Identification and characterization of a novel allele of Escherichia coli dnaB helicase that compromises the stability of plasmid P1

Bacteriophage P1 lysogenizes Escherichia coli cells as a plasmid with approximately the same copy number as the copy number of the host chromosome. Faithful inheritance of the plasmids relies upon proper DNA replication, as well as a partition system that actively segregates plasmids to new daughter cells. We genetically screened for E. coli chromosomal mutations that influenced P1 stability and identified a novel temperature-sensitive allele of the dnaB helicase gene (dnaB277) that replaces serine 277 with a leucine residue (DnaB S277L). This allele conferred a severe temperature-sensitive phenotype to the host; dnaB277 cells were not viable at temperatures above 34 degrees C. Shifting dnaB277 cells to 42 degrees C resulted in an immediate reduction in the rate of DNA synthesis and extensive cell filamentation. The dnaB277 allele destabilized P1 plasmids but had no significant influence on the stability of the F low-copy-number plasmid. This observation suggests that there is a specific requirement for DnaB in P1 plasmid maintenance in addition to the general requirement for DnaB as the replicative helicase during elongation.     

Separate roles of Escherichia coli replication proteins in synthesis and partitioning of pSC101 plasmid DNA

We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid replicons from half of the bacterial population during a single cell division. As was observed previously for DnaA, overexpression of the wild-type DnaB protein conversely stabilized the inheritance of partition-defective plasmids while not increasing plasmid copy number. The identification of dnaA mutations that selectively affected either replication or partitioning further demonstrated the separate roles of DnaA in these functions. The partition-related actions of DnaA were localized to a domain (the cell membrane binding domain) that is physically separate from the DnaA domain that interacts with other host replication proteins. Our results identify bacterial replication proteins that participate in partitioning of the pSC101 plasmid and provide evidence that these proteins mediate plasmid partitioning independently of their role in DNA synthesis.     

DnaA box sequences as the site for helicase delivery during plasmid RK2 replication initiation in Escherichia coli

DnaA box sequences are a common motif present within the replication origin region of a diverse group of bacteria and prokaryotic extrachromosomal genetic elements. Although the origin opening caused by binding of the host DnaA protein has been shown to be critical for the loading of the DnaB helicase, to date there has been no direct evidence presented for the formation of the DnaB complex at the DnaA box site. For these studies, we used the replication origin of plasmid RK2 (oriV), containing a cluster of four DnaA boxes that bind DnaA proteins isolated from different bacterial species (Caspi, R., Helinski, D. R., Pacek, M., and Konieczny, I. (2000) J. Biol. Chem. 275, 18454-18461). Size exclusion chromatography, surface plasmon resonance, and electron microscopy experiments demonstrated that the DnaB helicase is delivered to the DnaA box region, which is localized approximately 200 base pairs upstream from the region of origin opening and a potential site for helicase entry. The DnaABC complex was formed on both double-stranded superhelical and linear RK2 templates. A strict DnaA box sequence requirement for stable formation of that nucleoprotein structure was confirmed. In addition, our experiments provide evidence for interaction between the plasmid initiation protein TrfA and the DnaABC prepriming complex, formed at DnaA box region. This interaction is facilitated via direct contact between TrfA and DnaB proteins.     

Autonomous plasmid‐like replication of a conjugative transposon

Integrative and conjugative elements (ICEs), a.k.a. conjugative transposons, are mobile genetic elements involved in many biological processes, including pathogenesis, symbiosis and the spread of antibiotic resistance. Unlike conjugative plasmids that are extra‐chromosomal and replicate autonomously, ICEs are integrated in the chromosome and replicate passively during chromosomal replication. It is generally thought that ICEs do not replicate autonomously. We found that when induced, Bacillus subtilis ICEBs1 undergoes autonomous plasmid‐like replication. Replication was unidirectional, initiated from the ICEBs1 origin of transfer, oriT, and required the ICEBs1‐encoded relaxase NicK. Replication also required several host proteins needed for chromosomal replication, but did not require the replicative helicase DnaC or the helicase loader protein DnaB. Rather, replication of ICEBs1 required the helicase PcrA that is required for rolling circle replication of many plasmids. Transfer of ICEBs1 from the donor required PcrA, but did not require replication, indicating that PcrA, and not DNA replication, facilitates unwinding of ICEBs1 DNA for horizontal transfer. Although not needed for horizontal transfer, replication of ICEBs1 was needed for stability of the element. We propose that autonomous plasmid‐like replication is a common property of ICEs and contributes to the stability and maintenance of these mobile genetic elements in bacterial populations.     

Roles of long and short replication initiation proteins in the fate of IncP-1 plasmids

Broad-host-range IncP-1 plasmids generally encode two replication initiation proteins, TrfA1 and TrfA2. TrfA2 is produced from an internal translational start site within trfA1. While TrfA1 was previously shown to be essential for replication in Pseudomonas aeruginosa, its role in other bacteria within its broad host range has not been established. To address the role of TrfA1 and TrfA2 in other hosts, efficiency of transformation, plasmid copy number (PCN), and plasmid stability were first compared between a mini-IncP-1β plasmid and its trfA1 frameshift variant in four phylogenetically distant hosts: Escherichia coli, Pseudomonas putida, Sphingobium japonicum, and Cupriavidus necator. TrfA2 was sufficient for replication in these hosts, but the presence of TrfA1 enhanced transformation efficiency and PCN. However, TrfA1 did not contribute to, and even negatively affected, long-term plasmid persistence. When trfA genes were cloned under a constitutive promoter in the chromosomes of the four hosts, strains expressing either both TrfA1 and TrfA2 or TrfA1 alone, again, generally elicited a higher PCN of an IncP1-β replicon than strains expressing TrfA2 alone. When a single species of TrfA was produced at different concentrations in E. coli cells, TrfA1 maintained a 3- to 4-fold higher PCN than TrfA2 at the same TrfA concentrations, indicating that replication mediated by TrfA1 is more efficient than that by TrfA2. These results suggest that the broad-host-range properties of IncP-1 plasmids are essentially conferred by TrfA2 and the intact replication origin alone but that TrfA1 is nonetheless important to efficiently establish plasmid replication upon transfer into a broad range of hosts.     

Amelioration of the cost of conjugative plasmid carriage in Eschericha coli K12

Although plasmids can provide beneficial functions to their host bacteria, they might confer a physiological or energetic cost. This study examines how natural selection may reduce the cost of carrying conjugative plasmids with drug-resistance markers in the absence of antibiotic selection. We studied two plasmids, R1 and RP4, both of which carry multiple drug resistance genes and were shown to impose an initial fitness cost on Escherichia coli. To determine if and how the cost could be reduced, we subjected plasmid-containing bacteria to 1100 generations of evolution in batch cultures. Analysis of the evolved populations revealed that plasmid loss never occurred, but that the cost was reduced through genetic changes in both the plasmids and the bacteria. Changes in the plasmids were inferred by the demonstration that evolved plasmids no longer imposed a cost on their hosts when transferred to a plasmid-free clone of the ancestral E. coli. Changes in the bacteria were shown by the lowered cost when the ancestral plasmids were introduced into evolved bacteria that had been cured of their (evolved) plasmids. Additionally, changes in the bacteria were inferred because conjugative transfer rates of evolved R1 plasmids were lower in the evolved host than in the ancestral host. Our results suggest that once a conjugative bacterial plasmid has invaded a bacterial population it will remain even if the original selection is discontinued.     

The evolution of a conjugative plasmid and its ability to increase bacterial fitness

Conjugative plasmids are extra-chromosomal DNA elements that are capable of horizontal transmission and are found in many natural isolated bacteria. Although plasmids may carry beneficial genes to their bacterial host, they may also cause a fitness cost. In this work, we studied the evolution of the R1 plasmid and we found that, in spite of the R1 plasmid conferring an initial cost to its host, after 420 generations the cost disappeared in all five independent evolution experiments. In fact, in two of these five experiments evolved conjugative plasmids actually conferred a fitness advantage to their hosts. Furthermore, the relative fitness of the ancestral clone bearing one of the evolved plasmids is significantly higher than both the plasmid-free ancestral cells and the evolved cells carrying the evolved plasmid. Given that the R1 plasmid may spread among different species of enterobacteria, we wondered what the effect of the evolved plasmid would be inside Salmonella enterica cells. We found that the evolved plasmid is also able to dramatically increase the relative fitness of these cells. Our results suggest that even if general usage of antibiotics is halted, conjugative plasmids that have been selected with antibiotics in previous years can still persist among bacterial populations or even invade new strains.     

Replication origin mutations affecting binding of pSC101 plasmid-encoded Rep initiator protein.

To investigate the role of binding sites for Rep initiation protein in the replication of pSC101, a series of plasmids was constructed which carried different combinations of mutations in three binding sites within the minimal origin of replication. Mutation of all three sites reduced the affinity of purified Rep protein for the origin by 100-fold, as measured by a competition binding assay. Mutations in individual binding sites prevented binding of Rep protein to the mutant site but not to adjacent wild-type sites. Transformation efficiency, copy number, and stability over 150 generations were measured for each of the mutant plasmids. Unlike other similar plasmids related to pSC101, the Rep binding sites were found not to be equivalent. A mutation in the site RS1, proximal to repeated sequences which serve as DnaB helicase entry sites in oriC, had a severe effect on replication activity. A similar mutation in the distal site RS3 caused a reduction in copy number, but the mutant plasmid was stably maintained despite a broadened distribution of copy number within the population. A mutation in the middle RS2 site had no significant effect on pSC101 replication.     

A specific region in the N terminus of a replication initiation protein of plasmid RK2 is required for recruitment of Pseudomonas aeruginosa DnaB helicase to the plasmid origin

Broad host range plasmid RK2 encodes two versions of its essential replication initiation protein, TrfA, using in-frame translational starts spaced 97 amino acids apart. The smaller protein, TrfA-33, is sufficient for plasmid replication in many bacterial hosts. Efficient replication in Pseudomonas aeruginosa, however, specifically requires the larger TrfA-44 protein. With the aim of identifying sequences of TrfA-44 required for stable replication of RK2 in P. aeruginosa, specific deletions and a substitution mutant within the N terminus sequence unique to TrfA-44 were constructed, and the mutant proteins were tested for activity. Deletion mutants were targeted to three of the four predicted helical regions in the first 97 amino acids of TrfA-44. Deletion of TrfA-44 amino acids 21-32 yielded a mutant protein, TrfA-44Delta2, that had lost the ability to bind and load the DnaB helicase of P. aeruginosa or Pseudomonas putida onto the RK2 origin in vitro and did not support stable replication of an RK2 mini-replicon in P. aeruginosa in vivo. A substitution of amino acid 22 within this essential region resulted in a protein, TrfA-44E22A, with reduced activity in vitro, particularly with the P. putida helicase. Deletion of amino acids 37-55 (TrfA-44Delta3) slightly affected protein activity in vitro with the P. aeruginosa helicase and significantly with the P. putida helicase, whereas deletion of amino acids 71-88 (TrfA-44Delta4) had no effect on TrfA activity in vitro with either helicase. These results identify regions of the TrfA-44 protein that are required for recruitment of the Pseudomonas DnaB helicases in the initiation of RK2 replication.     

The phenotypes of temperature-sensitive mini-RK2 replicons carrying mutations in the replication control gene trfA are suppressed nonspecifically by intragenic cop mutations.

The minimal replicon of the broad-host-range plasmid RK2 consists of the origin of vegetative replication (oriV) and a gene (trfA) encoding an essential replication protein that binds to short repeats in oriV. We report here the results of a DNA sequence analysis of seven unique mutants that are temperature sensitive for replication in Escherichia coli. The mutations (designated rts) were distributed throughout 40% of the downstream part of the trfA gene. Spontaneous revertants of the rts mutants were isolated, and further analysis of four such revertants demonstrated that the new phenotypes resulted from intragenic second-site copy up (cop) mutations. Subcloning experiments showed that all tested intragenic combinations of rts and cop mutations resulted in elimination or strong reduction of the temperature sensitivity of replication. This suppression was also observed under conditions where the mutant TrfA protein was provided in trans with respect to oriV, indicating that the reduction in temperature sensitivity could not be a TrfA protein dosage effect. The phenotypes of two of the cop mutants in Pseudomonas aeruginosa were analyzed; the results demonstrated that the mutants were either not functional or poorly functional in this host. The rts mutant plasmids were also reduced in their ability to replicate in P. aeruginosa, and the intragenic cop mutations did not improve the functionality of these mutants. The significance of the results is discussed in relation to current models of the mechanism of action of the TrfA protein.     

Experimental Evolution of Gene Duplicates in a Bacterial Plasmid Model

The fate of gene duplicates subjected to diversifying selection was tested experimentally in a bacterial system. The wild-type TEM-1 β-lactamase gene confers resistance to ampicillin but not to cefotaxime. Point mutations confer cefotaxime resistance, but they compromise ampicillin resistance. Thus, selection for both drug resistances in a bacterium with two copies of β-lactamase should favor the divergence of one copy to improve cefotaxime resistance while maintaining the other copy to preserve ampicillin resistance. This selection was performed on a bacterium with identical sequences of β-lactamase on two separate, compatible plasmids. As expected, one plasmid evolved increased cefotaxime resistance when appropriately strong cefotaxime selection was applied. However, the cefotaxime-resistant plasmid maintained sufficient ampicillin resistance to tolerate the concentration of ampicillin used, and the other plasmid was lost. Hosts carrying both the cefotaxime-resistant and wild-type plasmids were then subjected to various higher concentrations of both drugs to find conditions that would ensure the maintenance of both plasmids. In a striking contradiction to our model, no such conditions were found. The fitness cost of carrying both plasmids increased dramatically as antibiotic levels were raised, and either the wild-type plasmid was lost or the cells did not grow. This study highlights the importance of the cost of duplicate genes and the quantitative nature of the tradeoff in the evolution of gene duplication through functional divergence. Reviewing Editor: Dr. Margaret Riley     

A broad host range replicon with different requirements for replication initiation in three bacterial species.

Plasmid RK2 is unusual in its ability to replicate stably in a wide range of Gram-negative bacteria. The replication origin (oriV) and a plasmid-encoded initiation protein (TrfA; expressed as 33 and 44 kDa forms) are essential for RK2 replication. To examine initiation events in bacteria unrelated to Escherichia coli, the genes encoding the replicative helicase, DnaB, of Pseudomonas putida and Pseudomonas aeruginosa were isolated and used to construct protein expression vectors. The purified proteins were tested for activity along with E.coli DnaB at RK2 oriV. Each helicase could be recruited and activated at the RK2 origin in the presence of the host-specific DnaA protein and the TrfA protein. Escherichia coli or P.putida DnaB was active with either TrfA-33 or TrfA-44, while P.aeruginosa DnaB required TrfA-44 for activation. Moreover, unlike the E.coli DnaB helicase, both Pseudomonas helicases could be delivered and activated at oriV in the absence of an ATPase accessory protein. Thus, a DnaC-like accessory ATPase is not universally required for loading the essential replicative helicase at a replication origin.     

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. In this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity.     

Cloning and mapping of the genetic determinants for microcin C51 production and immunity

Microcin C51 is a small peptide antibiotic produced by Escherichia coli cells harbouring the 38 kb low copy number plasmid pC51, which codes for microcin production and immunity. The genetic determinants for microcin synthesis and immunity were cloned into the vectors pBR325, pUC19 and pACYC184. Physical and phenotypic analysis of deletion derivatives and mutant plasmids bearing insertions of transposon Tn5 showed that a DNA fragment of about 5 kb is required for microcin C51 synthesis and expression of complete immunity to microcin. Partial immunity can be provided by a 2 kb DNA fragment. Mutant plasmids were tested for their ability to complement Mic^– mutations. Results of these experiments indicate that at least three plasmid genes are required for microcin production. The host OmpR function is also necessary for microcin C51 synthesis.     

Frequent conjugative transfer accelerates adaptation of a broad-host-range plasmid to an unfavorable Pseudomonas putida host

IncP-1 plasmids are known to be promiscuous, but it is not understood if they are equally well adapted to various species within their host range. Moreover, little is known about their fate in bacterial communities. We determined if the IncP-1beta plasmid pB10 was unstable in some Proteobacteria, and whether plasmid stability was enhanced after long-term carriage in a single host and when regularly switched between isogenic hosts. Plasmid pB10 was found to be very unstable in Pseudomonas putida H2, and conferred a high cost (c. 20% decrease in fitness relative to the plasmid-free host). H2(pB10) was then evolved under conditions that selected for plasmid maintenance, with or without regular plasmid transfer (host-switching). When tested in the ancestral host, the evolved plasmids were more stable and their cost was significantly reduced (9% and 16% for plasmids from host-switched and nonswitched lineages, respectively). Our findings suggest that IncP-1 plasmids can rapidly adapt to an unfavorable host by improving their overall stability, and that regular conjugative transfer accelerates this process.     

Conformation of a plasmid replication initiator protein affects its proteolysis by ClpXP system

Proteins from the Rep family of DNA replication initiators exist mainly as dimers, but only monomers can initiate DNA replication by interaction with the replication origin (ori). In this study, we investigated both the activation (monomerization) and the degradation of the broad‐host‐range plasmid RK2 replication initiation protein TrfA, which we found to be a member of a class of DNA replication initiators containing winged helix (WH) domains. Our in vivo and in vitro experiments demonstrated that the ClpX‐dependent activation of TrfA leading to replicationally active protein monomers and mutations affecting TrfA dimer formation, result in the inhibition of TrfA protein degradation by the ClpXP proteolytic system. These data revealed that the TrfA monomers and dimers are degraded at substantially different rates. Our data also show that the plasmid replication initiator activity and stability in E. coli cells are affected by ClpXP system only when the protein sustains dimeric form.     

Adaptive plasmid evolution results in host-range expansion of a broad-host-range plasmid

Little is known about the range of hosts in which broad-host-range (BHR) plasmids can persist in the absence of selection for plasmid-encoded traits, and whether this "long-term host range" can evolve over time. Previously, the BHR multidrug resistance plasmid pB10 was shown to be highly unstable in Stenotrophomonas maltophilia P21 and Pseudomonas putida H2. To investigate whether this plasmid can adapt to such unfavorable hosts, we performed evolution experiments wherein pB10 was maintained in strain P21, strain H2, and alternatingly in P21 and H2. Plasmids that evolved in P21 and in both hosts showed increased stability and decreased cost in ancestral host P21. However, the latter group showed higher variability in stability patterns, suggesting that regular switching between distinct hosts hampered adaptive plasmid evolution. The plasmids evolved in P21 were also equally or more stable in other hosts compared to pB10, which suggested true host-range expansion. The complete genome sequences of four evolved plasmids with improved stability showed only one or two genetic changes. The stability of plasmids evolved in H2 improved only in their coevolved hosts, not in the ancestral host. Thus a BHR plasmid can adapt to an unfavorable host and thereby expand its long-term host range.     

克雷伯氏菌属(Klebsiella)细菌比较基因组学分析揭示多复制子抗性质粒介导广泛的抗性基因传播

[目的] 研究克雷伯氏菌与多复制子抗性质粒间的关系,分析细菌携带多复制子质粒对抗生素环境的响应机制。[方法] 以2018-2020年分离的56株不同来源克雷伯氏菌（Klebsiella sp.）分离株为研究对象,利用微量肉汤稀释法评估其多重耐药表型,对分离菌株进行全基因组测序（WGS）,通过细菌全基因组关联分析（BGWAS）技术和比较基因组学方法深入解析多复制子抗性质粒形成的机制。[结果] 耐药表型分析发现野生动物来源的菌株具有更广的耐药谱系,总体Klebsiella sp.对氨苄西林表现出很高的耐药率（80.36%）,尤其是马来穿山甲来源菌株对头孢类抗生素高度耐受,同时对氯霉素、左氧氟沙星和复方新诺明等药物耐受,基因组分析发现这些菌株携带了抗性质粒和更多的抗生素抗性基因。进一步对69个质粒序列分析,发现有28个质粒为多复制子质粒,主要携带bla_CTX-M-15、bla_CTX-M-14、bla_CTX-M-55、bla_OXA-1和bla_TEM-1等β-内酰胺酶基因。细菌携带质粒类型分析认为Klebsiella pneumoniae可能是多复制子质粒的重要宿主,质粒骨架与结构分析发现多复制子质粒多由2个或2个以上单个质粒融合而成,携带此类质粒的菌株不仅获得了更广的耐药表型,而且在全球传播扩散分布逐年增加,因此产生对抗生素环境更强的适应性。[结论] 多重耐药性细菌呈现的表型与携带的多复制子质粒有关,相比较下多复制子质粒比非多复制子质粒有更强的抗性基因携带能力,或许是细菌在强大的抗生素压力下产生的重要响应机制。本研究对于未来探索细菌抗性基因的传播扩散机制具有重要意义。     

Replication of mini RK2 plasmid in extracts of Escherichia coli requires plasmid-encoded protein TrfA and host-encoded proteins DnaA, B, G DNA gyrase and DNA polymerase III

Soluble extracts of Escherichia coli capable of carrying out replication of the mini-RK2 derivative pCT461 have been prepared from cells carrying this plasmid or from plasmid-free bacteria. The latter are dependent upon exogenously added plasmid-encoded replication protein (TrfA) and require additional DnaA protein for optimum activity. This dependence upon DnaA was confirmed by the failure of DnaA-deficient cell extracts to support replication of pCT461 in the absence of added DnaA protein. Replication is unidirectional and begins at or near oriV, the vegetative replication origin of RK2. DNase I protection studies with purified TrfA indicate that this protein acts by binding to short (17 base-pairs) directly repeated DNA sequences present in oriV. The in vitro replication is resistant to rifampicin but can be abolished by antibodies against DnaG protein (E. coli primase) or DnaB protein (helicase) and by DNA gyrase inhibitors. Inhibition by arabinosyl-CTP suggests that DNA polymerase III is responsible for elongation of nascent DNA strands. These results are discussed in relation to the mechanism of RK2 replication and in the context of the host range of the plasmid.     

Host components required for the replication of the resistance plasmid R124 and a copy mutant derivative

The replication of R124, and a copy mutant derivative of it, was measured with respect to dependence on the host DnaA, DnaB, DnaC, DnaE, DnaG, and PolA gene products. Both plasmids replicated under conditions where the DnaA gene product was inactivated or where the polymerising activity of the PolA gene product was reduced. In contrast, neither plasmid replicated to any appreciable extent, if the DnaB, DnaC, DnaE or DnaG gene products were inactivated. R124 integratively suppressed the lesion of the dnaA mutant but the copy mutant derivative had only a very weak suppressing effect. Neither plasmid suppressed the lesions of any of the other dna mutants.