Peter Strike

R391: a Conjugative Integrating Mosaic Comprised of Phage, Plasmid, and Transposon Elements

The conjugative, chromosomally integrating element R391 is the archetype of the IncJ class of mobile genetic elements. Originally found in a South African Providencia rettgeri strain, R391 carries antibiotic and mercury resistance traits, as well as genes involved in mutagenic DNA repair. While initially described as a plasmid, R391 has subsequently been shown to be integrated into the bacterial chromosome, employing a phage-like integration mechanism closely related to that of the SXT element from Vibrio cholerae O139. Analysis of the complete 89-kb nucleotide sequence of R391 has revealed a mosaic structure consisting of elements originating in bacteriophages and plasmids and of transposable elements. A total of 96 open reading frames were identified; of these, 30 could not be assigned a function. Sequence similarity suggests a relationship of large sections of R391 to sequences from Salmonella, in particular those corresponding to the putative conjugative transfer proteins, which are related to the IncHI1 plasmid R27. A composite transposon carrying the kanamycin resistance gene and a novel insertion element were identified. Challenging the previous assumption that IncJ elements are plasmids, no plasmid replicon was identified on R391, suggesting that they cannot replicate autonomously.

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.

Plasmid genes Affecting DNA Repair and Mutation

SUMMARY Many bacterial plasmids have the effect of increasing the ultraviolet (u.v.) resistance of host cells that harbour them, apparently by an error-prone repair mechanism that leads to a high level of mutation amongst the survivors. These plasmid systems are apparently analogues of the Escherichia coli umuD/C operon, which is absolutely required in this organism for mutation induced by u.v. light and by many chemical mutagens. This article reviews the extensive and sometimes conflicting literature relating to this phenomenon, and describes the further characterization of one such plasmid system, the imp (I group mutation and protection) operon of the I1 group plasmid TP110. It is demonstrated that each of the protection mutation systems well characterized to date shows a similar genetic arrangement, and that significant homology can be detected at the amino acid level between the proteins encoded by these different systems.

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.

Complete nucleotide sequence and gene organization of plasmid NTP16

We have determined the complete nucleotide sequence of the 8.3-kb multicopy plasmid NTP16 and produced a functional map of its gene organization. Sixty percent of the plasmid DNA comprises transposon-derived sequences; in the remaining 3320 bp, we have identified three protein coding regions. NTP16 has a ColE1-type replication system, a cis-acting stability locus and a mobilization system comprising an oriT site and one mobilization protein. The roles of the other two protein products of this plasmid are unknown, but they are possibly involved in the plasmid incompatibility system.

Functional complementation between chromosomal and plasmid mutagenic DNA repair genes in bacteria

SummaryThe umuDC operons of Escherichia coli and Salmonella typhimurium and the analogous plasmid operons mucAB and impCAB have been previously characterized in terms of their roles in DNA repair and induced mutagenesis by radiation and many chemicals. The interrelationships of these mutagenic DNA repair operons were examined in vivo in functional tests of interchangeability of operon subunits in conferring UV resistance and UV mutability phenotypes to wild-type S. typhimurium and umu mutants of E. coli. This approach was combined with DNA and protein sequence comparisons between the four operons and a fifth operon, samAB, from the S. typhimurium LT2 cryptic plasmid. Components of the E. coli and S. typhimurium umu operons were reciprocally interchangeable whereas impCA and mucA could not function with umuC in either of these species. mucA and impB could also combine to give a mutagenic response to UV. These active combinations were associated with higher degrees of conservation of protein sequence than in other heterologous gene combinations and related to specific regions of sequence that may specify subunit interactions. The dominance of the E. coli umuD44 mutation over umuD was revealed in both wild-type E. coli and S. typhimurium and also demonstrated against impCAB. Finally interspecies transfer showed that the apparently poor activity of the S. typhimurium umuD gene in situ is not the result of an inherent defect in umuD but is due to the simultaneous presence of the S. typhimurium umuC sequence. It is suggested that the limitation of umuD activity by umuC in S. typhimurium is the basis of the poor induced mutability of this organism.

Efficiency of Escherichia coli repair processes on uv-damaged transforming plasmid DNA

Abstract A comparison has been made of the efficiencies with which the dark repair processes of Escherichia coli act on ultraviolet irradiated bacterial chromosomal DNA and ultraviolet damaged transforming plasmid DNA. It is shown that postreplicational repair pathways act very inefficiently on transforming plasmid DNA, and that the majority of repair is carried out by excision repair pathways. However, even excision repair pathways act less efficiently on damaged plasmid DNA than they do on chromosomal DNA. The large effect of mutations in recB on plasmid survival suggests that the product of this gene may be essential for the excision repair pathways which act on plasmid DNA, but not for those which act on chromosomal DNA.

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.