Inhibition of the multiple antibiotic resistance (mar) operon in Escherichia coli by antisense DNA analogs.

The multiple antibiotic resistance operon (marORAB) in Escherichia coli controls intrinsic susceptibility and resistance to multiple, structurally different antibiotics and other noxious agents. A plasmid construct with marA cloned in the antisense direction reduced LacZ expression from a constitutively expressed marA::lacZ translational fusion and inhibited the induced expression of LacZ in cells bearing the wild-type repressed fusion. The marA antisense construction also decreased the multiple antibiotic resistance of a Mar mutant. Two antisense phosphorothioate oligonucleotides, one targeted to marO and the other targeted to marA of the mar operon, introduced by heat shock or electroporation reduced LacZ expression in the strain having the marA::lacZ fusion. One antisense oligonucleotide, tested against a Mar mutant of E. coli ML308-225, increased the bactericidal activity of norfloxacin. These studies demonstrate the efficacy of exogenously delivered antisense oligonucleotides targeted to the marRAB operon in inhibiting expression of this chromosomal regulatory locus.

Construction of derivatives of the F plasmid pOX-tra715: characterization of traY and traD mutants that can be complemented in trans.

A derivative of the F plasmid, pOX38-tra715, expresses the entire F tra operon from a foreign promoter (PT7) derived from phage T7. A series of plasmids related to pOX38-tra715 were constructed which carry either deletion mutations or point mutations in traY. When the PT7 promoter was induced, these plasmids expressed the F pilus but were transfer deficient unless TraY was supplied In trans from compatible plasmids. Insertion of a kanamycin-resistance cassette in the traY gene of the pOX38 plasmid, which contains the wild-type PY promoter, resulted in loss of F piliation and transfer ability. Introduction of TraY in trans partially restored piliation and transfer suggesting that TraY has a role in positively regulating the PY promoter, pOX38-tra719-traD411, which contains a chloramphenicol-resistance cassette in place of the kanamycin-resistance cassette in pOX38-tra715 and a mutation in traD, was constructed to demonstrate the utility of this series of plasmids in studying the long (30 kb) F tra operon.

Identification for mar mutants among quinolone-resistant clinical isolates of Escherichia coli.

Quinolone-resistant clinical Escherichia coli isolates were examined for mutations in the marRAB operon of the multiple antibiotic resistance (mar) locus. Among 23 strains evaluated, 8 were chosen for further study: 3 that showed relatively high levels of uninduced, i.e., constitutive, expression of the operon and 5 with variable responses to induction by salicylate or tetracyclines. The marR genes, specifying the repressor of the operon, cloned from the three strains constitutively expressing the operon did not reduce the level of expression of beta-galactosidase from a marO::lacZ transcriptional fusion and were therefore mutant; however, marR genes cloned from the five other clinical strains repressed LacZ expression and were wild type. All three mutant marR genes contained more than one mutation: a deletion and a point mutation. Inactivation of the mar locus in the three known marR mutant strains with a kanamycin resistance cassette introduced by homologous recombination reduced resistance to quinolones and multiple antibiotics. These findings indicate that mar operon mutations exist in quinolone-resistant clinical E. coli isolates and contribute to quinolone and multidrug resistance.

Characterization of traX, the F plasmid locus required for acetylation of F-pilin subunits.

Acetylation of F-pilin subunits has previously been shown to depend upon expression of the F plasmid transfer operon gene traX. To assess the requirement for pilin acetylation in conjugative transfer of F, we constructed traX::kan insertion mutations and crossed them onto the transmissible F derivative pOX38. Under standard conditions, the function of traX seemed to be dispensable. Although pilin synthesized by mutant plasmids pOX38-traX482 and pOX38-traX483 was not acetylated, F-pilus production and F-pilus-specific phage infection appeared to be normal and transfer occurred at wild-type frequency. Analysis of labeled products showed that TraX+ plasmids expressed two approximately 24- (TraX1) and 22-kDa (TraX2) polypeptides that localized in the cytoplasmic membranes of cells. No product that was similar in size to the product predicted from the traX open reading frame (27.5 kDa) was detected. Therefore, we used site-directed mutagenesis, stop codon linker insertions, and phoA fusion analysis to investigate traX expression. Both TraX1 and TraX2 appeared to be encoded by the traX open reading frame. Insertion of a stop codon linker into the traX C-terminal coding region led to synthesis of two correspondingly truncated products, and fusions to phoA indicated that only the traX reading frame was translated. Expression was also very dependent on the traX M1 start codon; when this was altered, no protein products were observed. However, pilin acetylation activity was still detectable, indicating that some other in-frame start codon(s) can also be used. All sequences that are essential for activity are contained between traX codons 29 and 225. Sequence analysis indicated that traX mRNA is capable of forming a variety of base-paired structures. We suggest that traX expression is translationally controlled and that F-pilin acetylation activity may be regulated by physiological conditions in cells.

The pKSM710 vector cassette provides tightly regulated lac and T7lac promoters and strategies for manipulating N-terminal protein sequences.

We describe a set of plasmid vectors that are very useful for cloning, expressing, mutagenizing, deleting, and sequencing DNA fragments. A strategy for using one (pKSM717) to obtain mutant protein products that contain deletions of N-terminal amino acids is also presented. Desirable sequences were first combined in plasmid pKSM710 in a manner that facilitates construction of similar vectors carrying alternative selectable markers or replication origins: a cassette that includes LacI-regulated T7 (T7lac) and lacUV5 promoters, a multiple cloning site (MCS)/lacZ alpha sequence, a set of transcription terminators (T phi, rrnBT1, rrnBT2, and Tfd), and an fd origin of replication can be moved as a single unit. Alternative restriction sites permit a lambda PL promoter and/or the sequence of the pMB1 replicon to be included in this unit when desired. With vectors containing the cassette, inserts in the MCS can be identified by their lack of lacZ alpha peptide complementing activity and expressed from the dually regulated T7 (T7lac) and/or lacUV5 promoter. We found expression from this pair of promoters to be very tightly regulated in appropriate hosts; the degree of repression obtainable in the absence of inducer (IPTG) should allow these constructs to be useful for engineering and expressing gene products that are potentially toxic to the cell. Using the pKSM710 cassette, we made derivatives carrying kan (KmR) (pKSM711, pKSM712), kan and lacI (pKSM715), kan and lacIq (pKSM713, pKSM714), and amp (pKSM717, pKSM718). One can use pKSM717 to obtain deletion derivatives that lack the original amino-terminal coding region of a cloned gene sequence but express the polypeptide encoded by the portion of the gene that remains.

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

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.

Construction and analysis of F plasmid traR, trbJ, and trbH mutants.

F plasmid derivatives carrying kan insertion mutations in the transfer region genes traR, trbJ, and trbH were constructed. Standard tests indicated that these loci are not essential for F pilus production or F transfer among Escherichia coli K-12 hosts. Among the traR and trbH mutants tested, the orientation of the kan cassette had no effect on the mutant phenotype. In each case, there was no significant effect on the appearance of F pili, the transfer frequency, or the plating efficiency of F-pilus-specific phages. The trbJ insertion carrying a kan gene oriented in the direction opposite to tra transcription had very little effect on phage sensitivity but markedly reduced the plasmid transfer efficiency. However, the kan insertion mutation at the same site, in the tra orientation, did not seem to affect either property. Analysis of clones carrying trbJ sequences regulated by a phage T7 promoter showed that trbJ expresses an approximately 11-kDa protein product. The TrbJ protein was not expressed from clones carrying a kan insertion or stop codon linker insertion in the trbJ sequence. However, it was expressed from clones that did not include sequences at the beginning of the 113-codon open reading frame in this region. Our data indicated that translation of trbJ must be initiated at the more distal GUG codon in this frame. This would result in expression of a 93-amino-acid polypeptide.

Synthesis of F pilin.

Transfer of the Escherichia coli fertility plasmid, F, is dependent on expression of F pili. Synthesis of F-pilin subunits is known to involve three F plasmid transfer (tra) region products: traA encodes the 13-kDa precursor protein, TraQ permits this to be processed to the 7-kDa pilin polypeptide, and TraX catalyzes acetylation of the pilin amino terminus. Using cloned tra sequences, we performed a series of pulse-chase experiments to investigate the effect of TraQ and TraX on the fate of the traA product. In TraQ- cells, the traA gene product was found to be very unstable. While traA polypeptides of various sizes were detected early in the chase period, almost all were degraded within 5 min. Rapid traA product degradation was also observed in TraX+ cells, although an increased percentage of these products persisted during the chase. In TraQ+ cells, most of the traA product was processed to the 7-kDa pilin polypeptide within the 1-min pulse period; this product [7(Q)] was not degraded but was increasingly converted to an 8-kDa form [8(Q)] as the chase continued, suggesting that host enzymes can modify the pilin polypeptide. Similar results were observed in TraQ+ TraX+ cells, but the primary 7-kDa product appeared to be N-acetylated pilin (Ac-7). An 8-kDa product (Ac-8) was also detected, but this band did not increase in intensity during the chase. We suggest a pathway in which TraQ prevents the traA product from folding to a readily degradable conformation and assists its entry into the membrane, Leader peptidase I cleaves the traA product signal sequence, and a subset of the pilin polypeptides becomes modified by host enzymes; TraX then acetylates the N terminal of both the modified and unmodified pilin polypeptides.

Sequence alterations affecting F plasmid transfer gene expression: a conjugation system dependent on transcription by the RNA polymerase of phage T7.

We constructed derivatives of the Escherichia coli conjugative plasmid F that carry altered sequences in place of the major transfer operon promoter, PY. Replacement of PY with a promoter-deficient sequence resulted in a transfer-deficient, F-pilus-specific phage-resistant plasmid (pOX38-tra701) that could still express TraJ and TraT; TraY, F-pilin, TraD, and TraI were not detectable on Western blots. On a second plasmid (pOX38-tra715) we replaced PY with a phage T7 late promoter sequence. In hosts carrying a lacUV5-promoter-regulated T7 RNA polymerase gene, all transfer-associated properties of pOX38-tra715 could be regulated with IPTG. After induction, pOX38-tra715 transferred at the wild-type frequency, expressed normal numbers of F-pili and conferred sensitivity to pilus-specific phages. No adverse effects on cell viability were apparent, and additional mutations could easily be crossed onto pOX38-tra715. A traJ deletion (pOX38-tra716) had no effect on the IPTG-induced transfer phenotype. Insertion of cam into trbC, resulted in a mutant (pOX38-tra715trbC33) which, after induction, exhibited the same phenotype associated with other trbC mutants; it could also be complemented by expression of trbC in trans. With pOX38-tra715 or its derivatives, we were able to label specifically the products of tra genes located throughout the long tra operon, by using rifampicin. This feature can be used to investigate transfer protein interactions and to follow changes in these proteins that are associated with conjugal mating events.

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

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 of traW 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 traW546 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.

Characterization of trbC, a new F plasmid tra operon gene that is essential to conjugative transfer.

We have characterized a previously unidentified gene, trbC, which is contained in the transfer region of the Escherichia coli K-12 fertility factor, F. Our data show that the trbC gene is located between the F plasmid genes traU and traN. The product of trbC was identified as a polypeptide with an apparent molecular weight (Ma) of 23,500 that is processed to an Ma-21,500 mature protein. When ethanol was present, the Ma-23,500 polypeptide accumulated; the removal of ethanol resulted in the appearance of the processed mature protein. Subcellular fractionation experiments demonstrated that the processed, Ma-21,500 mature protein was located in the periplasm. DNA sequence analysis showed that trbC encodes a 212-amino-acid Mr-23,432 polypeptide that could be processed to a 191-amino-acid Mr-21,225 mature protein through the removal of a typical amino-terminal signal sequence. We also constructed two different Kmr gene insertion mutations in trbC and crossed these onto the transmissible F plasmid derivative pOX38. We found that cells carrying pOX38 trbC mutant plasmids were transfer deficient and resistant to infection by F-pilus-specific phages. Transfer proficiency and bacteriophage sensitivity were restored by complementation when a trbC+ plasmid clone was introduced into these cells. These results showed that trbC function is essential to the F plasmid conjugative transfer system and suggested that the TrbC protein participates in F-pilus assembly.

Characterization of the F-plasmid conjugative transfer gene traU.

We characterized the traU gene of the Escherichia coli K-12 conjugative plasmid F. Plasmids carrying segments of the F transfer operon were tested for their capacity to complement F lac traU526. The protein products of TraU+ clones were identified, and the nucleotide sequence of traU was determined. traU mapped between traW and trbC. It encodes a 330-amino-acid, Mr36,786 polypeptide that is processed. Ethanol caused accumulation of a precursor polypeptide; removal of ethanol permitted processing of the protein to occur. Because F lac traU526 strains appear to be resistant to F-pilus-specific phages, traU has been considered an F-pilus assembly gene. However, electron microscopic analysis indicated that the traU526 amber mutation caused only a 50% reduction in F-piliation. Since F lac traU526 strains also retain considerable transfer proficiency, new traU mutations were constructed by replacing a segment of traU with a kanamycin resistance gene. Introduction of these mutations into a transfer-proficient plasmid caused a drastic reduction in transfer proficiency, but pilus filaments remained visible at approximately 20% of the wild-type frequency. Like traU526 strains, such mutants were unable to plaque F-pilus-specific phages but exhibited a slight sensitivity on spot tests. Complementation with a TraU+ plasmid restored the wild-type transfer and phage sensitivity phenotypes. Thus, an intact traU product appears to be more essential to conjugal DNA transfer than to assembly of pilus filaments.