Purification of KorA Protein from Broad Host Range Plasmid RK2: Definition of a Hierarchy of KorA Operators

Jagura-Burdzy, Grażyna; Thomas, Christopher M.

doi:10.1006/jmbi.1995.0534

Cited by 55 publications

(71 citation statements)

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“…His-tagged polypeptides purified on Niagarose columns were cross-linked by use of glutaraldehyde (23) and separated by SDS-PAGE with a PHAST gel system.…”

Section: Methodsmentioning

confidence: 99%

“…An NdeI-EcoRI fragment of 284 nucleotides (nt) containing the palindrome was purified, dephosphorylated, and 5Ј-end labeled with polynucleotide kinase (Bethesda Research Laboratories) and [␥- 32 (23). The gels were dried and autoradiographed by using a Phosphor Imager system.…”

Section: Methodsmentioning

confidence: 99%

“…The blood collected from the rabbit was allowed to clot at room temperature before antiserum was collected. To decrease cross-reactivity with E. coli proteins, 1 ml of antiserum was pretreated with E. coli extracts as previously described (23) and diluted in 20 ml of phosphate-buffered saline (PBS; phosphate buffer [pH 6.5]-150 mM NaCl).…”

Section: Methodsmentioning

confidence: 99%

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ParB ofPseudomonas aeruginosa: Interactions with Its Partner ParA and Its TargetparSand Specific Effects on Bacterial Growth

et al. 2004

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“…His-tagged polypeptides purified on Niagarose columns were cross-linked by use of glutaraldehyde (23) and separated by SDS-PAGE with a PHAST gel system.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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ParB ofPseudomonas aeruginosa: Interactions with Its Partner ParA and Its TargetparSand Specific Effects on Bacterial Growth

et al. 2004

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“…Rabbit polyclonal antibodies against KorB and TrbA were prepared as described previously for antibodies against KorA (5).…”

Section: Methodsmentioning

confidence: 99%

Cooperativity between KorB and TrbA Repressors of Broad-Host-Range Plasmid RK2

et al. 2001

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The KorB and TrbA proteins of broad-host-range plasmid RK2 are key regulators of the plasmid genes required for conjugative transfer. trbBp is the primary promoter responsible for expression of mating pair formation genes. We show that despite the targets for KorB and TrbA at trbBp being about 165 bp apart, 189 bp upstream of the transcription start point and overlapping the ؊10 region, respectively, these two proteins show up to 10-fold cooperativity for the repression of trbBp. Deletion analysis of TrbA showed that the Cterminal domain (CTD), which has a high degree of sequence conservation with the CTD of KorA, is required for this cooperativity with KorB. Western blotting demonstrated that the apparently mutual enhancement of repression is not due simply to elevation of repressor level by the presence of the second protein, suggesting that the basis for cooperativity is interaction between KorB and TrbA bound at their respective operators.The self-transmissible, broad-host-range plasmid RK2 (indistinguishable from RP4 and RP1) has been studied in great detail (17). Its replication, partitioning, and transfer functions show many features of interest, but its most striking aspect is the complex regulatory circuitry which coordinates expression of the genes for all these basic functions. The regulatory proteins responsible for this coordination are KorA and KorB, encoded in the central control operon; KorC, encoded in the klc operon; and TrbA, encoded before the trb operon. The study described in this paper focused on the interactions between KorB and TrbA at trbBp (Fig.

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“…These regulons span the entire 60-kb genome of the plasmid and are controlled by global regulatory proteins such as KorA, KorB, KorF, and TrbA (10,20,34). The trfA and trb operons specify vegetative replication and plasmid transfer functions, respectively, and their expression is determined by a bidirectional switch under the regulation of KorA (10,11,28). KorA also represses transcription from seven promoters on RK2 (10).…”

Section: Discussionmentioning

confidence: 99%

Characterization of a region of the IncHI2 plasmid R478 which protects Escherichia coli from toxic effects specified by components of the tellurite, phage, and colicin resistance cluster

1997

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The IncHI2 plasmid R478 specifies resistance to potassium tellurite (Te r ), to some bacteriophages (Phi), and to pore-forming colicins (PacB). The genes encoding the three phenotypes are linked, and an 8.4-kb fragment of R478 DNA encoding them cannot be subcloned unless cocloned with a second section of the plasmid. Subclone pKFW4A contains a 5.9-kb BamHI-EcoRI fragment which caused some toxicity when present in Escherichia coli cells. Bacterial cells containing freshly transformed pKFW4A, examined by light microscopy and electron microscopy, had a filamentous morphology consistent with a block in septation. Insertion of transposon Tn1000 into terZ, -A, -B, and -C genes of pKFW4A resulted in the loss of the filamentation phenotype. Deletion of several regions of the clone confirmed that these latter components are involved in the filamentation phenotype. The region specifying protection from toxicity caused by the larger 8.4-kb fragment (encompassing this cluster and the entire 5.9-kb section of pKFW4A) was sequenced and analyzed by T7 polymerase expression and Tn1000 mutagenesis. Three open reading frames, terW, terY, and terX, were identified in a 2.6-kb region. Two polypeptides with approximate molecular masses of 18 and 28 kDa were expressed in CSRDE3 cells and were consistent with TerW (17.1 kDa; 155 amino acids [aa]) and TerY (26.9 kDa; 248 aa), whereas a protein of 213 aa deduced from terX was not observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The terX gene product shows strong identity with the previously identified TerE, TerD, and TerZ polypeptides, and there is a conserved motif of 13 residues, GDN(R/L)TG(E/A)GDGDDE, within this group of polypeptides. Complementation analysis indicated that terW, located approximately 6.0 kb upstream of terZ, brings about protection of cells from toxic effects of components of the Te r , Phi, and PacB cluster.R478 is a 272-kb self-transmissible plasmid of the incompatibility subgroup IncHI2 which mediates resistance to kanamycin, chloramphenicol, tetracycline, mercuric chloride, potassium tellurite (Te r ), and arsenic compounds, as well as resistance to some bacteriophages (Phi) and to pore-forming colicins (PacB) (18,23,30,32,33,42,43). During physical and genetic mapping of R478 (42), several plasmid subclones which expressed Te r but not PacB were obtained, whereas other subclones encoded Phi but not Te r . The Te r , Phi, and PacB phenotypes were linked by insertion mutagenesis to a 6.2-kb DNA fragment which contained seven genes, terZ, -A, -B, -C, -D, -E, and -F (43). Insertion mutagenesis within terZ, terC, or terD reduced or abolished resistance to phage T5, colicins A, B, and K, and potassium tellurite. The amino acid sequences deduced from terD, terE, and terZ were found to be related. Identity was also observed between the amino acid sequences of terA and terF; in addition, highly related amino acid domains were noted among various subsets of the five latter putative polypeptides (43).Subcloning and internal deletion within the Te r ...

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Purification of KorA Protein from Broad Host Range Plasmid RK2: Definition of a Hierarchy of KorA Operators

Cited by 55 publications

References 0 publications

ParB ofPseudomonas aeruginosa: Interactions with Its Partner ParA and Its TargetparSand Specific Effects on Bacterial Growth

ParB ofPseudomonas aeruginosa: Interactions with Its Partner ParA and Its TargetparSand Specific Effects on Bacterial Growth

Cooperativity between KorB and TrbA Repressors of Broad-Host-Range Plasmid RK2

Characterization of a region of the IncHI2 plasmid R478 which protects Escherichia coli from toxic effects specified by components of the tellurite, phage, and colicin resistance cluster

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