Biomedical subjects
Publications and source records attributed to D A Ritchie.
Increasing firearms violence has produced much public disquiet in recent months and Liverpool has seen a particularly well publicized spate of shootings. This is a case report of an initially occult intracranial injury which illustrates the unpredictable nature of missile trauma and the importance of computerised tomography in all cases of gunshot injury to the head.
The bacterial mercury resistance determinant carried on the IncJ plasmid pMERPH has been characterized further by DNA sequence analysis. From the sequence of a 4097 bp Bg/II fragment which confers mercury resistance, it is predicted that the determinant consists of the genes merT, merP, merC and merA. The level of DNA sequence similarity between these genes and those of the mer determinant of Tn21 was between 56 center dot 4 and 62 center dot 4%. A neighbour-joining phylogenetic tree of merA gene sequences was constructed which suggested that pMERPH bears the most divergent Gram-negative mer determinant characterized to date. Although the determinant from pMERPH has been shown to be inducible, no regulatory genes have been found within the Bg/II fragment and it is suggested that a regulatory gene may be located elsewhere on the plasmid. The cloned determinant has been shown to express mercury resistance constitutively. Analysis of the pMERPH mer operator/promoter (O/P) region in vivo has shown constitutive expression from the mer PTCPA promoter, which could be partially repressed by the presence of a trans-acting MerR protein from a Tn21-like mer determinant. This incomplete repression of mer PTCPA promoter activity may be due to the presence of an extra base between the -35 and -10 sequences of the promoter and/or to variation in the MerR binding sites in the O/P region. Expression from the partially repressed mer PTCPA promoter could be restored by the addition of inducing levels of Hg2+ ions. Using the polymerase chain reaction with primers designed to amplify regions in the merP and merA genes, 1 center dot 37 kb pMERPH-like sequences have been amplified from the IncJ plasmid R391, the environmental isolate SE2 and from DNA isolated directly from non-cultivated bacteria in River Mersey sediment. This suggests that pMERPH-like sequences, although rare, are nevertheless persistent in natural environments.
The gene encoding the principal sigma factor from Synechococcus sp. strain PCC 7002 was isolated and characterized. The Synechococcus sp. strain PCC 7002 sigA gene encodes a protein of 375 amino acids (43 center dot 7 kDa) that is required for viability under normal growth conditions. The SigA protein was overproduced in Escherichia coli and the purified protein was used to raise polyclonal antiserum in rabbits. This antiserum was used in immunoblot analyses of partially purified RNA polymerase from Synechococcus sp. strain PR6000. The probable in vivo translational start site was identified by a comparison of amino acid sequencing results obtained with SigA proteins overproduced in E. coli with immunoblot analyses of SigA protein in crude preparations of RNA polymerase from the cyanobacterium. The sigA gene is encoded on a transcript of 1700 bases that initiates 496 nucleotides upstream from the probable in vivo translational start site. The abundance of sigA transcripts decreases rapidly after the removal of combined nitrogen from the growth medium.
The presence of methanogenic bacteria was assessed in peat and soil cores taken from upland moors. The sampling area was largely covered by blanket bog peat together with small areas of red-brown limestone and peaty gley. A 30-cm-deep core of each soil type was taken, and DNA was extracted from 5-cm transverse sections. Purified DNA was subjected to PCR amplification with primers IAf and 1100Ar, which specifically amplify 1.1 kb of the archaeal 16S rRNA gene, and ME1 and ME2, which were designed to amplify a 0.75-kb region of the alpha-subunit gene for methyl coenzyme M reductase (MCR). Amplification with both primer pairs was obtained only with DNA extracted from the two deepest sections of the blanket bog peat core. This is consistent with the notion that anaerobiosis is required for activity and survival of the methanogen population. PCR products from both amplifications were cloned, and the resulting transformants were screened with specific oligonucleotide probes internal to the MCR or archaeal 16S rRNA PCR product. Plasmid DNA was extracted from probe-positive clones of both types and the insert was sequenced. The DNA sequences of 8 MCR clones were identical, as were those of 16 of the 17 16S rRNA clones. One clone showed marked variation from the remainder in specific regions of the sequence. From a comparison of these two different 16S rRNA sequences, an oligonucleotide was synthesized that was 100% homologous to a sequence region of the first 16 clones but had six mismatches with the variant. This probe was used to screen primary populations of PCR clones, and all of those that were probe negative were checked for the presence of inserts, which were then sequenced. By using this strategy, further novel methanogen 16S rRNA variants were identified and analyzed. The sequences recovered from the peat formed two clusters on the end of long branches within the methanogen radiation that are distinct from each other. These cannot be placed directly with sequences from any cultured taxa for which sequence information is available.
Southern hybridization was performed on 30 gram-negative, mercury-resistant soil bacteria isolated from three terrestrail sites in Great Britain; two of these sites were mercury polluted (SO and SE), and one was pristine (SB). Most of the isolates (20 of 30) hybridized to probes encoding regions of the transposase (tnpA) and resolvase (tnpR) genes from Tn501 and Tn21. Isolates SE9 and SB3 hybridized to the Tn21 but not the Tn501 tnpA probe; however, they differed in that SB3 hybridized to both Tn501 and Tn21 tnpR probes while SE9 did not hybridize to either tnpR probe. The remaining isolates (7 of 30) did not hybridize to any of the transposon gene probes under the conditions used. tnpA and tnpR regions were PCR amplified from most of the hybridizing isolates and from Tn501 and Tn21, and variation was assessed by restriction fragment length polymorphism analysis. On the basis of these data, tnpA regions were divided into eight restriction fragment length polymorphism classes and tnpR regions were divided into five classes. Similarity coefficients were calculated between classes and used to construct dendrograms showing percent similarity. A compilation of the data from this study on tnpA and tnpR regions and a previous study on merRT delta P regions (A. M. Osborn, K. D. Bruce, P. Strike, and D. A. Ritchie, Appl. Environ. Microbiol. 59:4024-4030, 1993) indicates the presence of hybrid transposons and provides evidence for extensive recombination, both between transposon genes and between transposon and mer genes, within these natural populations of bacteria.
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RNA isolated at intervals during fermentation from the novobiocin-producing wild-type strain of Streptomyces niveus and from a series of novobiocin-non-producing (Nov-) mutants was hybridized to DNA probes containing sequences which specify novobiocin resistance. The probes were made from inserts contained in the clones pGL101 and pGL103 which increase the level of novobiocin resistance of S. lividans transformants from 10 micrograms ml-1 to 50 micrograms ml-1 and 150 micrograms ml-1, respectively. No hybridization was detected with the pGL101 probe. The pGL103 probe hybridized to RNA extracted during the later stages of growth--a pattern corresponding to the transition from low to high level novobiocin resistance during growth of S. niveus wild-type cultures. Neither probe hybridized to RNA extracted from four Nov- mutants. These mutants showed variable levels of novobiocin resistance but none expressed the high wild-type levels. The authors conclude that expression of the DNA sequence in pGL103 is associated with high level novobiocin resistance.
Individual merRT delta P regions were amplified from DNA directly isolated from soil and sediment samples using consensus primers derived from the conserved mer sequences of Tn501, Tn21 and pMER419. Soil and sediment samples were taken from four sites in the British Isles; one 'pristine' (SB) and three polluted (SO, SE, T2) with respect to mercury. The sizes of the PCR products amplified (approximately 1 kb) were consistent with their generation from mer determinants related to the archetypal elements found in Gram negative bacteria. Forty-five individual clones of sequences obtained from these four sites were isolated which hybridized (> 70% homology) to a merRT delta P probe from Tn501. The diversity of these amplified mer genes was analysed using Restriction Fragment Length Polymorphism (RFLP) profiling. Fourteen RFLP classes were distinguished, 12 of which proved to be novel and only two of which had been identified in an earlier study of 40 Gram negative mercury resistant bacteria cultured from the same four sites. UPGMA analysis was used to examine the relationships between the 22 classes of determinant identified. The T2 site, which has the longest history of mercury exposure, was found to have the greatest level of diversity in terms of numbers of classes of determinant, while the SO site, which had the highest mercury levels showed relatively low variation. Variation of mer genes within and between the sequences from cultivated bacteria and from total bacterial DNA shows clearly that analysing only sequences from cultivated organisms results in a gross underestimation of genetic variation.
Three different, independently isolated mercury-resistance-conferring plasmids, pMER327/419, pMER330 and pMER05, from cultures originating from the river Mersey (UK), contain identical regulatory merR genes and transposon ends. The mer determinant from pMER327/419 contains an additional potential ORF (ORF F) located between merP and merA when compared with the archetypal Tn501. Although these plasmids confer narrow-spectrum resistance (resistance to Hg2+, but not organomercurials) their merR genes encode a potential organomercurial-sensing protein. Transposition of the mer of pMER05 into plasmid RP4 was demonstrated and, as with Tn502 and Tn5053, insertion occurred at a specific region. The sequence of pMER05 is identical at the 'left' and 'right' termini and across merR to Tn5053, which was independently isolated from the chromosome of a Xanthomonas sp. bacteria from the Khaidarkan mercury mine in Kirgizia, former Soviet Union [Kholodii et al., J. Mol. Biol. 230 (1993a) 1103-1107]. The transpositional unit of pMER05 is, like that of Tn5053, bounded by DNA homologous to the imperfect 25-bp inverted repeats (IR) of the In2 integron, which brackets antibiotic-resistance cassettes in Tn21 subgroup transposons. At one end of the transposable element, and internal to the In2-like IR, is a 38-bp IR which closely resembles the IR that bounds Tn21.
A gene library constructed from large (approximately 20 kb) fragments of total DNA from the geldananmycin-producing strain Streptomyces hygroscopicus 3602 cloned in the plasmid vector pIJ61 were used to transform S. lividans TK24. Three transformants of about 800 tested were found to have acquired the ability to produce an antibiotic lethal to a geldanamycin-sensitive strain of Bacillus subtilis. The plasmids isolated from these transformants, pIA101, pIA102 and pIA103, each contained an insert of approximately 15 kb. A 4.5 kb DNA fragment from the insert in pIA102 hybridised to DNA from S. hygroscopicus 3602 and to DNA encoding part of the erythromycin polyketide synthase but not to S. lividans TK24 DNA. The integration-defective phage vector phi C31 KC515 containing this 4.5 kb fragment was able to lysogenise S. hygroscopicus 3602 to produce lysogens defective in geldanamycin production. Loss of the prophage restored the ability to produce geldanamycin. Extracts of fermentation broth cultures of S. lividans containing pIA101, pIA102 and pIA102 and pIA103 analysed by thin-layer chromatography (TLC) contained compounds identical or very similar to purified geldanamycin, which were not present in S. lividans. These compounds showed a mass spectrum indistinguishable from geldanamycin. The evidence suggests that the clones contain DNA sequences encoding functions required for geldanamycin biosynthesis including components of the polyketide synthase.
The tellurite accumulation properties of three Escherichia coli strains containing different tellurium-resistance determinants of Gram-negative origin, from plasmids pMER610, pHH1508a and RK2, were compared. In all three cases membrane-associated tellurium crystallization was observed, and neither reduced uptake nor increased export contributed to the resistance. Specific membrane-proximal reduction is proposed as the mechanism of resistance to tellurite coded by all three determinants, despite their lack of sequence homology.
During growth of Streptomyces niveus wild-type in the novobiocin production medium CDM the resistance of mycelia to novobiocin rises from about 25 micrograms/ml to over 200 micrograms/ml. (S. lividans, a novobiocin-sensitive strain, is resistant to approx. 10 micrograms/ml novobiocin.) The initial period of low level resistance extends from the time of inoculation of the culture until approx. 70 h when the culture is still in the growth phase. High level resistance is initiated before the start of novobiocin production and rises rapidly to a maximum level beyond the end of the growth phase. The rise in pH of the unbuffered CDM medium which occurs during S. niveus fermentation was shown not to be the cause of the change in novobiocin resistance. However, mycelia-free CDM from S. niveus cultures expressing high level novobiocin resistance was shown to contain a factor which induced high level novobiocin resistance in germinating S. niveus spores. Kinetic studies revealed that the inducer first appears in the culture medium before the switch to high level resistance begins and reaches its highest concentration before resistance reaches its maximum level.
The transpositional phage MudI 1734 lacZ was used to construct transcriptional fusions within the plasmid pMJ611, which contains the cloned tellurite resistance (TeR) determinant of the IncHI-2 plasmid pMER610. A series of 70 MudI insertions, in both orientations, causing loss of tellurite resistance in pMJ611, mapped within a 4.3 kb region which included the genes terA-terD and a 0.4 kb region upstream of the site previously reported as the 5' limit of the TeR determinant. Expression of beta-galactosidase from these transcriptional fusions, including those involving the 5' upstream region, occurred only from inserts transcribed in the direction terA-terD, confirming the transcriptional orientation of the TeR determinant deduced from DNA sequence analysis. Sixteen of the tellurite-sensitive MudI fusions, distributed over the entire determinant and in both orientations, showed the same pattern of expression when transferred by conjugation and homologous recombination to pMER610, except that the beta-galactosidase levels were consistently 2- to 3-fold higher in the parent plasmid. Northern analysis with a DNA probe spanning the TeR determinant identified five transcripts of 4.8, 4.0, 2.7, 1.5 and 1.0 kb synthesised by pMER610. Further hybridisations with DNA probes defining sub-sections of the TeR determinant, together with DNA sequence analysis, suggested the presence of three transcriptional start sites, at approximately 0.9 and 0.1 kb upstream of terA, and near the junction between terC and terD. Three transcriptional termination sites, located within terA, near the terC-terD junction and at the 3' end of terE are also indicated. Both the expression of beta-galactosidase from the MudI fusions and the synthesis of ter gene transcripts are constitutive and were not affected by prior exposure of cultures to sub-toxic levels of tellurite. Further DNA sequence analysis reveals that the extensive homology between terD and terE extends to a section of terA.
Actinomycetes have the genetic capability to synthesize many different biologically active secondary metabolites and of these compounds, antibiotics predominate in therapeutic and commercial importance. Intensive research often centres on the use of molecular techniques to investigate the physiology and genetics of antibiotic biosynthesis with a view to improving production. The isolation of clones of Streptomyces hygroscopicus, the producer of geldanamycin, which synthesizes geldanamycin in S. lividans, is reported. Molecular approaches using genes for elongation factors (tuf) were used in attempts to increase the fermentation yield of kirromycin, whilst probes for aphD and sph, genes for streptomycin phosphotransferases, were used to gather information on streptomycin genes in soil. Actinomycete populations in soil and earthworms may help in developing a strategy for discovering additional antimicrobials in soil. The relationship of proline metabolism to the secondary metabolite undecylprodigiosin and the carbon regulation of spiramycin biosynthesis in S. ambofaciens is also reported.
Mercury resistant (Hgr) bacteria were isolated from four terrestrial sites: three containing high levels of mercury (sites T2, SE, and SO) and one uncontaminated site (SB). The frequencies of Hgr bacteria in the total cultivable populations were 0.05% (SB), 0.69% (SO), 4.8% (SE), and 25% (T2). Between 35 and 100% of the isolates from the four sites contained DNA sequences homologous to a DNA probe from the mercury resistance (mer) operon of the Tn501 Hgr determinant. The mer sequences of 10 Tn501-homologous Hgr determinants from each site were amplified by the polymerase chain reaction, with primers designed to consensus sequences of the mer determinants of Tn501, Tn21, and pMJ100, and were classified on the basis of the size of the amplified product and the restriction fragment length polymorphism pattern. Two main groups of amplification product were identified. The first, represented by the T2 and SB isolates and one SE isolate, gave an amplification product indistinguishable in size from that amplified from Tn501 (approximately 1,010 bp). The second group, represented by the SO isolates and the majority of the SE isolates, produced larger amplification products of 1,040 or 1,060 bp. Restriction fragment length polymorphism analysis revealed that each amplification product size group could be further subdivided into five subgroups.
The clinical and radiological features of 14 patients with ilio-psoas bursal enlargement associated with articular disease of the hip are presented. The condition primarily affects patients over the range of 50 years with pre-existing hip disease, usually of long duration. A clinical diagnosis before investigation was never made in our series as the symptoms were non-specific and varied, consisting of pain (40%), a palpable groin mass (40%) and lower limb swelling (28%). The diagnosis can be made pre-operatively and the extent along the path of the ilio-psoas accurately delineated. The bursal contents, although predominantly fluid in nature, do include a varying proportion of solid components. Ultrasound is the simplest, quickest and most cost effective means of demonstrating the hip effusion, the bursa, its contents and extent. Although the compressive effects are well seen sonographically, computed tomography (CT) and magnetic resonance imaging (MRI) provide a better appreciation of the regional anatomy and hip joint disease.
Specific DNA sequences from native bacterial populations present in soil, sediment, and sand samples were amplified by using the polymerase chain reaction with primers for either "universal" eubacterial 16S rRNA genes or mercury resistance (mer) genes. With standard amplification conditions, 1.5-kb rDNA fragments from all 12 samples examined and from as little as 5 micrograms of soil were reproducibly amplified. A 1-kb mer fragment from one soil sample was also amplified. The identity of these amplified fragments was confirmed by DNA-DNA hybridization.
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