D.A. Ritchie

Molecular Ecological Analysis of Methanogens and Methanotrophs in Blanket Bog Peat

A bstractMethane production and methane oxidation potential were measured in a 30 cm peat core from the Moorhouse Nature Reserve, UK. The distribution of known groups of methanogens and methane oxidizing bacteria throughout this peat core was assessed. Using 16S rRNA gene retrieval and functional gene probing with genes encoding key proteins in methane oxidation and methanogenesis, several major groups of microorganisms were detected. Methane production and oxidation was detected in all depths of the peat core. PCR amplification and oligonucleotide probing experiments using DNA isolated from all sections of the peat core detected methanotrophs from the groups Methylosinus and Methylococcus and methanogens from the groups Methanosarcinaceae, Methanococcaceae, and Methanobacteriaceae. 16S rDNA sequences amplified with the Methylosinus-specific primer were shown to have a high degree of identity with 16S rDNA sequences previously detected in acidic environments. However, no methanogen sequences were detected by the probes available in this study in the sections of the peat core (above 7 cm) where the majority of methanogenesis occurred, either because of low methanogen numbers or because of the presence of novel methanogen sequences.

Genetic and physical analysis of plasmid genes expressing inducible resistance of tellurite in Escherichia coli

SummaryA large (>250 kb) conjugative plasmid, pMER610, specifying resistance to tellurium and mercury was isolated from an Alcaligenes strain and transferred by conjugation to Escherichia coli AB1157. The acquisition of pMER610 by AB1157 increased the resistance to both tellurite and tellurate by 100-fold. Expression of tellurite resistance by pMER610 and the cloned Ter determinant was inducible by prior exposure to tellurite at levels sub-toxic to the sesitive AB1157. Physical analysis of the cloned Ter fragment located the resistance determinant to a 3.55 kb region. Insertion of Tn 1000 (γδ) into this region produced two classes of sensitive mutations, fully sensitive and intermediate or hyposensitive, which map in adjacent regions and form two complementation groups. Maxicell analysis identified four polypeptides (15.5, 22, 23 and 41 kDa) expressed by the Ter clone. The 23 kDa polypeptide may not be required for resistance since tellurium-sensitive γδ insertion mutations were not detected in the 23 kDa coding region.

Analysis of Methanogen Diversity in a Hypereutrophic Lake Using PCR-RFLP Analysis of mcr Sequences

The incidence and diversity of methanogens in Priest Pot, a dynamic and active lake, were monitored by analysing mcrA gene sequences generated from total DNA samples obtained at different times of the year and amplified using the polymerase chain reaction. A number of mcrA clones were analysed by developing an RFLP-based protocol to generate a number of restriction patterns that were assigned to a number of classes. The RFLP patterns for each class were compared with published sequence information for mcrA from cultured methanogens as well as with those from other experimental studies. They could be used to assign tentative identification for some of the Priest Pot clones and also revealed the presence of a number of clones that could not be affiliated to any known methanogens. The limitations of using RFLP profiles of mcrA gene sequences for studying methanogen ecology are discussed.

Genetic diversity within mer genes directly amplified from communities of noncultivated soil and sediment bacteria

Individual merRTΔP 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 (= 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 merRTΔP 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.

Novel mercury resistance determinants carried by INcJ plasmids pMERPH and R391

SummaryHgCl2 resistance (Hgr) in a strain of Pseudomonas putrefaciens isolated from the River Mersey was identified as plasmid-borne by its transfer to Escherichia coli in conjugative matings. This plasmid, pMERPH, could not be isolated and was incompatible with the chromosomally integrated IncJ Hgr plasmid R391. pMERPH and R391 both express inducible, narrow-spectrum mercury resistance and detoxify HgCl2 by volatilization. The cloned mer determinants from pMERPH (pSP100) and R391 (pSP200) have very similar restriction maps and express identical polypeptide products. However, these features show distinct differences from those of the Tn501 family of mer determinants. pSP100 and pSP200 failed to hybridize at moderate stringency to merRTPA and merC probes from Tn501 and Tn21, respectively. We conclude that the IncJ mer determinants are only distantly related to that from Tn501 and its closely homologous relatives and that it identifies a novel sequence which is relatively rare in bacteria isolated from natural environments.

Sequence conservation between regulatory mercury resistance genes in bacteria from mercury polluted and pristine environments

Summary The regulatory gene merR , and the adjacent operator/promoter region was amplified from the mercury resistance (Hg R ) determinants from 10 Gram-negative bacterial isolates from mercury polluted and pristine environments using the polymerase chain reaction. These mer regions showed polymorphism in size of PCR amplification products with those from isolates SE3, SE11, SE12, SE31, SO1 and T217 being of 557 base pairs in size, whilst those from isolates SE20, T238, SB3, SB4 and the positive control (Tn 501 ) were 536 base pairs in size. From the sequence analysis of these mer regions and comparison with previously sequenced Hg R determinants an evolutionary tree was constructed which showed there to be a significant difference between Gram- negative merR genes and those found in Gram-positive organisms. With the exception of the Thiobacillus Hg R determinants, merR genes from Gram negative bacteria were strongly conserved and could be grouped closely around the previously sequenced determinants of Tn 501 , Tn 21 , Tn 5053 (pMER327/419) and pKLH2. Only the merR genes of pDU1358 and T238 showed significant variation from these subgroups. The regions of greatest variation were the carboxyl terminal coding region of the merR gene and the operator/promoter region. It is suggested that, due to the global nature of inducible mercury resistance and its strong sequence conservation across large geographical distances, bacterial resistance to mercury is an ancient genetic character.

The mercury resistance operon of the IncJ plasmid pMERPH exhibits structural and regulatory divergence from other Gram-negative mer operons

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.

A physical map of the permuted genome of bacteriophage T1.

SummaryA restriction map has been constructed for the DNA of coliphage T1 which locates the cleavage sites of the restriction endonucleases, BglI (6 cuts), BglII (16 cuts), EcoRI (2 cuts), HindIII (2 cuts) and PstI (2 cuts). Digestions with BglI and BglII reveal fragments which are present in sub-molar quantities. Two methods, one using the selective removal of molecular ends with exonuclease III and the other involving the comparison of digestion patterns of concatemeric and virion DNA, have shown that the submolar fragments are at or close to the ends of the molecules. Digestions with BglI show that one terminal fragment has a very precise molecular weight whereas all the others are of heterogenous molecular weight. These results are consistent with the model for DNA packaging in which maturation is initiated at a precise site on a concatemeric precursor and proceeds by the encapsidation of up to four successive ‘headfuls’ of 1.065 genome equivalents (MacHattie and Gill 1977).

Functional expression of the tellurite resistance determinant from the IncHI-2 plasmid pMER610.

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 β-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 β-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 β-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.

Cloning and analysis of DNA sequences from Streptomyces hygroscopicus encoding geldanamycin biosynthesis

A gene library constructed from large (∼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 ∼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 ϕC31 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.