Construction and characterization of derivatives carrying insertion mutations in F plasmid transfer region genes, trbA, artA, traQ, and trbB

Kathir, Pushpa; Ippen‐Ihler, Karin

doi:10.1016/0147-619x(91)90035-u

Cited by 32 publications

(36 citation statements)

References 21 publications

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“…7. Each was then crossed onto pOX38, using the triparental mating procedure and selections described previously (11,15). Restriction analysis of four representative recombinant plasmids, pOX38-trbI463, pOX38-trbI464, pOX38-trbI472, and pOX38-trbI473, confirmed that they carry the four trbI::kan insertions made in reasonably normal numbers of F pili that could adsorb RNA phages (Table 3).…”

Section: Resultsmentioning

confidence: 99%

“…We investigated the function of trbI by constructing trbI kanamycin insertion mutations in pOX38, a transmissible F-deletion derivative that retains all tra region sequences. The approach used has been effective in analysis of other tra operon gene functions (11,13,15,18). In this case, we first constructed pKI457, a clone carrying the 1.7-kb HincII fragment that includes trbI and traW.…”

Section: Resultsmentioning

confidence: 99%

“…The Tn9O3 kanamycin resistance (kan) gene cassette used came from plasmid pUC4K (Pharmacia Inc., Molecular Biology Division, Piscataway, N.J.). Matings and selections used for in vivo construction of pOX38 mutant derivatives were as previously described (11,15). pOX38 is a transmissible F replicon made by recircularization of the large F HindIII fragment and lacks transposable sequences (6).…”

Section: Methodsmentioning

confidence: 99%

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Characterization, localization, and sequence of F transfer region products: the pilus assembly gene product TraW and a new product, TrbI

Maneewannakul

Ippen‐Ihler

1992

J Bacteriol

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The traW gene of the Escherichia coli K-12 sex factor, F, encodes one of the numerous proteins required for conjugative transfer of this plasmid. We have found that the nucleotide sequence of traW encodes a 210-amino-acid, 23,610-Da polypeptide with a characteristic amino-terminal signal peptide sequence; in DNA from the F lac traW546 amber mutant, the traW open reading frame is interrupted at codon 141. Studies oftraW expression in maxicells in the presence and absence of ethanol demonstrate that the traW product does undergo signal sequence processing. Cell fractionation experiments additionally demonstrated that mature TraW is a periplasmic protein. Electron microscopy also showed that F lac traWS46 hosts do not express F pili, confirming that TraW is required for F-pilus assembly. Our nucleotide sequence also revealed the existence of an additional gene, trbI, located between traC and traW. The trbI gene encodes a 128-amino-acid polypeptide which could be identified as a 14-kDa protein product. Fractionation experiments demonstrated that TrbI is an intrinsic inner-membrane protein. Hosts carrying the pOX38-trbI::kan insertion mutant plasmids that we constructed remained quite transfer proficient but exhibited increased resistance to F-pilus-specific phages. Mutant plasmids pOX38-trbI472 and pOX38-trbI473 expressed very long F pili, suggestive of a pilus retraction deficiency. Expression of an excess of TrbI in hosts carrying a wild-type pOX38 plasmid also caused F-pilus-specific phage resistance. The possibility that TrbI influences the kinetics of pilus outgrowth and/or retraction is discussed.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Characterization, localization, and sequence of F transfer region products: the pilus assembly gene product TraW and a new product, TrbI

Maneewannakul

Ippen‐Ihler

1992

J Bacteriol

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“…Table 2 also summarizes the results of JEL92 and JEL93 Western blot assays with inner membranes prepared from these strains. F pilin that was reactive with both antibodies was present in all cases but two: as expected, membranes from a traQ mutant failed to react with either JEL92 or JEL93, since this gene is essential for expression of the 7-kDa pilin polypeptide (22). Samples from the only Flac traY mutant strain available (Flac traY244) also did not react positively with either antibody; however, this mutation may affect tra operon expression (21).…”

Section: Resultsmentioning

confidence: 99%

“…Instead, the major traA product was a 7-kDa polypeptide [named traA product 7(Q) or Ap7(Q)] that was expressed very efficiently (23). Laine et al also detected small amounts of an 8-kDa traA product (Ap8) (23 F pili or detectable quantities of F-pilin subunits (22). Thus, the traQ product, a 94-amino-acid inner membrane protein (48,49), appears to be required for efficient utilization and processing of the pilin precursor.…”

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confidence: 99%

The Escherichia coli K-12 F plasmid gene traX is required for acetylation of F pilin

et al. 1993

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The Escherichia coli F plasmid gene required for amino-terminal acetylation of F-pilin subunits was identified. Using Western blots (immunoblots), we assayed the reaction of monoclonal antibodies with F-pilin polypeptides in inner membrane preparations from various F mutant strains. It was known that JEL92 recognizes an internal pilin epitope and JEL93 recognizes the acetylated amino-terminal sequence (L.S. Frost, J.S. Lee, D.G. Scraba, and W. Paranchych, J. Bacteriol. 168:192-198, 1986). As expected, neither antibody reacted with inner membranes from F- cells or Flac derivatives that do not synthesize pilin. Mutations that affected the individual activities of F tra genes traA, -B, -C, -D, -E, -F, -G, -H, -I, -J, -K, -L, -M, -N, -P, -R, -U, -V and -W or trb genes trbA, -B, -C, -D, -E, -G, -H, and -I did not prevent JEL92 or JEL93 recognition of membrane pilin. However, Hfr deletion mutants that lacked the most-distal transfer region genes did not express pilin that reacted with JEL93. Nevertheless, all strains that retained traA and traQ did express JEL92-reactive pilin polypeptides. Analysis of strains expressing cloned tra segments showed that traA and traQ suffice for synthesis of JEL92-reactive pilin, but synthesis of JEL93-reactive pilin is additionally dependent on traX. We concluded that the traX product is required for acetylation of F pilin. Interestingly, our data also showed that TraA+ TraQ+ cells synthesize two forms of pilin which migrate at approximately 7 and 8 kDa. In TraX+ cells, both become acetylated and react with JEL93. Preparations of wild-type F-pilus filaments contain both types of subunits.

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Genetic Organization of Transfer-Related Determinants on the Sex Factor F and Related Plasmids

Ippen‐Ihler

Skurray

1993

Bacterial Conjugation

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Construction and characterization of derivatives carrying insertion mutations in F plasmid transfer region genes, trbA, artA, traQ, and trbB

Cited by 32 publications

References 21 publications

Characterization, localization, and sequence of F transfer region products: the pilus assembly gene product TraW and a new product, TrbI

Characterization, localization, and sequence of F transfer region products: the pilus assembly gene product TraW and a new product, TrbI

The Escherichia coli K-12 F plasmid gene traX is required for acetylation of F pilin

Genetic Organization of Transfer-Related Determinants on the Sex Factor F and Related Plasmids

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