Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2

Roberts, R. C.; Burioni, Roberto; Helinski, Donald R.

doi:10.1128/jb.172.11.6204-6216.1990

Cited by 110 publications

(151 citation statements)

References 56 publications

(65 reference statements)

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“…We tested this hypothesis directly using a temperature-sensitive, low-copy RK2 plasmid derivative, pRR10ts97. This plasmid contains a mutation in the essential TrfA replication protein that leads to rapid loss of the plasmid from actively growing bacteria at 378C (Roberts et al, 1990). We transformed pRR10ts97 into S. typhimurium 14028s and grew the inoculum with selection at the permissive temperature of 308C.…”

Section: Detached Macrophages Contain Proliferating Bacteriamentioning

confidence: 99%

The Salmonella virulence plasmid spv genes are required for cytopathology in human monocyte-derived macrophages

et al. 2000

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SummaryThe pathogenesis of serious systemic Salmonella infections is characterized by survival and proliferation of bacteria inside macrophages. Infection of human monocyte-derived macrophages in vitro with S. typhimurium or S. dublin produces cytopathology characterized by detachment of cells that contain large numbers of proliferating bacteria. This cytopathology is dependent on the expression of the bacterial spv genes, a virulence locus previously shown to markedly enhance the ability of Salmonella to produce systemic disease. After 24 h of infection, macrophage cultures contain two populations of bacteria: (i) proliferating organisms present in a detached cell fraction; and (ii) a static bacterial population in macrophages remaining attached to the culture well. Mutations in either the essential transcriptional activator SpvR or the key SpvB protein markedly reduce the cytopathic effect of Salmonella infection. The spvdependent cytopathology in macrophages exhibits characteristics of apoptosis, with release of nucleosomes into the cytoplasm, nuclear condensation and DNA fragmentation. The current ®ndings suggest that the mechanism of the spv effect is through induction of increased cytopathology in host macrophages.

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Section: Detached Macrophages Contain Proliferating Bacteriamentioning

confidence: 99%

The Salmonella virulence plasmid spv genes are required for cytopathology in human monocyte-derived macrophages

et al. 2000

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“…Data were subjected to the G-squared test for significance (P 0n01). Escherichia coli strains containing plasmids pRR10 (5n0 kb), pRR54 (8n3 kb) (Roberts et al, 1990), pRK290 (20 kb) (Ditta et al, 1980), R6K (38 kb) (Stalker et al, 1979) and RK2 (60 kb) (Pansegrau et al, 1994), and Sinorhizobium meliloti 102F34 containing three plasmids of 100, 150 and 220 kb, respectively, were used as plasmid size standards.…”

Section: Methodsmentioning

confidence: 99%

Differentiation of plasmids in marine diazotroph assemblages determined by randomly amplified polymorphic DNA analysis

et al. 2002

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Nitrogen fixation by diazotrophic bacteria is a significant source of new nitrogen in salt marsh ecosystems. Recent studies have characterized the physiological and phylogenetic diversity of oxygen-utilizing diazotrophs isolated from the rhizoplanes of spatially separated intertidal macrophyte habitats. However, there is a paucity of information regarding the traits encoded by and the diversity of plasmids occurring in this key ecological functional group. Five-hundred and twenty-one isolates cultivated from the rhizoplanes of Juncus roemarianus, Spartina patens and different growth forms (short-form and tall-form) of Spartina alterniflora were screened for the presence of plasmids. One-hundred and thirty-four diazotrophs carrying plasmids that ranged in size from 2 to S 100 kbp were identified. The majority of the marine bacteria contained one plasmid. Diazotrophs from the shortform S. alterniflora rhizoplane contained significantly fewer plasmids relative to isolates from tall-form S. alterniflora, J. roemarianus and S. patens. Although some plasmids exhibited homology to a nifH gene probe, the majority of the plasmids were classified as cryptic. Two oligonucleotide primers were developed to facilitate genotypic typing of the endogenously isolated marine plasmids by the randomly amplified polymorphic DNA (RAPD)-PCR technique. These primers proved to be more effective than 21 commercially available primers tested to generate RAPD-PCR patterns. Analysis of the RAPD-PCR patterns indicated as many as 71 different plasmid genotypes occurring in diazotroph bacterial assemblages within and between the four different salt marsh grass rhizoplane habitats investigated in this study.

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“…Plasmid Stability in Vivo-To test the ability of the TrfA-44 mutants to support replication in P. aeruginosa, the mutations were transferred into the RK2 mini-replicon pRR10 (32). The EcoRI-SfiI fragment containing the 5Ј end of trfA of plasmid pRR10 was replaced with the EcoRI-SfiI fragment containing the 5Ј end of trfA from plasmids pZZ23 (TrfA-44⌬4), pZZ25 (TrfA-44⌬3), pZZ28 (TrfA-44⌬2), pZZ29 (TrfA-44E22A), and pGC1 (TrfA-44), resulting in plasmids pRR10-98His⌬4, pRR10-98His⌬3, pRR10-98His⌬2, pRR10-98HisE22A, and pRR10-98His, respectively.…”

Section: Methodsmentioning

confidence: 99%

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

Zhong¹,

Helinski

Toukdarian

2003

Journal of Biological Chemistry

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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-44⌬2, 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-44⌬3) 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-44⌬4) 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.A critical step in the initiation of DNA replication is the recruitment, loading, and activation of the replicative helicase. Helicase activity is not only necessary for progression of the replication fork but, as studies with the Escherichia coli chromosomal origin oriC have revealed, the DnaB helicase makes essential contacts with other proteins in the replication complex. Recruitment of the helicase in E. coli requires association of the DnaB hexamer with the E. coli DnaC accessory protein (1-3). The DnaB-DnaC complex interacts with the host initiation protein, DnaA, bound to specific sequences (DnaA boxes) at oriC. This association results in the loading of the helicase onto unwound single-stranded DNA in the origin and the release of DnaC (4 -7). Once loaded onto the single-stranded DNA, DnaB interacts directly with DnaG to facilitate primase loading onto the single-stranded DNA at the replication fork (8,9). This interaction becomes the primary regulator of Okazaki fragment synthesis (10) and also ensures the proper placement of primers for leading strand synthesis (11). The subsequent association of DnaB with the Tau subunit of the DNA polymerase III holoenzyme results in rapid movement of the replication fork (12, 13).Studies on plasmids and phages that replicate in E. coli have revealed additional strategies for recruiting DnaB to a replication origin. The replication initiation...

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Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2

Cited by 110 publications

References 56 publications

The Salmonella virulence plasmid spv genes are required for cytopathology in human monocyte-derived macrophages

The Salmonella virulence plasmid spv genes are required for cytopathology in human monocyte-derived macrophages

Differentiation of plasmids in marine diazotroph assemblages determined by randomly amplified polymorphic DNA analysis

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

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