delay in diagnosis and a history of prior therapy (OR = 28.3) . CONCLUSIONS: The high proportion of patients resistant to antituberculosis therapy poses a public health problem and is a clear consequence of the problems arising from inadequate treatment. Genetics, 1995 Aug, 140(4), 1247 - 58 Alterations in ribosomal protein RPS28 can diversely affect translational accuracy in Saccharomyces cerevisiae; Anthony RA et al.; Three small-subunit ribosomal proteins shown to influence translational accuracy in Saccharomyces cerevisiae are conserved in structure and function with their procaryotic counterparts . One of these, encoded by RPS28A and RPS28B (RPS28), is comparable to bacterial S12 . The others, encoded by sup44 (RPS4) or, sup46 and YS11A (RPS13), are homologues of procaryotic S5 and S4, respectively . In Escherichia coli, certain alterations in S12 cause hyperaccurate translation or antibiotic resistance that can be counteracted by other changes in S5 or S4 that reduce translational accuracy . Using site-directed and random mutagenesis, we show that different changes in RPS28 can have diametrical influences on translational accuracy or antibiotic sensitivity in yeast . Certain substitutions in the amino-terminal portion of the protein, which is diverged from the procaryotic homologues, cause varying levels of nonsense suppression or antibiotic sensitivity . Other alterations, found in the more conserved carboxyl-terminal portion, counteract SUP44- or SUP46-associated antibiotic sensitivity, mimicking E . coli results . Although mutations in these different parts of RPS28 have opposite affects on translational accuracy or antibiotic sensitivity, additive phenotypes can be observed when opposing mutations are combined in the same protein. Gene, 1995 Jul 4, 160(1), 63 - 7 Improved antibiotic-resistance gene cassettes and omega elements for Escherichia coli vector construction and in vitro deletion/insertion mutagenesis; Alexeyev MF et al.; Several antibiotic-resistance gene cassettes and omega elements for Escherichia coli vector construction include the aacC1, aadA+, bla, cat, nptII and tet gene cassettes, and also the omega-Gm, omega-Sm, omega-Ap, omega-Cm, omega-Km and omega-Tc elements . Both cassettes and elements are flanked by pBluescriptII plasmid multiple cloning sites (MCS) duplicated in inverted (symmetric MCS) or direct (tandem MCS) orientation . Genes that were modified in order to remove sites for the most common restriction endonucleases from their coding regions (except aacC1 and aadA+) were used for cassette and omega-element construction. Microb Drug Resist, 1995 Summer, 1(2), 137 - 42 Epidemiologic aspects on antibiotic resistance; Hoiby N; The increasing usage of antibiotics has selected for resistant bacteria . Spread of such bacteria may follow mathematical models of infectious diseases, taking into consideration the number of infectious individuals, the number of susceptible individuals, and the effective contact rate between individuals from these two groups . According to calculations of the theoretical epidemic curves, the highest incidence of individuals infected with a resistant bacterial strain is present when the prevalence of patients and carriers is approximately 20-80% . Moreover, the rapidity of the spread of an epidemic increases drastically if the total number of individuals in the exposed group increases and also if the contact rate increases . This means that precautions to stop an epidemic spread of a resistant bacterial strain in a given group of individuals should be undertaken early when the prevalence is below 20% . Efficient precautions consist of cohort isolation, decrease of the number of individuals in exposed groups by subdivision into several smaller groups, and decrease of contact rates by hygienic precautions . Examples are given where such precautions have proven efficient. J Bacteriol, 1995 Jul, 177(14), 4176 - 8 Regulation of the multiple antibiotic resistance (mar) regulon by marORA sequences in Escherichia coli; Martin RG et al.; The mar operon and adjacent sequences were subcloned on a low-copy-number plasmid to identify essential regulatory elements . A 1.1-kbp fragment containing 133 bp of the operator-promoter region (marO), the full marRA gene sequences, and only 10 of 72 marB codons provided a dela mar strain with normal repressibility and inducibility and the ability to beget mar constitutive mutants. Yeast, 1995 Jun 15, 11(7), 629 - 40 In vivo cloning by homologous recombination in yeast using a two-plasmid-based system; Degryse E et al.; In order to reduce the number of classical DNA manipulation and ligation steps in the generation of yeast expression plasmids, a series of vectors is described which facilitate the assembly of such plasmids by the more efficient 'recombination in vivo' technique . Two sets of vectors were developed . The first set, called 'expression vectors', contains an expression cassette with a yeast promoter and the PGK terminator separated by a polylinker, and an Escherichia coli replicon . Subcloning in these vectors of a DNA fragment generates a 'transfer vector' which is compatible with the second set of E . coli-yeast shuttle vectors . This set of 'recombination vectors' contains a cassette for a functional copy of a gene complementing a host strain auxotrophy or a bacterial gene conferring an antibiotic resistance to the plasmid-bearing host . Plasmid copy numbers can be modulated through the use of URA3 or URA3-d as the selective marker together with an ARS/CEN and the 2 microns replicon . Integration of the cloned DNAs into the yeast linearized replicative vectors occurs by recombination between homologous flanking sequences during transformation in yeast or E . coli . All the vectors contain the origin of replication of phage f1 and allow the generation of single-stranded DNA in E . coli for sequencing or site-directed mutagenesis. Proc Natl Acad Sci U S A, 1995 Jun 6, 92(12), 5456 - 60 Binding of purified multiple antibiotic-resistance repressor protein (MarR) to mar operator sequences; Martin RG et al.; Elevated expression of the marORAB multiple antibiotic-resistance operon enhances the resistance of Escherichia coli to various medically significant antibiotics . Transcription of the operon is repressed in vivo by the marR-encoded protein, MarR, and derepressed by salicylate and certain antibiotics . The possibility that repression results from MarR interacting with the marO operator-promoter region was studied in vitro using purified MarR and a DNA fragment containing marO . MarR formed at least two complexes with marO DNA, bound > 30-fold more tightly to it than to salmon sperm DNA, and protected two separate 21-bp sites within marO from digestion by DNase I . Site I abuts the downstream side of the putative -35 transcription-start signal and includes 4 bp of the -10 signal . Site II begins 13 bp downstream of site I, ending immediately before the first base pair of marR . Site II, approximately 80% homologous to site I, is not required for repression since a site II-deleted mutant (marO133) was repressed in trans by wild-type MarR . The absence of site II did not prevent MarR from complexing with the site I of marO133 . Salicylate bound to MarR (Kd approximately 0.5 mM) and weakened the interaction of MarR with sites I and II . Thus, repression of the mar operon, which curbs the antibiotic resistance of E . coli, correlates with the formation of MarR-site I complexes . Salicylate appears to induce the mar operon by binding to MarR and inhibiting complex formation, whereas tetracycline and chloramphenicol, which neither bind MarR nor inhibit complex formation, must induce by an indirect mechanism. Afr J Med Med Sci, 1995 Jun, 24(2), 125 - 30 The menace of beta-lactamase production on antibiotic prescription in community acquired-infections in Nigeria; Oyelese AO et al.; Antibiotic resistance is a major clinical problem in the management of infectious diseases . The production of beta-lactamases by pathogens of all grades which has spread extensively during the last decade has further narrowed down the choice of antibiotics . Those antibiotics that are efficacious are costly and not readily available . The purposes of this study therefore were: To evaluate the incidence of beta-lactamase producing organisms responsible for common community-acquired infections . To evaluate the incidence of bacterial resistance to commonly prescribed antibiotics in the general practice . Nitrocefin strip was used to test each isolate for beta-lactamase production . All isolates were tested against five commonly prescribed antibiotics and a new oral cephalosporin . A nationwide survey revealed that 78% of community-acquired pathogens produced beta-lactamases while more than 50% of most community isolated showed in-vitro resistance to most commonly prescribed antibiotics . We conclude that treatment of bacterial infections are becoming more difficult and more costly . There is need therefore to continually review the susceptibility profiles of community-acquired pathogens. J Bacteriol, 1995 Jun, 177(12), 3414 - 9 Characterization of MarR, the repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli; Seoane AS et al.; The marRAB operon is one of two operons in the mar locus of Escherichia coli that are divergently transcribed from a central regulatory region, marO . The marRAB operon, transcribed from marOII, controls intrinsic resistance or susceptibility to multiple antibiotics and is inducible by structurally unrelated compounds such as tetracycline and chloramphenicol (S . P . Cohen, H . Hachler, and S . B . Levy, J . Bacteriol . 175:1484-1492, 1993) . To clarify the role of the operon in response to environmental signals, its transcription was studied under different conditions, using a marOII-lacZ transcriptional fusion introduced into the chromosome of wild-type or mar-deleted cells . In wild-type cells, uncoupling agents (such as carbonyl cyanide m-chlorophenylhydrazone) and different redox-cycling compounds (e.g., menadione and plumbagin) induced expression from the marOII-lacZ fusion two- to sevenfold . In the mar-deleted strain, LacZ expression from the fusion was 10-fold higher than in wild-type cells . This activity was temperature sensitive (3-fold lower at 42 than at 30 degrees C) and decreased 20-fold with the introduction of the gene for MarR . Structurally different compounds which induce the mar operon in wild-type cells reversed the MarR repression of marOII-lacZ expression . To determine the size of MarR, it was fused to MalE as a MarR fusion protein of 144 amino acids {MarR(144)} or of 125 amino acids (deleted of 19 amino acids at the N terminus) {MarR(125)} . Only the MarR(144) fusion showed repressor ability . The purified MarR(144) fusion, but not the MarR(125) fusion, bound specifically to marO in vitro, as revealed by gel retardation, with an apparent dissociation constant of 5 x 10(-9) M . MarR, therefore, controls expression of the marRAB operon presumably by binding to marO . MarR repression in cells can be reversed by different compounds, facilitating the response of bacteria to multiple environmental stress conditions. Pediatr Clin North Am, 1995 Jun, 42(3), 497 - 507 Mechanisms of bacterial resistance; Burns JL; Antibiotic resistance is a growing problem in clinical pediatrics . Many of the agents traditionally used to treat pediatric pathogens are becoming less effective because of increasing bacterial resistance . In addition, many more children are immunocompromised because of primary or acquired immunodeficiencies and because of advances in cancer chemotherapy and transplantation . These children are being admitted to hospitals where they may be exposed to multiply resistant nosocomial pathogens . An improved understanding of the mechanisms of antibiotic resistance and the development of treatment strategies to prevent the emergence of resistance will be increasingly required in pediatrics. J Hosp Infect, 1995 Jun, 30 Suppl, 15 - 25 How best to utilize limited resources; Forder AA; South Africa's new health policy embraces the primary health care (PHC) approach for all its peoples and will include good primary, secondary and tertiary care . The policy will hope to provide the highest possible standards of care, yet be of a scale and complexity that the country can sustain into the future . There will almost certainly be rationalization of many of the tertiary teaching hospitals, with inevitable cut-backs in their budgets . This in turn could carry the risk of damage to the fabric of these institutions, which might be impossible to repair . Medicines offer a simple, cost-effective answer to many health problems in Africa, provided they are available, accessible, affordable and properly used . A looming problem in African drug markets is inefficiency and waste . The use of counterfeit medicines has reached unparalleled heights . It is vital that there should be a competent, honest, accountable and independent national drug regulatory authority, secured in law, to provide the necessary infrastructure for the acquisition of sound medicines . Medicines are central to a sound national health policy, but there is great public concern about their costs . Anti-infective drugs are amongst the most widely used class of drugs in the world . Inappropriate use of these agents is widespread and guidelines need to be established for their correct use . The control of all medicines in South Africa is governed by the Medicines & Related Substance Act of 1965 . The Medicines Control Council is mandated to ensure that all medicines (including antibiotics) available to the public are efficacious, safe and of high quality . An informally-constituted Antibiotic Study Group has been established in order to monitor aspects of antibiotic therapy that impinge on more general issues of public health, country-wide . The Antibiotic Study Group has instituted an Antibiotic Surveillance Programme to monitor the development of antibiotic resistance nationally . In addition the majority of the tertiary teaching hospitals have comparable in-house antibiotic control policies to help prevent such resistance and to cut costs . These issues need to be debated and resolved . Once in place and working effectively, they will in the long-term supply the most cost-effective means of providing health care for all. Gene, 1995 May 26, 158(1), 9 - 14 Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant; Cherepanov PP et al.; Two cassettes with tetracycline-resistance (TcR) and kanamycin-resistance (KmR) determinants have been developed for the construction of insertion and deletion mutants of cloned genes in Escherichia coli . In both cassettes, the resistance determinants are flanked by the short direct repeats (FRT sites) required for site-specific recombination mediated by the yeast Flp recombinase . In addition, a plasmid with temperature-sensitive replication for temporal production of the Flp enzyme in E . coli has been constructed . After a gene disruption or deletion mutation is constructed in vitro by insertion of one of the cassettes into a given gene, the mutated gene is transferred to the E . coli chromosome by homologous recombination and selection for the antibiotic resistance provided by the cassette . If desired, the resistance determinant can subsequently be removed from the chromosome in vivo by Flp action, leaving behind a short nucleotide sequence with one FRT site and with no polar effect on downstream genes . This system was applied in the construction of an E . coli endA deletion mutation which can be transduced by P1 to the genetic background of interest using TcR as a marker . The transductant can then be freed of the TcR if required. Mol Med, 1995 May, 1(4), 436 - 46 The MarR repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli: prototypic member of a family of bacterial regulatory proteins involved in sensing phenolic compounds; Sulavik MC et al.; BACKGROUND: The marR gene of Escherichia coli encodes a repressor of the marRAB operon, a regulatory locus controlling multiple antibiotic resistance in this organism . Inactivation of marR results in increased expression of marA, which acts at several target genes in the cell leading to reduced antibiotic accumulation . Exposure of E . coli to sodium salicylate (SAL) induces marRAB operon transcription and antibiotic resistance . The mechanism by which SAL antagonizes MarR repressor activity is unclear . MATERIALS AND METHODS: Recombinant plasmid libraries were introduced into a reporter strain designed to identify cloned genes encoding MarR repressor activity . Computer analysis of sequence databases was also used to search for proteins related to MarR . RESULTS: A second E . coli gene, MprA, that exhibits MarR repressor activity was identified . Subsequent database searching revealed a family of 10 proteins from a variety of bacteria that share significant amino acid sequence similarity to MarR and MprA . At least four of these proteins are transcriptional repressors whose activity is antagonized by SAL or by phenolic agents structurally related to SAL . CONCLUSIONS: The MarR family is identified as a group of regulatory factors whose activity is modulated in response to environmental signals in the form of phenolic compounds . Many of these agents are plant derived . Some of the MarR homologs appear more likely to control systems expressed in animal hosts, suggesting that phenolic sensing by bacteria is important in a variety of environments and in the regulation of numerous processes. J Bacteriol, 1995 Apr, 177(7), 1655 - 61 Activation of multiple antibiotic resistance and binding of stress-inducible promoters by Escherichia coli Rob protein; Ariza RR et al.; Multiple antibiotic resistance in Escherichia coli can be mediated by induction of the SoxS or MarA protein, triggered by oxygen radicals (in the soxRS regulon) or certain antibiotics (in the marRAB regulon), respectively . These small proteins (SoxS, 107 residues; MarA, 127 residues) are homologous to the C terminus of the XylS-AraC family of proteins and are more closely related to a approximately 100-residue segment in the N terminus of Rob protein, which binds the right arm of the replication origin, oriC . We investigated whether the SoxS-MarA homology in Rob might extend to the regulation of some of the same inducible genes . Overexpression of Rob indeed conferred multiple antibiotic resistance similar to that known for SoxS and MarA (against chloramphenicol, tetracycline, nalidixic acid, and puromycin), as well as resistance to the superoxide-generating compound phenazine methosulfate . The Rob-induced antibiotic resistance depended only partially on the micF antisense RNA that down-regulates the OmpF outer membrane porin to limit antibiotic uptake . Similar antibiotic resistance was conferred by expression of a Rob fragment containing only the N-terminal 123 residues that constitute the SoxS-MarA homology . Both intact Rob and the N-terminal fragment activated expression of stress genes (inaA, fumC, sodA) but with a pattern distinct from that found for SoxS and MarA . Purified Rob protein bound a DNA fragment containing the micF promoter (50% bound at approximately 10(-9) M Rob) as strongly as it did oriC, and it bound more weakly to DNA containing the sodA, nfo, or zwf promoter (50% bound at 10(-8) to 10(-7) M) . Rob formed multiple DNA-protein complexes with these fragments, as seen previously for SoxS . These data point to a DNA-binding gene activator module used in different protein contexts. Infect Immun, 1995 Apr, 63(4), 1521 - 8 Virulence of a Porphyromonas gingivalis W83 mutant defective in the prtH gene; Fletcher HM et al.; In a previous study we cloned and determined the nucleotide sequence of the prtH gene from Porphyromonas gingivalis W83 . This gene specifies a 97-kDa protease which is normally found in the membrane vesicles produced by P . gingivalis and which cleaves the C3 complement protein under defined conditions . We developed a novel ermF-ermAM antibiotic resistance gene cassette, which was used with the cloned prtH gene to prepare an insertionally inactivated allele of this gene . This genetic construct was introduced by electroporation into P . gingivalis W83 in order to create a protease-deficient mutant by recombinational allelic exchange . The mutant strain, designated V2296, was compared with the parent strain W83 for proteolytic activity and virulence . Extracellular protein preparations from V2296 showed decreased proteolytic activity compared with preparations from W83 . Casein substrate zymography revealed that the 97-kDa proteolytic component as well as a 45-kDa protease was missing in the mutant . In in vivo experiments using a mouse model, V2296 was dramatically reduced in virulence compared with the wild-type W83 strain . A molecular survey of several clinical isolates of P . gingivalis using the prtH gene as a probe suggested that prtH gene sequences were conserved and that they may have been present in multiple copies . Two of 10 isolates did not hybridize with the prtH gene probe . These strains, like the V2296 mutant, also displayed decreased virulence in the mouse model . Taken together, these results suggest an important role for P . gingivalis proteases in soft tissue infections and specifically indicate that the prtH gene product is a virulence factor. Microbiology, 1995 Apr, 141 ( Pt 4), 831 - 41 Inducible expression of heterologous genes targeted to a chromosomal platform in the cyanobacterium Synechococcus sp . PCC 7942; Geerts D et al.; High-level, inducible expression of heterologous genes in the cyanobacterium Synechococcus sp . strain PCC 7942 was obtained using the Escherichia coli trc promoter and lacI repressor . The petE gene of Anabaena sp . strain PCC 7937 encoding plastocyanin precursor protein and the E . coli uidA gene encoding beta-glucuronidase were initially placed under the control of the trc promoter and lacI repressor by cloning into the E . coli pTrc99C expression vector and were introduced into the chromosomal platform for integration in metF (PIM) of the Synechococcus R2-PIM9 recipient strain . These pTrc99C-derived constructs often gave rise to transformants that did not contain a complete insert gene, probably because of gene conversion events . Selection of the desired Synechococcus R2-PIM9 transformants was vastly improved using the new pTrcIS vector that contains the aadA gene encoding streptomycin resistance as an extra antibiotic resistance marker . The influence of IPTG concentration and induction time on gene expression with the E . coli trc/lacI system in Synechococcus was determined using beta-glucuronidase as a reporter . The Anabaena PCC 7937 petE gene in Synechococcus was expressed to a high level upon induction with IPTG as shown by RNA and immunoblot analysis . The general usability of pTrcIS as a cloning vector for inducible heterologous gene expression in Synechococcus was confirmed by the introduction of several more genes. J Mol Biol, 1995 Mar 10, 246(5), 595 - 608 Determination of the binding sites of RepA, a replication initiator protein of the basic replicon of the IncN group plasmid pCU1; Papp PP et al.; A 2kb DNA region of the broad-host-range plasmid pCU1 carries all of the information essential for the stable maintenance of the plasmid and to express the same host-range specificity . It was predicted that the protein required to initiate replication from at least one of the three origins of the plasmid is encoded by the longest open-reading frame (ORF239) of the three overlapping in-frame ORFs located within the 2 kb region . The product of ORF239 has been named RepA . The initiator protein was overexpressed, purified and used for in vitro binding studies . Gel mobility shift experiments were performed to localize RepA binding sites . The DNA sequence protected by the bound RepA molecule(s) was determined by DNase I footprinting and 19 of a 20 bp long sequence that is part of the protected sequence were located in two clusters flanking the repA gene . A plasmid created by linking a 310 bp fragment (nucleotides 238 to 547) of the 2 kb region to the antibiotic resistance genes carried by the omega fragment, can be maintained stably if the RepA protein is supplied in trans . We conclude that this 310 bp DNA fragment, which consists of a short G+C and a long A+T rich region and the cluster of five RepA binding sites, carries a functional origin of the plasmid-protein dependent replication . The position of this origin indicates that it is oriB, one of the three origins previously identified by electron microscopy . The second cluster of RepA binding sites is downstream of the repA gene and consists of 14 sites that are in inverted orientation compared with the binding sites located in the oriB region . They are part of the region that was shown formerly to be involved in controlling the copy number of the plasmid . In contrast to oriB, binding of RepA to neither the oriS nor oriV region was detected. Mol Biochem Parasitol, 1995 Mar, 70(1-2), 45 - 58 Expression of GARP, a major surface glycoprotein of Trypanosoma congolense, on the surface of Trypanosoma brucei: characterization and use as a selectable marker; Hehl A et al.; Procyclic and epimastigote forms of Trypanosoma congolense express an immunodominant glutamic acid/alanine-rich protein (GARP) that covers the parasite surface . Although GARP shows no sequence similarity to procyclins from T . brucei, the general characteristics of the two sets of surface glycoproteins suggest that they have analogous functions, in much the same way that variant surface glycoproteins with unrelated primary sequences fulfil the same function in bloodstream form trypanosomes . Since T . brucei and T . congolense do not follow the same pathway through the tsetse fly, one possible function of procyclins might be to direct parasites to the correct compartments . As a first step towards testing this hypothesis, we have produced stably transformed procyclic forms of T . brucei in which the GARP coding region has been integrated into a procyclin expression site . GARP can be detected on the surface of these transgenic trypanosomes, uniformly distributed within the endogenous procyclin coat, but there are differences in post-translational modification when it is expressed in T . brucei rather than in T . congolense . The fact that GARP is readily accessible to antibodies which were raised against a bacterial fusion protein led us to examine its potential as a selectable surface marker for transfection . We have established a rapid and simple procedure for isolating stable transformants that provides an alternative to conventional methods of selection for antibiotic resistance. J Bacteriol, 1995 Feb, 177(3), 530 - 5 Identification of new genes regulated by the marRAB operon in Escherichia coli; Seoane AS et al.; Random TnphoA and TnlacZ translational fusions were introduced into an Escherichia coli strain with a deletion of the multiple antibiotic resistance (mar) locus, complemented in trans by a temperature-sensitive plasmid bearing the mar locus with a constitutively expressed mar operon . Five gene fusions (two with lacZ and three with phoA) regulated by the mar operon were identified by increased or decreased marker enzyme activity following loss of the complementary plasmid at the restrictive temperature . Expression of LacZ from both lacZ fusions increased in the presence of the mar operon; expression from the three phoA fusions was represented by the mar operon . The lacZ fusions were mapped at 31.5 and 14 min on the Escherichia coli chromosome . One of the phoA fusions was located at 51.6 min while the two others mapped at 77 min . Cloning and sequencing of a portion of the fused genes showed all of them to be different . The phoA fusions at 77 min were located in a recently identified gene, slp, a lipoprotein of unknown function (D.M . Alexander and A . C . St . John, Mol . Microb . 11:1059-1071, 1994) . The others showed no homology with any known genes of E . coli . The insertions caused small but reproducible changes in the antibiotic susceptibility profile . This approach has enabled the identification of new genes in E . coli which are regulated by the marRAB operon and involved in the Mar phenotype. Mol Microbiol, 1995 Feb, 15(4), 593 - 600 Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination; Hall RM et al.; An integron is a genetic unit that includes the determinants of the components of a site-specific recombination system capable of capturing and mobilizing genes that are contained in mobile elements called gene cassettes . An integron also provides a promoter for expression of the cassette genes, and integrons thus act both as natural cloning systems and as expression vectors . The essential components of an integron are an int gene encoding a site-specific recombinase belonging to the integrase family, an adjacent site, attI, that is recognized by the integrase and is the receptor site for the cassettes, and a promoter suitably oriented for expression of the cassette-encoded genes . The cassettes are mobile elements that include a gene (most commonly an antibiotic-resistance gene) and an integrase-specific recombination site that is a member of a family of sites known as 59-base elements . Cassettes can exist either free in a circularized form or integrated at the attI site, and only when integrated is a cassette formally part of an integron . A single site-specific recombination event involving the integron-associated attI site and a cassette-associated 59-base element leads to insertion of a free circular cassette into a recipient integron . Multiple cassette insertions can occur, and integrons containing several cassettes have been found in the wild . The integrase also catalyses excisive recombination events that can lead to loss of cassettes from an itegron and generate free circular cassettes . Due to their ability to acquire new genes, integrons have a clear role in the evolution of the genomes of the plasmids and transposons that contain them. Biochim Biophys Acta, 1995 Jan 19, 1246(2), 109 - 27 Contribution of mutant analysis to the understanding of enzyme catalysis: the case of class A beta-lactamases; Matagne A et al.; Class A beta-lactamases represent a family of well studied enzymes . They are responsible for many antibiotic resistance phenomena and thus for numerous failures in clinical chemotherapy . Despite the facts that five structures are known at high resolution and that detailed analyses of enzymes modified by site-directed mutagenesis have been performed, their exact catalytic mechanism remains controversial . This review attempts to summarize and to discuss the many available data. J Biol Chem, 1995 Jan 13, 270(2), 775 - 80 Substitution of Asp for Asn at position 132 in the active site of TEM beta-lactamase . Activity toward different substrates and effects of neighboring residues; Osuna J et al.; Using a random, combinatorial scheme of mutagenesis directed against the conserved SDN region of TEM beta-lactamase, and selective screening in ampicillin-plates, we obtained the N132D mutant enzyme . The kinetic characterization of this mutant indicated relatively small effects compared to the wild-type . Both pK1 and pK2 for catalysis were decreased about 1 unit relative to the pK's for the wild type . This effect was predominantly due to changes in Km . In contrast to the wild-type, the pH-rate profiles of the mutant showed that Km for several side chain-containing penicillin substrates increases when the pH is above 5.5 . 6-Aminopenicillanic acid, which lacks a side chain, did not show this effect . With benzylpenicillin, ampicillin, and carbenicillin, kcat for the mutant showed a similar pH dependence as the wild type . With 6-aminopenicillanic acid, kcat for the mutant was greater than that for the wild type . The nature of the 104 side chain may affect the environment of Asp132; double mutants N132D/E104X (where X can be Q or N) are unable to confer antibiotic resistance to bacterial cells . The computed contact interactions from modeling substrate complexes between benzylpenicillin or 6-aminopenicillanic acid with the N132D mutant confirmed the importance of the protonation state of residue Asp132 for the complex stability with side chain-containing substrates . The data indicate that the contact between the side chain of residue 132 and the substrate is relevant for the ground state recognition, but because of close contact with several important groups in its neighborhood, residue 132 is also indirectly involved in the catalytic step of the wild-type enzyme. J Bacteriol, 1995 Jan, 177(2), 459 - 61 D-serine deaminase is a stringent selective marker in genetic crosses; Maas WK et al.; The presence of the locus for D-serine deaminase (dsd) renders bacteria resistant to growth inhibition by D-serine and enables them to grow with D-serine as the sole nitrogen source . The two properties permit stringent selection in genetic crosses and make the D-serine deaminase gene an excellent marker, especially in the construction of strains for which the use of antibiotic resistance genes as selective markers is not allowed. Antimicrob Agents Chemother, 1995 Jan, 39(1), 185 - 91 PCR mapping of integrons reveals several novel combinations of resistance genes; Levesque C et al.; The integron is a new type of mobile element which has evolved by a site-specific recombinational mechanism . Integrons consist of two conserved segments of DNA separated by a variable region containing one or more genes integrated as cassettes . Oligonucleotide probes specific for the conserved segments have revealed that integrons are widespread in recently isolated clinical bacteria . Also, by using oligonucleotide probes for several antibiotic resistance genes, we have found novel combinations of resistance genes in these strains . By using PCR, we have determined the content and order of the resistance genes inserted between the conserved segments in the integrons of these clinical isolates . PCR mapping of integrons can be a useful epidemiological tool to study the evolution of multiresistance plasmids and transposons and dissemination of antibiotic resistance genes. Antimicrob Agents Chemother, 1995 Jan, 39(1), 155 - 62 Expression of antibiotic resistance genes in the integrated cassettes of integrons; Collis CM et al.; Plasmids containing cloned integron fragments which differ only with respect to either the sequence of the promoter(s) or the number and order of inserted cassettes were used to examine the expression of resistance genes encoded in integron-associated gene cassettes . All transcripts detected commenced at the common promoter P(ant), and alterations in the sequence of P(ant) affected the level of resistance expressed by cassette genes . When both P(ant) and the secondary promoter P2 were present, transcription from both promoters was detected . When more than one cassette was present, the position of the cassette in the array influenced the level of antibiotic resistance expressed by the cassette gene . In all cases, the resistance level was highest when the gene was in the first cassette, i.e., closest to P(ant), and was reduced to different extents by the presence of individual upstream cassettes . In Northern (RNA) blots, multiple discrete transcripts originating at P(ant) were detected, and only the longer transcripts contained the distal genes . Together, these data suggest that premature transcription termination occurs within the cassettes . The most abundant transcripts appeared to contain one or more complete cassettes, and is possible that the 59-base elements found at the end of the cassettes (3' to the coding region) not only function as recombination sites but may also function as transcription terminators. Immunogenetics, 1995, 42(3), 181 - 7 Efficient nonhomologous and homologous recombination in scid cells; Buhler B et al.; The severe combined immunodeficiency (scid) mutation affects both coding joint formation during immunoglobulin and T-cell receptor V(D)J recombination and double-strand break repair . We analyzed scid cells for their ability to undergo other types of DNA end joining: nonhomologous and homologous recombination . Using plasmid constructs carrying antibiotic resistance genes, we observed that the efficiency of nonhomologous integration in scid cells was equal to that in wildtype cell lines . In addition, there was no obvious difference in the fidelity of the integration and in the expression of the resistance genes . Moreover, scid cells were able to carry out homologous recombination of extrachromosomal substrates just as well as wildtype cells . These results suggest a mechanistic difference between nonhomologous integration and homologous recombination on the one hand and V(D)J recombination and double-strand break repair on the other. Prog Brain Res, 1995, 105, 273 - 81 The transport of neurotransmitters into synaptic vesicles; Peter D et al.; Using selection in the neurotoxin MPP+, we have isolated a cDNA encoding vesicular amine transport . The transporter protects against MPP+ by sequestering the toxin in vesicles, away from its primary site of action in mitochondria . Unexpectedly, two distinct but highly related genes encode vesicular amine transport in the adrenal gland and the central nervous system . The sequence of both predicts twelve transmembrane domains and weak homology to a class of bacterial antibiotic resistance proteins . The two human genes occur on different chromosomes . In addition, the two transporters show a number of differences in function, including substrate specificity and the interaction with one inhibitor and the amphetamines. Arch Intern Med, 1994 Dec 12-26, 154(23), 2666 - 77 Efficacy of pneumococcal vaccination in adults . A meta-analysis of randomized controlled trials; Fine MJ et al.; BACKGROUND: Because of the prevalence of pneumococcal pneumonia, the substantial morbidity and mortality associated with many pneumococcal infections, and an increase in the incidence of antibiotic resistance among pneumococcal isolates, considerable efforts for disease prevention have been made using a polyvalent polysaccharide pneumococcal vaccine . Despite numerous clinical trials of the vaccine, its efficacy in the prevention of pneumococcal infections and other clinically relevant medical outcomes in adults remains uncertain . METHODS: To assess quantitatively the efficacy of pneumococcal vaccination, a MEDLINE literature search, manual reviews of article bibliographies, and communications with pneumococcal vaccine investigators were used to identify randomized controlled trials of the pneumococcal vaccine . Independent review of 594 articles revealed nine randomized trials with 12 vaccine and control study groups that evaluated clinically relevant outcomes in adults . To estimate a summary effect size for all outcomes, Mantel-Haenszel odds ratios (ORs) and Dersimonian and Laird rate differences (RDs) and their associated 95% confidence intervals (CIs) were computed . RESULTS: Summary ORs demonstrated a statistically significant protective effect of the vaccine for four pneumococcal infection-related outcomes: definitive pneumococcal pneumonia (OR = 0.34; 95% CI = 0.24 to 0.48), definitive pneumococcal pneumonia for vaccine-containing pneumococcal antigen types only (vaccine types only) (OR = 0.17; 95% CI = 0.09 to 0.33), presumptive pneumococcal pneumonia (OR = 0.47; 95% CI = 0.35 to 0.63), and presumptive pneumococcal pneumonia (vaccine types only) (OR = 0.39; 95% CI = 0.26 to 0.59) . The summary RDs, which account for heterogeneity among studies, confirmed a statistically significant protective effect for two of these same outcomes: definitive pneumococcal pneumonia (RD = 4/1000; 95% CI = 0/1000 to 7/1000) and definitive pneumococcal pneumonia (vaccine types only) (RD = 8/1000; 95% CI = 1/1000 to 16/1000) . Summary ORs and RDs failed to demonstrate a protective effect for pneumonia (all causes), bronchitis, and mortality (all causes) or mortality due to pneumonia or pneumococcal infection . Subgroup analyses showed that for all four pneumococcal infection-related outcomes, vaccine efficacy differed for high- and low-risk subjects, demonstrating efficacy for low-risk subjects and lack of efficacy for high-risk subjects . CONCLUSIONS: Pneumococcal vaccination appears efficacious in reducing bacteremic pneumococcal pneumonia in low-risk adults . However, evidence from randomized controlled trials fails to demonstrate vaccine efficacy for pneumococcal infection-related or other medical outcomes in the heterogeneous group of subjects currently labeled as high risk. J Bacteriol, 1994 Dec, 176(24), 7754 - 6 Analysis of the genetic requirements for inducible multiple-antibiotic resistance associated with the mar locus in Escherichia coli; Sulavik MC et al.; A series of novel genetic constructs derived from the marRAB operon was used to determine the role of this gene cluster in salicylate-inducible multiple-antibiotic resistance in Escherichia coli . Our findings indicate that regulated antibiotic resistance associated with this locus requires only the products of marR and marA, without any neighboring genes. J Bacteriol, 1994 Dec, 176(24), 7735 - 9 The incN plasmid replicon: two pathways of DNA polymerase I-independent replication; Kim HY et al.; The 2,053-bp broad-host-range incompatibility group N replicon of plasmid pCU1 has two components: a region of 1,200 bp that is sufficient for its replication in Escherichia coli PolA+ and PolA- hosts and a regulatory region called the group I iteron region that contains 13 39-bp iterons . Within the 1,200-bp region, there are three replication origins, two of which, called oriB and oriS, function in PolA+ and PolA- hosts and a third, called oriV, which functions only in PolA+ hosts . The region also specifies a protein called RepA . We now show that both oriB and oriS can function in a delta polA strain but that in such a strain, only oriB has an absolute requirement for RepA . oriS can function without RepA and polymerase I provided that the iteron region is deleted and that in this circumstance, it is the only origin, the usage of which is detected . The requirements for oriB usage can thus be distinguished from those for oriS usage . The oriB region can be recovered as a plasmid only if RepA is provided in trans . These complex features of this replicon are also shown to be shared by the IncN replicons of other antibiotic resistance plasmids . Functionally distinguishable origins in a small replicon may be a way of endowing such a replicon with a broad host range. Eur J Clin Microbiol Infect Dis, 1994 Dec, 13(12), 1015 - 22 Non-canonical mechanisms of antibiotic resistance; Martinez JL et al.; Although the current in vitro methods used for detection and analysis of the phenotypes of antibiotic resistance in the laboratory are well established, other resistance mechanisms of resistance exist which may escape detection using the standard approach . The present article reviews some of these mechanisms which are grouped under the term 'non-canonical mechanisms' of antibiotic resistance . Such mechanisms include gene dosage, heterologous induction or selection, populational resistance and synergism between mechanisms of low resistance . The role of these mechanisms in the failure of therapy is discussed. Southeast Asian J Trop Med Public Health, 1994 Dec, 25(4), 698 - 701 The use of surgical antibiotic prophylaxis in seven Malaysian hospitals; Lim VK et al.; A survey on the use of antibiotics in surgical prophylaxis was carried out in seven Malaysian hospitals . Details of antibiotic prescriptions were obtained through questionnaires completed by the prescriber . A total of 430 such prescriptions was analysed . A large number of different antibiotic regimens were used for a variety of surgical procedures . The majority of prescriptions (70%) were issued for procedures where such prophylaxis was probably not necessary . Antibiotics were also often prescribed for durations that were longer than necessary . There is an urgent need to educate surgeons and standardize surgical prophylactic regimens in order to reduce cost and combat the emergence of antibiotic resistance. J Bacteriol, 1994 Oct, 176(20), 6262 - 9 Dual regulation of inaA by the multiple antibiotic resistance (mar) and superoxide (soxRS) stress response systems of Escherichia coli; Rosner JL et al.; The roles of the marRAB (multiple antibiotic resistance) operon and soxRS (superoxide response) genes in the regulation of inaA, an unlinked weak-acid-inducible gene, were studied . inaA expression was estimated from the beta-galactosidase activity of a chromosomal inaA1::lacZ transcriptional fusion . marR mutations that elevate marRAB transcription and engender multiple antibiotic resistance elevated inaA expression by 10- to 20-fold over that of the wild-type . Similarly, one class of inaA constitutive mutants that mapped to the mar region were multiply antibiotic resistant . Overexpression of marA alone on a multicopy plasmid caused high constitutive expression of inaA in a strain with an extensive (39-kbp) marRAB deletion . Salicylate, an inducer of marRAB and of an unidentified mar-independent antibiotic resistance system, induced inaA by 6-fold . A portion of this induction was also mar independent . Two soxRS constitutive mutants that were tested showed elevated levels of inaA . Paraquat, an inducer of the soxRS system, elevated inaA expression by 6- to 9-fold . This induction was soxRS dependent and not mar dependent, whereas induction of inaA by salicylate was not dependent on soxRS . Paraquat induced resistance to norfloxacin in the mar-deleted strain but not in a soxRS-deleted strain . Thus, induction of multiple antibiotic resistance and inaA by salicylate occurs via mar and an unidentified pathway, while induction by paraquat occurs via soxRS. Trends Microbiol, 1994 Oct, 2(10), 416 - 21 Resistance to drugs targeting protein synthesis in mycobacteria; Bottger EC; Many antibiotics exert their effects by interfering with protein synthesis . Studies of the molecular mechanisms of antibiotic resistance in clinical strains of mycobacteria have revealed mutations in ribosomal RNAs . This type of acquired resistance was previously unknown in bacterial pathogens and was made possible because mycobacteria have only a single set of rRNA genes. Genetika, 1994 Sep, 30(9), 1141 - 5 {Cloning and expression of the B1 hordein gene of barley (Hordeum vulgare L.) in cells of the cyanobacterium Synechocystis sp . PCC6803 and Escherichia coli}; Chemeresiuk NN et al.; The hordein B1 gene of Hordeum vulgare L . (variety Donetskii 4) was cloned in cells of Escherichia coli and cyanobacterium Synechocystis sp . PCC6803 . For cloning in E . coli, a 2.3-kb Hpa I/EcoR I DNA fragment carrying the hordein B1 gene was inserted into plasmid pBSM13(-), controlled by a lac promoter . The constructed recombinant plasmid pBSB1 provides hordein B1 gene expression in transformed E . coli cells . To introduce the hordein B1 gene into the genome of Synechocystis sp . PCC6803, an integrative vector, containing a fragment of cyanobacterium chromosomal DNA with inserted Tn5 antibiotic resistance genes (KmR, BleoR, StpR) was constructed . A Pvu II-fragment of pBSB1, containing hordein B1 gene under control of the lac promoter, was cloned in the Sma I-site of gene BleoR . The resulting recombinant plasmid was used to transform cyanobacterial cells . One KmR transformants contained a DNA fragment yielding a positive signal during Southern blotting with a {32P}-labeled DNA fragment carrying hordein B1 gene . Western blotting with polyclonal antibodies to barley hordein revealed hordein B1 gene expression in Synechocystis sp . PCC6803. Heart Lung, 1994 Sep-Oct, 23(5), 363 - 7 Antibiotic use and antibiotic resistance in the intensive care unit: are we curing or creating disease? Kollef MH. Antibiotics represent one of the most commonly prescribed medical therapies for hospitalized patients . The practice of "spiralling empiricism" has increasingly led to the unnecessary administration of antibiotics, resulting in the emergence of infections with antibiotic-resistant bacteria that are associated with increased rates of patient mortality . Ventilator-associated pneumonia due to antibiotic-resistant bacteria has become recognized as an important problem resulting from prior antibiotic exposure . Health professionals must be aware of this problem and avoid the unnecessary administration of these drugs . Future research efforts should be aimed at improving our ability to diagnose and exclude infections and to develop better strategies for antibiotic administration in the intensive care unit setting. Todays OR Nurse, 1994 Sep-Oct, 16(5), 7 - 12 Bugs and drugs: antibiotic resistance in the 1990s; Rickman LS; 1 . Bacteria have been versatile in circumventing the mechanisms by which antibiotics work; in the 1990s there has been a world-wide resurgence of antibiotic-resistant bacterial diseases . 2 . Resistance to certain antibiotics develops in bacteria through three basic processes: mutation, transduction, and conjugation . 3 . Infection-control programs, if combined with intensive infection-control measures and control of antibiotic use, may be effective at reducing the transmission of resistant pathogens. Plasmid, 1994 Sep, 32(2), 222 - 7 DNA sequence of direct repeats of the sulI gene of plasmid pSa; Valentine CR et al.; The restriction enzyme and genetic map of the antibiotic-resistance region of plasmid pSa is related to Tn21 integrons by the insertion of 5.4 kb containing a chloramphenicol resistance gene (catII) and a 1.1-kb direct repeat . We report here the nucleotide sequences of both copies of the repeat with adjoining sequences . They were identical for 1065 bp and contained the entire coding sequence of the sulfanilamide resistance gene, sulI . Since only the first copy of the repeat confers sulfonamide resistance, this leads to the conclusion that no promoter was available for the second copy . The sequence of the pSa sulI gene was identical to several published sulI sequences from other plasmids . The first junction point of the catII-containing insert was identical to the sequence for pDG0100; the second junction occurred farther into the 3'-conserved segment of integrons than does that of pDG0100 . A recent report of these junction sequences for pSa and pDG0100 differs from our sequences by one nucleotide . Two additional differences were an insert of 41 bases and a single base insertion between sulI and ORF341 in our sequence . Our sequenced regions have been assigned GenBank Accession Nos . UO4277 and UO4278 for the first and second sulI genes of pSa, respectively. Curr Genet, 1994 Aug, 26(2), 179 - 83 Highly-efficient transformation of the homobasidiomycete Schizophyllum commune to phleomycin resistance; Schuren FH et al.; Regulatory sequences of the glyceraldehyde-3-phosphate-dehydrogenase (GPD) gene from the homobasidiomycete Schizophyllum commune were fused to the coding sequence of the ble gene from Streptoalloteichus hindustanus, which codes for a phleomycin-binding protein . The resulting construct transformed S . commune to phleomycin resistance at a high frequency (up to 10(4) transformants/microgram DNA per 10(7) protoplasts) when regeneration was done in 0.5 M MgSO4 . A similar construct with regulatory sequences from Aspergillus nidulans failed to give transformants, showing the importance of homologous regulatory sequences for the expression of genes in S . commune . The homologous GPD promoter could be deleted up to position -130 without any effect on the number of phleomycin-resistant transformants . This is the first effective stable transformation system in a homobasidiomycete employing antibiotic resistance. Antimicrob Agents Chemother, 1994 Aug, 38(8), 1773 - 9 Genetic relationship between soxRS and mar loci in promoting multiple antibiotic resistance in Escherichia coli; Miller PF et al.; Multiple antibiotic resistance in Escherichia coli has typically been associated with mutations at the mar locus, located at 34 min on the E . coli chromosome . A new mutant, marC, isolated on the basis of a Mar phenotype but which maps to the soxRS (encoding the regulators of the superoxide stress response) locus located at 92 min, is described here . This mutant shares several features with a known constitutive allele of the soxRS gene, prompting the conclusion that it is a highly active allele of this gene . The marC mutation has thus been given the designation soxR201 . This new mutant was used to examine the relationship between the mar and sox loci in promoting antibiotic resistance . The results of these studies indicate that full antibiotic resistance resulting from the soxR201 mutation is partially dependent on an intact mar locus and is associated with an increase in the steady-state level of mar-specific mRNA . In addition, paraquat treatment of wild-type cells is shown to increase the level of antibiotic resistance in a dose-dependent manner that requires an intact soxRS locus . Conversely, overexpression of MarA from a multicopy plasmid results in weak activation of a superoxide stress response target gene . These findings are consistent with a model in which the regulatory factors encoded by the marA and soxS genes control the expression of overlapping sets of target genes, with MarA preferentially acting on targets involved with antibiotic resistance and SoxS directed primarily towards components of the superoxide stress response . Furthermore, compounds frequently used to induce the superoxide stress response, including paraquat, menadione, and phenazine methosulfate, differ with respect to the amount of protection provided against them by the antibiotic resistance response. Zh Mikrobiol Epidemiol Immunobiol, 1994 Aug-Sep, Suppl 1, 87 - 91 {A comparative evaluation of the persistence characteristics of Escherichia isolated from different econiches}; Gritsenko VA et al.; The multiple evaluation of the persistence characteristics, including antilysozyme, anti-interferon and anticomplement activity, as well as other biological properties, such as adhesiveness, colicinogenicity and resistance to antibiotics, was carried out in 173 E . coli strains isolated from water, healthy and sick children . This evaluation revealed that each group of E . coli, depending on the source of its isolation, had its characteristic set of properties (or bioprofiles) to be analyzed, making it different from other bacterial populations . The comparative intergroup analysis showed differences between E . coli isolated from children with pathological conditions (enteric coli-bacteriosis, pyelonephritis) and E . coli isolated from water and feces of healthy children . These differences were manifested by more pronounced persistence characteristics . Dispersion analysis, having confirmed this feature, revealed that the most labile characteristics of E.coli, subject to the influence of ecological conditions, were their markers of persistence and antibiotic resistance . The results of factor analysis made it possible to unite the above mentioned properties which determined, together with adhesiveness, pathogenic potential of these bacteria. Mol Gen Genet, 1994 Jul 25, 244(2), 189 - 96 Chloroplast targeting of spectinomycin adenyltransferase provides a cell-autonomous marker for monitoring transposon excision in tomato and tobacco; Scofield SR et al.; Antibiotic resistance genes can act as either cell autonomous or non-cell autonomous genetic markers with which to monitor the excision of plant transposons . To convert spectinomycin resistance from a non-cell autonomous resistance to cell autonomous resistance, a transit peptide for chloroplast localization from a petunia ribulose bisphosphate carboxylase (rbcS) gene was fused in-frame to the aadA gene, which confers spectinomycin and streptomycin resistance . Constructs were generated in which the expression of this chimeric gene was prevented by the presence, in the 5' untranslated leader, of the maize transposons Activator (Ac) or Dissociation (Ds) . When progeny of tobacco or tomato plants transformed with these constructs were germinated on spectinomycin-containing medium, germinally revertant and somatically variegated individuals could be distinguished. Infect Control Hosp Epidemiol, 1994 Jul, 15(7), 472 - 7 Antibiotic resistance is selected primarily in our patients; Pechere JC; The potential for bacterial resistance probably existed prior to the arrival of humans on earth and bacterial populations isolated before the antibiotic era surely contained antibiotic-resistant organisms . Antibiotic resistance has undergone an explosive development following the introduction of antibiotics in medical practice and in agriculture, and there is no doubt that the higher prevalence of bacterial resistance is closely related to human activities . Strict infection control policies limit the risk of patient-to-patient transmission of resistant as well as susceptible bacteria. Nucleic Acids Res, 1994 Jun 11, 22(11), 2071 - 8 Characterisation of specific and secondary recombination sites recognised by the integron DNA integrase; Recchia GD et al.; Integrons determine a site-specific recombination system which is responsible for the acquisition of genes, particularly antibiotic resistance genes . The integrase encoded by integrons recognises two distinct classes of recombination sites . The first is the family of imperfect inverted repeats, known as 59-base elements, which are associated with the mobile gene cassettes . The second consists of a single site into which the cassettes are inserted . This site, here designated attI, is located adjacent to the int gene in the recipient integron structure . The attI site has none of the recognisable features of members of the 59-base element family except for a seven-base core site, GTTRRRY, at the recombination crossover point . Using a conduction assay to quantitate site activity, the sequence required for maximal attI site activity was confined to a region of > 39 and < or = 70 bases . Both integrative and excisive site-specific recombination events involving attI and a 59-base element site were demonstrated, but no evidence for events involving two attI sites was obtained . Integrase-mediated recombination between a 59-base element and several secondary sites in pACYC184 with the consensus GNT occurred at low frequency, and such events could potentially lead to insertion of gene cassettes at many non-specific sites. J Bacteriol, 1994 Jun, 176(11), 3257 - 68 Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements; Radstrom P et al.; Integrons confer on bacterial plasmids a capability of taking up antibiotic resistance genes by integrase-mediated recombination . We show here that integrons are situated on genetic elements flanked by 25-bp inverted repeats . The element carrying the integron of R751 has three segments conserved with similar elements in Tn21 and Tn5086 . Several characteristics suggest that this element is a transposon, which we call Tn5090 . Tn5090 was shown to contain an operon with three open reading frames, of which two, tniA and tniB, were predicted by amino acid similarity to code for transposition proteins . The product of tniA (559 amino acids) is a probable transposase with 25% amino acid sequence identity to TnsB from Tn7 . Both of these polypeptides contain the D,D(35)E motif characteristic of a protein family made up of the retroviral and retrotransposon IN proteins and some bacterial transposases, such as those of Tn552 and of a range of insertion sequences . Like the transposase genes in Tn552, Mu, and Tn7, the tniA gene was followed by a gene, tniB, for a probable ATP-binding protein . The ends of Tn5090, like those of most other elements producing D,D(35)E proteins, begin by 5'-TG and also contains a complex structure with four 19-bp repeats at the left end and three at the right end . Similarly organized repeats have been observed earlier at the termini of both Tn7 and phage Mu, where they bind their respective transposases and have a role in holoenzyme assembly . Another open reading frame observed in Tn5090, tniC, codes for a recombinase of the invertase/resolvase family, suggesting a replicative transposition mechanism . The data presented here suggest that Tn5090, Tn7, Tn552, and Mu form a subfamily of bacterial transposons which in parallel to many insertion sequences are related to the retroelements. FASEB J, 1994 Jun, 8(9), 639 - 45 Bcl-2 inhibits glucocorticoid-induced apoptosis but only partially blocks calcium ionophore or cycloheximide-regulated apoptosis in S49 cells; Caron-Leslie LA et al.; Many non-Hodgkins B-cell lymphomas possess a deregulated bcl-2 gene resulting in a phenotype that is apparently resistant to programmed cell death (apoptosis) . We have used a mouse lymphoma cell line (S49.1) that undergoes apoptosis in response to a variety of stimuli to determine the effect of bcl-2 expression on induction of apoptosis . S49 cells were stably transfected with recombinant amphotrophic retroviruses carrying either a G418 antibiotic resistance gene alone (S49-NEO) or this gene in combination with a bcl-2 complementary DNA (S49-Bcl-2) . Three different agents previously shown to activate apoptosis by different pathways in S49 cells (dexamethasone, the calcium ionophore A23187, and cycloheximide) were used to examine the effect of bcl-2 expression on cell growth and apoptosis caused by multiple signal transduction pathways . Dexamethasone (DEX) treatment inhibited cell growth and stimulated cell death in S49-NEO cells . Although S49-Bcl-2 cells exhibited a similar antiproliferative response, they failed to die in response to steroid treatment . Western blot analysis revealed no difference in the levels of glucocorticoid receptor protein in the two cell lines, and both responded to glucocorticoid with a profound inhibition of protein synthesis . Cycloheximide (CX) and A23187 also had antiproliferative and cell killing effects in both cell types, although higher concentrations of each agent were needed to kill S49-Bcl-2 cells . To determine whether the loss of viability in response to these drugs was due to apoptosis, cells were examined morphologically and DNA integrity was examined by gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS) EMBO J, 1994 Jun 1, 13(11), 2483 - 92 Crystal structure and site-directed mutagenesis of a bleomycin resistance protein and their significance for drug sequestering; Dumas P et al.; The antibiotic bleomycin, a strong DNA cutting agent, is naturally produced by actinomycetes which have developed a resistance mechanism against such a lethal compound . The crystal structure, at 2.3 A resolution, of a bleomycin resistance protein of 14 kDa reveals a structure in two halves with the same alpha/beta fold despite no sequence similarity . The crystal packing shows compact dimers with a hydrophobic interface and involved in mutual chain exchange . Two independent solution studies (analytical centrifugation and light scattering) showed that this dimeric form is not a packing artefact but is indeed the functional one . Furthermore, light scattering also showed that one dimer binds two antibiotic molecules as expected . A crevice located at the dimer interface, as well as the results of a site-directed mutagenesis study, led to a model wherein two bleomycin molecules are completely sequestered by one dimer . This provides a novel insight into antibiotic resistance due to drug sequestering, and probably also into drug transport and excretion. Gene, 1994 May 16, 142(2), 225 - 30 Three selectable markers for transformation of Ustilago maydis; Gold SE et al.; Although Ustilago maydis is readily amenable to molecular genetic experimentation, few antibiotic-resistance markers are available for DNA-mediated transformation . This poses constraints on experiments involving targeted gene disruption and complementation . To address this problem, we constructed vectors using one of three additional genes as dominant selectable markers for transformation . Two genes, sat-1 (encoding streptothricin acetyltransferase) and Sh-ble (encoding a phleomycin-resistance polypeptide), are of bacterial origin and have been engineered for expression in Ustilago sp . The third gene encodes an allele of U . maydis beta-tubulin that confers resistance to the fungicide benomyl. Gene, 1994 May 3, 142(1), 49 - 54 Diversity and relative strength of tandem promoters for the antibiotic-resistance genes of several integrons; Levesque C et al.; The integron is a new type of mobile element containing one or more antibiotic-resistance-encoding genes site-specifically integrated as cassettes . The integrated genes are expressed from a common promoter region located in an adjacent conserved segment . Sequence analysis has revealed the existence of four versions of the integron promoters . In this study, we have determined the relative strength of the different integron promoters and compared their activity with that of the tac promoter . Each version of the promoter was cloned upstream from a promoter-less chloramphenicol acetyltransferase-encoding gene (cat) in plasmid pKK232-8 . CAT activity was used to measure transcriptional expression from the promoters of the antibiotic-resistance operon . The strongest promoter is the version (TTGACAN17TAAACT) found in plasmid R388 and in transposon Tn1696 . This promoter is six times more efficient than the derepressed tac promoter. J Clin Microbiol, 1994 May, 32(5), 1318 - 21 Oligonucleotide (GTG)5 as a marker for Mycobacterium tuberculosis strain identification; Wiid IJ et al.; Culture of Mycobacterium tuberculosis provides no information on the identity of a strain or the distribution of such a strain in the community . Strain identification of M . tuberculosis can help to address important epidemiological questions, e.g., the origin of an infection in a patient's household or community, whether reactivation of infection is endogenous or exogenous in origin, and the spread and early detection of organisms with acquired antibiotic resistance . To research this problem, strain identification must be reliable and accurate . Although genetic identification techniques already exist, it is valuable to have genetic identification techniques based on a number of genetic markers to improve the accurate identification of M . tuberculosis strains . We show that oligonucleotide (GTG)5 can be successfully applied to the identification of M . tuberculosis strains . This technique may be particularly useful in cases in which M . tuberculosis strains have few or no insertion elements (e.g., IS6110) or in identifying other strains of mycobacteria when informative probes are lacking. J Bacteriol, 1994 May, 176(9), 2525 - 31 Lipid synthesis in mycobacteria: characterization of the biotin carboxyl carrier protein genes from Mycobacterium leprae and M . tuberculosis; Norman E et al.; The causative agents of leprosy and tuberculosis, Mycobacterium leprae and Mycobacterium tuberculosis, have a lipid-rich cell envelope which contributes to virulence and antibiotic resistance . Acyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis, consists in mycobacteria of two subunits, one of which is biotinylated . Genes from M . leprae and M . tuberculosis encoding a biotinylated protein have been cloned and sequenced . Analysis of the derived protein sequences demonstrated the presence of biotin-binding sites and putative ATP-bicarbonate interactions sites, consistent with the proteins having a biotin carboxylase function as well as their being biotin carrier proteins. Med J Aust, 1994 Apr 4, 160(7), 395 - 7 Tuberculosis: medical students at risk; Wilkins D et al.; In 1979 an outbreak of tuberculosis occurred in medical students at the University of Sydney . Eight of 35 Mantoux-negative students who attended the autopsy of an immunosuppressed patient with unsuspected active tuberculosis became infected and one developed clinical disease . A report of the incident was prepared for publication because it supported the then controversial University policy of recommending BCG vaccination to medical and dental students in a country where the reported prevalence of tuberculosis is very low . The report was never published, mainly in order to protect the privacy of the individual students involved, but also because it was felt by the administration of the time that it might undermine confidence in infection control procedures in the autopsy room . The original report, updated and reproduced here, suggested that tuberculosis might be an emerging nosocomial problem . This has been all too clearly realised since its re-emergence as an opportunistic infection in AIDS patients . Worldwide, the problem of antibiotic resistance in Mycobacterium tuberculosis provides an added risk of a return to the situation which prevailed early this century when tuberculosis was a major occupational risk for young health care workers . Infection often restricted career choices, even in those whose disease was relatively benign . Our purpose in bringing this incident to light after so many years is to point out the relevance of the extensive studies of the problem which were conducted in the 1930s and 1940s to the current situation and to suggest that health care students are vulnerable to airborne infections as well as those spread by inoculation injuries . In retrospect, our 1979 conclusions about prospects for preventing nosocomial tuberculosis appear optimistic. Mol Microbiol, 1994 Apr, 12(2), 217 - 29 Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of beta-lactamase; Palzkill T et al.; In order to understand how TEM-1 beta-lactamase substrate specificity can be altered by mutation, amino acid residues 161 through to 170 were randomly mutagenized to sample all possible amino acid substitutions . The 161-170 region includes a portion of an omega loop structure, which is involved in the formation of the active-site pocket . The percentage of random sequences that provide bacterial resistance to either ampicillin or to the extended-spectrum cephalosporin ceftazidime was determined . It was found that the sequence requirements for wild-type levels of ampicillin resistance are much more stringent than the sequence requirements for ceftazidime resistance . Surprisingly, more than 50% of all amino acid substitutions in the 161-170 region result in levels of ceftazidime resistance at least three times greater than wild type . In addition, by increasing the level of the selection for ceftazidime resistance, substitutions that result in a greater than 100-fold increase in ceftazidime resistance were identified . Characterization of altered beta-lactamase enzymes indicated that while their catalytic efficiency (kcat/Km) for ceftazidime hydrolysis is higher, the enzymes are poorly expressed relative to wild-type TEM-1 beta-lactamase. Biotechniques, 1994 Apr, 16(4), 626 - 8, 630-3 Phagemid pSIT permits efficient in vitro mutagenesis and tightly controlled expression in E . coli; Andreansky M et al.; A new phagemid vector, pSIT, was constructed that allows both oligonucleotide-directed mutagenesis and tightly controlled, high-level expression of proteins in Escherichia coli . An efficient rate of mutagenesis is achieved by taking advantage of the double oligonucleotide primer technique . In addition to the mutagenic primer, a second oligonucleotide primer conferring antibiotic resistance to the mutant DNA strand is annealed to single-strand DNA . Selection for the antibiotic thus increases the frequency of mutants . High-level and tightly controlled expression of heterologous proteins is enabled by utilizing a very strong hybrid T7lac promoter and lac repressor in conjunction with T7 RNA polymerase, as well as a high copy number of the vector . The pSIT phagemid permits overexpression of proteins and their mutants without subclonings from mutagenic to expression constructs, which saves time, especially when multiple mutations of the same protein are proposed . A retroviral proteinase precursor, toxic for E . coli, was successfully expressed to a high level, and a series of mutants of this protein was readily obtained. Plasmid, 1994 Mar, 31(2), 166 - 83 Identification of a gene encoding the replication initiator protein of the Streptomyces integrating element, pSAM2; Hagege J et al.; pSAM2 is an 11-kilobase integrating element from Streptomyces ambofaciens which was previously shown to generate single-stranded DNA during replication, indicating that it probably replicates by a rolling-circle replication (RCR) mechanism . Two separate regions are involved in its replication, one of which was shown to contain the plus origin of replication (ds origin) . We report here the study of the second region . Its nucleotide sequence was determined and analysed for open reading frames (ORFs) . Three putative ORFs were identified: orf183 (183 amino acids (aa)), orf50 (50 aa), and repSA (459 aa) . orf183 is not necessary for replication . The function of orf50 is unknown . repSA is essential for pSAM2 replication; it could encode a protein, RepSA, presenting similarities to the replication initiator proteins (Rep) of elements that replicate by an RCR mechanism . A derivative consisting of repSA, the region containing ds origin, a Streptomyces antibiotic resistance marker, and pBR322, could replicate in Streptomyces, further demonstrating that this ORF encodes the major replication protein of pSAM2 . repSA might be co-transcribed with the genes involved in integration and excision of pSAM2. Genetics, 1994 Mar, 136(3), 867 - 77 A suppressor of a mating-type limited zygotic lethal allele also suppresses uniparental chloroplast gene transmission in Chlamydomonas monoica; VanWinkle-Swift K et al.; Uniparental inheritance of Chlamydomonas chloroplast genes is thought to involve modification of maternal (mt+) chloroplast genomes to protect against a nuclease that is activated after gamete fusion . The mating-type limited mtl-1 mutant strain of Chlamydomonas monoica is unable to protect mt(+)-derived chloroplast DNA . Zygotes homozygous for mtl-1 lose all chloroplast DNA and fail to germinate . We have selected for suppression of this zygote-specific lethality, and have obtained 20 mutant strains that produce viable homozygotes despite the continued presence of the mtl-1 allele . Gene