Papers by Christopher Thomas
Regulation of Replication and Maintenance Functions of Broad Host-Range Plasmid RK2
Plasmids in Bacteria, 1985
Replication of broad host-range plasmid RK2 depends on a cisacting vegatative replication origin ... more Replication of broad host-range plasmid RK2 depends on a cisacting vegatative replication origin oriVRK2 and the polypeptide product(s) of the trans-acting gene trfA as well as on host-specified products. The trfA gene is the second cistron in a polycistronic unit whose first cistron may be kilD, one of 4 known RK2-specified kil loci (kilA, B, C, and D) which are inhibitory for bacterial host or plasmid vector in the absence of kor functions which suppress in trans the effect of their respective kil genes. Transcription of the operon containing trfA is negatively regulated by the products of both the trfB locus (alias korD and korA) and korB. The loci, trfB and korB, are expressed from a single transcriptional unit which we propose to be negatively autoregulated by the products of both loci, although an additional, weaker and unregulated transcript may also express korB. While deletions in the oriVRK2 region have indicated the presence of copy number control elements adjacent to and possibly overlapping with the minimal oriVRK2 segment, the overriding control of copy number seems to reside in the trfB and korB loci which in conjunction appear to reduce expression of the trfA gene to levels limiting for replication. Coregulation of trfA with kil genes may indicate that kil genes play a role in plasmid maintenance other than replication.
BMC Systems Biology, 2011
Background: IncP-1 plasmids are broad host range plasmids that have been found in clinical and en... more Background: IncP-1 plasmids are broad host range plasmids that have been found in clinical and environmental bacteria. They often carry genes for antibiotic resistance or catabolic pathways. The archetypal IncP-1 plasmid RK2 is a well-characterized biological system, with a fully sequenced and annotated genome and wide range of experimental measurements. Its central control operon, encoding two global regulators KorA and KorB, is a natural example of a negatively self-regulated operon. To increase our understanding of the regulation of this operon, we have constructed a dynamical mathematical model using Ordinary Differential Equations, and employed a Bayesian inference scheme, Markov Chain Monte Carlo (MCMC) using the Metropolis-Hastings algorithm, as a way of integrating experimental measurements and a priori knowledge. We also compared MCMC and Metabolic Control Analysis (MCA) as approaches for determining the sensitivity of model parameters. We identified two distinct sets of parameter values, with different biological interpretations, that fit and explain the experimental data. This allowed us to highlight the proportion of repressor protein as dimers as a key experimental measurement defining the dynamics of the system. Analysis of joint posterior distributions led to the identification of correlations between parameters for protein synthesis and partial repression by KorA or KorB dimers, indicating the necessary use of joint posteriors for correct parameter estimation. Using MCA, we demonstrated that the system is highly sensitive to the growth rate but insensitive to repressor monomerization rates in their selected value regions; the latter outcome was also confirmed by MCMC. Finally, by examining a series of different model refinements for partial repression by KorA or KorB dimers alone, we showed that a model including partial repression by KorA and KorB was most compatible with existing experimental data. Conclusions: We have demonstrated that the combination of dynamical mathematical models with Bayesian inference is valuable in integrating diverse experimental data and identifying key determinants and parameters for the IncP-1 central control operon. Moreover, we have shown that Bayesian inference and MCA are complementary methods for identification of sensitive parameters. We propose that this demonstrates generic value in applying this combination of approaches to systems biology dynamical modelling.
Applied and Environmental Microbiology, Nov 1, 2006
Although it is generally assumed that mobile genetic elements facilitate the adaptation of microb... more Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1 replicon-specific sequences detected in total community DNA. The isolation of IncP-1 plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1 plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1 plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRT⌬P probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1 plasmids R751 and pB8. However, this is the first example of an IncP-1 plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.
BMC Microbiology, Apr 4, 2012
Background Understanding the survival of resistance plasmids in the absence of selective pressure... more Background Understanding the survival of resistance plasmids in the absence of selective pressure for the antibiotic resistance genes they carry is important for assessing the value of interventions to combat resistant bacteria. Here, several poorly explored questions regarding the fitness impact of IncP1 and IncN broad host range plasmids on their bacterial hosts are examined; namely, whether related plasmids have similar fitness impacts, whether this varies according to host genetic background, and what effect antimicrobial resistance gene silencing has on fitness. Results For the IncP1 group pairwise in vitro growth competition demonstrated that the fitness cost of plasmid RP1 depends on the host strain. For the IncN group, plasmids R46 and N3 whose sequence is presented for the first time conferred remarkably different fitness costs despite sharing closely related backbone structures, implicating the accessory genes in fitness. Silencing of antimicrobial resistance genes was fou...
PLOS ONE, Nov 20, 2012
The korAB operon in RK2 plasmids is a beautiful natural example of a negatively and cooperatively... more The korAB operon in RK2 plasmids is a beautiful natural example of a negatively and cooperatively self-regulating operon. It has been particularly well characterized both experimentally and with mathematical models. We have carried out a detailed investigation of the role of the regulatory mechanism using a biologically grounded mechanistic multi-scale stochastic model that includes plasmid gene regulation and replication in the context of host growth and cell division. We use the model to compare four hypotheses for the action of the regulatory mechanism: increased robustness to extrinsic factors, decreased protein fluctuations, faster response-time of the operon and reduced host burden through improved efficiency of protein production. We find that the strongest impact of all elements of the regulatory architecture is on improving the efficiency of protein synthesis by reduction in the number of mRNA molecules needed to be produced, leading to a greater than ten-fold reduction in host energy required to express these plasmid proteins. A smaller but still significant role is seen for speeding response times, but this is not materially improved by the cooperativity. The self-regulating mechanisms have the least impact on protein fluctuations and robustness. While reduction of host burden is evident in a plasmid context, negative self-regulation is a widely seen motif for chromosomal genes. We propose that an important evolutionary driver for negatively self-regulated genes is to improve the efficiency of protein synthesis.
Journal of Bacteriology, 1980
The sites of cleavage on the map of the broad-host-range plasmid RK2 (56 kilobases) were determin... more The sites of cleavage on the map of the broad-host-range plasmid RK2 (56 kilobases) were determined for the BglII, PstI, and SmaI restriction enzymes, and the determinants for tetracycline and ampicillin resistance were localized. The cleavage sites were clustered at or near the drug resistance genes. To localize regions required for plasmid replication and maintenance in Escherichia coli, we deleted nonessential regions of RK2 by partial digestion with the restriction endonuclease HaeII to produce small derivatives. The smallest stable replicon obtained contained five HaeII fragments of RK2 which total 5.4 kilobases. These fragments were derived from three regions of RK2 that are separated from each other by antibiotic resistance genes. One of these HaeII fragments (0.75 kilobases) has the properties expected of the origin of replication. The outer four fragments, located in two separate regions of RK2, were found to provide, in trans, functions that permit the replication of the H...
Journal of General Microbiology, 1978
Journal of General Microbiology, 1978
The properties of a temperature-sensitive mutant (ts39) of staphylococcus aureus NCTC 8235 are de... more The properties of a temperature-sensitive mutant (ts39) of staphylococcus aureus NCTC 8235 are described. After transfer to the restrictive temperature (42 "C), absorbance increased 10to 20-fold but DNA content did not increase beyond 150 to 200 and cell division continued at a greatly reduced rate. On transfer back to the permissive temperature, both cell division and DNA synthesis resumed if the transfer occurred after less than 120 min at 42 "C. Resumption of DNA replication was blocked by chloramphenicol(lO0 pg ml-l). The results are discussed with reference to possible defects in DNA replication. Replication of the plasmids PI,,, and pT10501 and the chromosome were affected to a similar extent in ts39. Growth at 42 "C resulted in the appearance of an increased amount of p1268 DNA in a form that sedimented slowly in a sucrose gradient.
Journal of Antimicrobial Chemotherapy, 2018
ObjectivesTo assess stability and contribution of a large ESBL-encoding IncI1 plasmid to intestin... more ObjectivesTo assess stability and contribution of a large ESBL-encoding IncI1 plasmid to intestinal colonization by Escherichia coli O104:H4 in two different mammalian hosts.MethodsSpecific-pathogen-free 3–4-day-old New Zealand White rabbits and conventionally reared 6-week-old weaned lambs were orally infected with WT E. coli O104:H4 or the ESBL-plasmid-cured derivative, and the recovery of bacteria in intestinal homogenates and faeces monitored over time.ResultsCarriage of the ESBL plasmid had differing impacts on E. coli O104:H4 colonization of the two experimental hosts. The plasmid-cured strain was recovered at significantly higher levels than WT during late-stage colonization of rabbits, but at lower levels than WT in sheep. Regardless of the animal host, the ESBL plasmid was stably maintained in virtually all in vivo passaged bacteria that were examined.ConclusionsThese findings suggest that carriage of ESBL plasmids has distinct effects on the host bacterium depending upon t...
The EMBO Journal, 1984
Broad host-range plasmid RK2 is able to replicate in a controlled manner in most Gram negative ba... more Broad host-range plasmid RK2 is able to replicate in a controlled manner in most Gram negative bacterial species. To analyze the elements of its control mechanism, we have measured the copy number in Escherichia coli of mini-RK2 replicons isogenic except for defined deletions in regions adjacent to the vegetative replication origin, ortVRK2, which have previously been implicated in copy number control because of their expression of plasmid incompatibility. The results in- dicate that while the previously defined 700-bp HaeII oriVRK2 fragment carries one copy control element (copA), a second (copB) lies at least partly outside this fragment towards the tetracycline resistance genes of RK2. Deletions affecting both these regions give a mini replicon with a copy number of 35-40 compared with 4-7 for parental RK2. Further in- compatibility experiments indicate that targets for both incA (copA) and incB (copB) lie within the 700-bp HaeII oriVRK2 fragment.
Journal of general microbiology, 1992
The nucleotide sequence of a gene encoding an L-2-haloalkanoic acid halidohydrolase from Pseudomo... more The nucleotide sequence of a gene encoding an L-2-haloalkanoic acid halidohydrolase from Pseudomonas putida strain AJ1 was determined. The ORF (hadL) codes for a polypeptide of 227 amino acids (Mr 25,687) which has significant homology to two other L-2-haloalkanoic acid halidohydrolases of Pseudomonas sp., DehcI and DehcII; these show 38% and 51% amino acid identity respectively to HadL. All three enzymes produce products of an opposite optical configuration to that of the substrates. Comparison of the three sequences shows several highly conserved motifs which indicate the possible position of the enzyme active site. The enzyme was purified to homogeneity and appears to exist as a tetramer.
PLoS ONE, 2014
IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation... more IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC-DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are ''hot spots'' of plasmids potentially carrying catabolic genes.
PLoS ONE, 2012
The korAB operon in RK2 plasmids is a beautiful natural example of a negatively and cooperatively... more The korAB operon in RK2 plasmids is a beautiful natural example of a negatively and cooperatively self-regulating operon. It has been particularly well characterized both experimentally and with mathematical models. We have carried out a detailed investigation of the role of the regulatory mechanism using a biologically grounded mechanistic multi-scale stochastic model that includes plasmid gene regulation and replication in the context of host growth and cell division. We use the model to compare four hypotheses for the action of the regulatory mechanism: increased robustness to extrinsic factors, decreased protein fluctuations, faster response-time of the operon and reduced host burden through improved efficiency of protein production. We find that the strongest impact of all elements of the regulatory architecture is on improving the efficiency of protein synthesis by reduction in the number of mRNA molecules needed to be produced, leading to a greater than ten-fold reduction in host energy required to express these plasmid proteins. A smaller but still significant role is seen for speeding response times, but this is not materially improved by the cooperativity. The self-regulating mechanisms have the least impact on protein fluctuations and robustness. While reduction of host burden is evident in a plasmid context, negative self-regulation is a widely seen motif for chromosomal genes. We propose that an important evolutionary driver for negatively self-regulated genes is to improve the efficiency of protein synthesis.
Nucleic Acids Research, 1987
The product of the korB gene of broad host range plaamid RK2 ia one of at least two proteins whic... more The product of the korB gene of broad host range plaamid RK2 ia one of at least two proteins which repress transcription of the essential replication gene trfA. We report here the nucleotide sequence of korB and the properties of its predicted polypeptide product KorB which has a molecular weight of 39 r 011 Da. KorB is likely to be a soluble protein with an overall net negative charge. However, consistent with a role in transcriptlonal regulation, there is a region with extensive homology to the ahelix-turn-ahelix motif of many DNA binding proteins. This region shows no significant homology to equivalent regions of the TrfB protein which is the primary transcriptional repressor of RX2 and which binds to an operator whose half sites show considerable homology to the half sites of the korB operator.
Nucleic Acids Research, 1990
Broad host range plasmid RK2 is capable of transfer between and stable maintenance in most Gram-n... more Broad host range plasmid RK2 is capable of transfer between and stable maintenance in most Gram-negative bacteria species (1). The sector of plasmid involved in replication and stable inheritance encodes a series of coordinately regulated operons (2). One of these, the tfA operon, encodes two genes the second of which, trfA, is essential for vegetative plasmid replication. Preceding tifA is an ORF encoding an abundant polypeptide of
Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2
Molecular and General Genetics MGG, 1981
A DNA sequence consisting of 617 base pairs (bp) from the region of the origin of replication of ... more A DNA sequence consisting of 617 base pairs (bp) from the region of the origin of replication of the broad-host range plasmid RK2 has been determined. Included within this sequence is a 393 bp HpaII restriction fragment that provides a functional origin or replication when other essential RK2 specified functions are provided in trans. Also contained in this sequence is a region, distinguished functionally from the replication origin, which is involved in the expression of inc2 incompatibility, i.e., the ability of derivatives of RK2 to eliminate a resident RK2 plasmid. The 617 bp sequence includes eight 17 base pair direct repeats with 5 located within the region required for a functional replication origin and 3 within the region involved in inc2 incompatibility. In addition, a 40 bp region rich in A-T followed by a 60 bp stretch having a high G + C content is present. Deletion evidence indicates that the A-T rich and possibly the G + C regions are required for a functional replication origin. Based on the evidence contained in this and the preceding paper (Thomas et al. 1980 b) a model will be presented for the involvement of these specific sequences in the initiation of RK2 DNA replication, plasmid maintenance and plasmid incompatibility.
Molecular Biology, 2006
Digestion with restriction enzymes ( Hae III, Rsa I, and Msp I) was carried out as recommended by... more Digestion with restriction enzymes ( Hae III, Rsa I, and Msp I) was carried out as recommended by Fermentas. Fragment sizes were determined with reference to 100-bp and 1-kb standard DNA marker ladders (Fermentas).
Microbiology, 1989
A library of Treponerna pallidurn genomic DNA fragments produced by partial Sau3A digestion was e... more A library of Treponerna pallidurn genomic DNA fragments produced by partial Sau3A digestion was established in Escherichia coli K12 using the plasmid vector pAT153. The library was screened using immune syphilitic rabbit serum and six recombinant phenotypes expressing eight treponemal polypeptides were detected. With two exceptions, all the recombinant gene products were the same size as polypeptides detected on Western immunoblots of T. pallidurn. The genes encoding three novel gene products, with molecular masses in SDS-PAGE of 42, 17 and 15.5 kDa, which had not been cloned previously from T. pallidurn were also identified. Monoclonal antibodies which reacted with four of the eight recombinant polypeptides were generated. * Only those polypeptides which could be stained with Coomassie Blue were classified by Norris et al. (1987).
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Papers by Christopher Thomas