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

Abstract

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 ...

Tellurite-mediated thiol oxidation in Escherichia coli

“…The majority of the tellurite-resistance research to date has centred on the ter operon from the IncH plasmids (Hill et al, 1993 ;Jobling & Ritchie, 1987Taylor et al, 1994 ;Whelan, 1992 ;Whelan et al, 1995Whelan et al, , 1997 and the kilA (or kla) operon from the IncPα plasmid RK2 (Bradley, 1985 ;Taylor & Bradley, 1987 ;Thomas et al, 1980 ;Turner et al, 1994aTurner et al, , b, 1995bWalter & Taylor, 1989). Research has also focused on the teh genes from the E. coli chromosome (Taylor et al, 1994 ;Turner et al, 1995aTurner et al, , b, 1997Walter et al, 1991b).…”

The Helicobacter pylori gene encoding phosphatidylserine synthase: sequence, expression, and insertional mutagenesis

“…The polypeptide encoded by the H. pylori pss gene was expressed in a derivative of maxicell strain CSR603 (40), namely, CSRDE3, carrying the P lac -controlled T7 RNA polymerase gene on the chromosome (52). Initially, the wild-type pss gene was amplified from pBSHpC10 and cloned into pBluescript KSϪ.…”

“…Nucleotide sequence was determined with thermocycling Sequenase purchased from Amersham Life Science, Inc. (Cleveland, Ohio), following the procedure specified in the supplier's manual. The pss gene was expressed in modified maxicell strain E. coli CSRDE3 cells (52) and analyzed by sodium dodecyl sulfate-polyacrylamide (15%) gel electrophoresis as described previously (10). Plasmid mutants were introduced into H. pylori UA802 by a natural transformation procedure (50).…”

The role of TerW protein in the tellurite resistance of uropathogenic Escherichia coli

“…These genes protect many Enterobacteria from the toxic effects of potassium tellurite but parts of the ter operon, especially terA and terZ, have also been shown to be toxic to host cells when overexpressed or expressed without the protective terW gene product. The unusual filamentous morphology of E. coli cells harbouring the ter genes cloned into the pUC13 plasmid (plasmid pKFW4A) was described by Whelan et al (1997), who divided the operon into protective (upstream of terZ gene) and toxic (downstream of, and including, the terZ gene) regions. They found that chromosomal DNA replication was not inhibited and proposed that the unusual filamentous morphology was caused by the inhibition of cell division.…”

“…(Jobling & Ritchie 1987); it has also been found on the plasmid pHH1508a (IncHII) from Klebsiella aerogenes (Walter & Taylor 1989), the large conjugative plasmid pR478 (IncHI2) from Serratia marcescens (Whelan et al 1997) and the chromosomes of Proteus mirabilis (Toptchieva et al 2003) and Escherichia coli O157:H7 (Perna et al 2001). Several enteric pathogens share a similar locus of ter genes, which provides the high level resistance that is currently used for the isolation and identification of many pathogens, including the emergent enterohemorrhagic E. coli O157:H7 strain (Zadik et al 1993).…”