Transcription of the transfer genes traY and traM of the antibiotic resistance plasmid R100-1 is linked.

Three separate traY deletion mutants of R100-1 were prepared by allele replacement. These mutants retained the ability to transfer at a level 100 times greater than R100 and 1/50 that of the parental R100-1. The mutants were complemented to normal R100-1 transfer levels by pDSP06, a multicopy traY clone. Comparison of transcripts initiated at the traY promoter, P(Y), by primer extension experiments showed that there was no detectable P(Y) activity in R100 and that the level of P(Y) activity in the traY deletion mutants was lower than that in R100-1. Similar measurements performed on RNA from a set of previously described traM deletion mutants showed that those traM deletion mutants that produced more traM and finM (M) transcripts than the parental R100-1 also produced more traY transcripts than R100-1 and that those traM mutants that produced fewer M transcripts than R100-1 also produced fewer traY transcripts than R100-1. We conclude that in R100, TraY regulates P(Y) activity and that transcripts originating in traM affect P(Y) activity.

The finM promoter and the traM promoter are the principal promoters of the traM gene of the antibiotic resistance plasmid R100.

finP multicopy repression and traJ multicopy derepression indicate that the ratio of sense to antisense transcripts is important in the regulation of R100 conjugation. The extension of R100 traM transcripts into traJ shows that promoters in traM can affect this ratio, making the regulation of traM transcription important in the regulation of R100 conjugation. Since R100 traM, traY and tral proteins bind to the traM promoter region, we examined traM transcription in R100-1 traM, traY and tral mutants and compared it with traM transcription in both R100-1 and R100. We verified that the traM and finM promoters provide virtually all the transcripts originating in the R100-1 traM gene. When either is deleted, as in VAR22 or VAR30, the remaining promoter is highly active. We show here that traY positively regulates R100-1 traM transcription, as has been found for F. We found that tral did not regulate R100-1 traM transcription. The measured activity of the native R100 traM promoter was 12% of that in R100-1, whereas the native R100 finM promoter was 45% of that in R100-1. These data and data from the R100-1 traY and tral mutants show that the activities of the two promoters varied independently.

Regulation of R100 conjugation requires traM in cis to traJ.

Deletion mutants of R100-1 were constructed by classical methods to remove various segments of the traM open reading frame, pTraM-binding sites and the traM promoters. Complementation tests showed that traM was efficiently complemented only when the trans-acting fragment contained both the complete traM gene and the adjacent traJ promoter and leader sequences. The conclusion is that traM and traJ constitute a complex operon. A deletion mutant lacking all of the traJ gene, and one containing a frameshifting traM deletion, retained the ability to transfer at a low level, thereby showing that neither pTraM nor pTraJ is absolutely essential for transfer.

traJ sense RNA initiates at two different promoters in R100-1 and forms two stable hybrids with antisense finP RNA.

RNase protection experiments show that the sizes of the two R100 finP molecules are 74 and 135 nucleotides. In an RNase III mutant, finP transcripts form stable double-stranded hybrids of 108 bp and 68 bp with traJ transcripts. RNase protection experiments also show that most R100-1 transcripts originating in traM cross the traM-traJ intergenic region and end inside the untranslated leader region of traJ. Some extend into the traJ open reading frame. These findings mean that the antisense finP RNA, thought to regulate traJ translation, must regulate traJ transcripts from both J and M promoters.

Suppression of insertions in the complex pdxJ operon of Escherichia coli K-12 by lon and other mutations.

Complementation analyses using minimal recombinant clones showed that all known pdx point mutations, which cause pyridoxine (vitamin B6) or pyridoxal auxotrophy, are located in the pdxA, pdxB, serC, pdxJ, and pdxH genes. Antibiotic enrichments for chromosomal transposon mutants that require pyridoxine (vitamin B6) or pyridoxal led to the isolation of insertions in pdxA, pdxB, and pdxH but not in pdxJ. This observation suggested that pdxJ, like pdxA, pdxB, and serC, might be in a complex operon. To test this hypothesis, we constructed stable insertion mutations in and around pdxJ in plasmids and forced them into the bacterial chromosome. Physiological properties of the resulting insertion mutants were characterized, and the DNA sequence of pdxJ and adjacent regions was determined. These combined approaches led to the following conclusions: (i) pdxJ is the first gene in a two-gene operon that contains a gene, temporarily designated dpj, essential for Escherichia coli growth; (ii) expression of the rnc-era-recO and pdxJ-dpj operons can occur independently, although the pdxJ-dpj promoter may lie within recO; (iii) pdxJ encodes a 26,384-Da polypeptide whose coding region is preceded by a PDX box, and dpj probably encodes a basic, 14,052-Da polypeptide; (iv) mini-Mud insertions in dpj and pdxJ, which are polar on dpj, severely limit E. coli growth; and (v) three classes of suppressors, including mutations in lon and suppressors of lon, that allow faster growth of pdxJ::mini-Mud mutants can be isolated. A model to account for the action of dpj suppressors is presented, and aspects of this genetic analysis are related to the pyridoxal 5'-phosphate biosynthetic pathway.

Integration host factor affects expression of two genes at the conjugal transfer origin of plasmid R100.

Integration host factor (IHF) binds to two sites near the origin of transfer of the conjugative antibiotic resistance plasmid, R100. DNase I footprinting shows that one site is immediately adjacent to oriT and the gene X promoter, and another is adjacent to the traM promoter. A third site, known only from retardation gels, is near the traJ promoter. The relative promoter activities of genes X, traJ and traM are reduced in himA mutants (IHF-), as measured by chloramphenicol-resistance assays. Transcript analyses by Northern blots showed a reduction in size of the principal gene X and traJ transcripts in the absence of IHF.

Derepression of conjugal transfer of the antibiotic resistance plasmid R100 by antisense RNA.

Conjugal transfer of the normally repressed antibiotic resistance plasmid R100 was derepressed by fragments of R100 that carried the traJ promoter and the traJ leader but lacked the finP promoter.

Sense and antisense transcripts of traM, a conjugal transfer gene of the antibiotic resistance plasmid R100.

The region of the antibiotic resistance plasmid R100 that encodes the plasmid-specific transfer gene traM has two tandemly aligned promoters separated by 145 nucleotides. The principal transcripts are 705 and 562 nucleotides long. Minor transcripts are 1550 and 1700 nucleotides long. The 705-base transcript appears to encode an 11 kD traM protein. The 562-base transcript does not encode a detectable protein. When subcloned on short fragments, the promoter for the 562-base transcript initiates efficiently but that for the 705 site does not. The 3' ends of the 705 and 562 base transcripts end inside the traJ ORF. Thus they provide additional sense RNA to compete with traJ for finP, the antisense translational regulator of traJ. A model is proposed for the participation of these sense and antisense transcripts in the control of expression of the traJ gene.

Transcript analysis of the plasmid R100 traJ and finP genes.

Single-stranded RNA probes were used to study the regulation of plasmid transfer in the infectious antibiotic resistance plasmid R100. Transcription of the positive transfer control gene traJ of R100 appears to be initiated continuously. In the presence of finO, the traJ transcript is 235 bases long, and in the absence of finO it is 1050. These sizes are strain specific. finO increases four-to tenfold the amount of the transcript from the finP gene that is detectable in cells containing R100, R136, or the sex factor F. The size of the principal finP transcript from R100 as determined on Northern blots is 105 bases. A secondary transcript with a size of 180 bases was detected in small amounts in R100 extracts. The finP transcript size was also determined by nuclease protection experiments. In this case the size was 74 bases. The 5' ends of the finP and traJ transcripts were located by primer extension experiments. A new model of FinO/P control is proposed.

Integration host factor and conjugative transfer of the antibiotic resistance plasmid R100.

Transfer of plasmid R100-1 was reduced 100-fold in the absence of integration host factor.

oriT sequence of the antibiotic resistance plasmid R100.

We present the nucleotide sequence of the oriT region from plasmid R100. Comparison to other IncF plasmids revealed homology around the proposed nick sites as well as conservation of inverted repeated sequences in the nonhomologous region. Three areas showed strong homology (eight of nine nucleotides) to the consensus sequence for binding of integration host factor, suggesting a role for this DNA-binding protein in nicking at oriT.

Fertility inhibition gene of plasmid R100.

The fin0 gene of R100 was isolated from the Fin0+ transducing phage VA lambda 57. The limits of the gene were determined by BAL31 digestions and by analysis of deletion mutations derived from an internal restriction site. The DNA sequence contained an open reading frame of 558 nucleotides that would encode a protein of 21,268 daltons. Synthesis of such a protein was observed only when the fragment was cloned in front of the TAC promoter. Deletions entering the large open reading frame from either end were Fin0-, while internal frame shift mutations retained high Fin0 activity. One such strain had a 13 bp internal deletion that would produce a protein of 63 amino acid residues of which 21 were basic. We were consequently unable to rigorously establish that the 558 base orf encoded a fin0 product. The strand opposite the large open reading frame contained several transcription termination signals, and it is possible that the active gene product is one or two small RNAs from this strand.

Cloning, mapping, and sequencing of plasmid R100 traM and finP genes.

The fertility control gene finP, the transfer gene traM, and the transfer origin, oriT, of plasmid R100 were isolated on a single 1.2-kilobase EcoRV fragment and were then subcloned as HaeIII fragments. The sequence of the 754-base-pair finP-containing fragment is reported here. In addition to the finP gene, the sequence includes all but two bases of the R100 traM open reading frame and apparently all of the leader mRNA sequence and amino end of the traJ gene of R100. The sequence contains two open reading frames which encode small proteins on the opposite strand from the traM and traJ genes. It also shows two sets of inverted repeats that have the characteristics of transcription terminators. One set is positioned as if it was the traM terminator, and the other set, which is downstream from the first, sits in the middle of the leader mRNA sequence for traJ. On the bottom strand, this inverted repeat has the structure of a rho-independent terminator. Other less-stable inverted repeats overlap this second terminator in the same way as is seen in attenuation sequences, and the two separate small open reading frames on the bottom strand also totally overlap the stem of the rho-independent terminator, suggesting that their translation would cause shifting of termination to the bottom strand homolog of the putative traM terminator. The finP gene product was not identified, but the gene was mapped to the sequence which contains the traJ gene. It either overlaps traJ or is antisense to it.

Low muscle levels of pyridoxine in McArdle's syndrome.

Pyridoxal phosphate is a covalently bound cofactor of glycogen phosphorylase. Phosphorylase is a major muscle protein and therefore represents a significant pool of pyridoxal phosphate. Muscle pyridoxine content was measured in three patients with myophosphorylase deficiency (McArdle's syndrome) in whom there was a marked diminution or absence of phosphorylase protein as determined by acrylamide gel electrophoresis. Total muscle pyridoxine in the patients with McArdle's syndrome (0.55 +/- 0.08 microgram/g wet weight, mean +/- SD) was markedly reduced compared with 11 human control subjects who had normal levels of muscle phosphorylase (total muscle B6 = 2.49 +/- 0.47). Despite such drastically low levels of muscle pyridoxine, these patients had no evidence of pyridoxine "deficiency." These results suggest that low muscle B6 in McArdle's syndrome represents the specific loss of pyridoxal phosphate normally bound to phosphorylase apoenzyme and imply that phosphorylase pyridoxal phosphate accounts for 75 to 80 percent of the total pyridoxine in normal human muscle.

The finO gene of antibiotic resistance plasmid R100.

Lambda phages carrying the R100 finO gene have been isolated from an R100:: lambda cointegrate in which lambda was inserted into the R100 traD gene at kb coordinate 72.1. Physical analyses of these phages place the finO gene within R100 SalI fragment D, near kb coordinate 82.0. Analysis of proteins synthesized by the phages did not identify the finO gene product, although a constitutive protein of m.w. 30,100 was encoded by R100 DNA between kb coordinates 78.7 and 81.2.

Threonine prevents derepression of pyridoxine synthesis in Escherichia coli B.

After 40 min of pyridoxal starvation, pyridoxine phosphate oxidase-less mutants of Escherichia coli B derepressed pyridoxine biosynthesis 13-fold to a rate of 1.7 X 10(-9) mol/h per mg of cells. Threonine at 100 mg/liter prevented this derepression but did not affect the continued synthesis of pyridoxine. Neither serine nor branched-chain amino acids altered the threonine effect.

Lambda transducing phages derived from a FinO- R100::lambda cointegrate plasmid: proteins encoded by the R100 replication/incompatibility region and the antibiotic resistance determinant.

Three lambda transducing phages have been isolated from pEDR20, an R100::lambda cointegrate plasmid in which the lambda insertion inactivated the R100 finO gene. Physical analysis of the three phages showed that the lambda is inserted at kilobase coordinate 81.3 of R100. All three phages carry different amounts of R100 DNA in the left arm of lambda. Each pahge contains ISlb, the mer genes and the region between coordinate 81.3 and 88.6; thus, all contain the genes necessary for R100 replication. One phage, VA lambda 73, contains the entire r-determination of R100 in addition to the above DNA. Five proteins coded by the region between 81.3 and 88.6 were detected. These had subunit molecular weights of 10,400; 12,200; 16,200; 19,600; and 38,300. The first was made constitutively and the other four only from a lambda promoter. Other constitutive proteins were one from the cml fus region with a molecular weight of 22,400 (cml) and two from the str sul region with molecular weights of 31,500 (str?) and 30,100 (sul?). Mercuric ion induced synthesis of at least 10 proteins. Six of these were known from earlier work. The total size of the proteins which appear to derive from the mer genes exceeds by a factor of 1.5, the coding capacity of this region without overlapping genes. Some, or all of these extra proteins may be chromosomal in origin, possibly derepressed in response to mercury gene products.

Properties of lambda transducing bacteriophages carrying R100 plasmid DNA: mercury resistance genes.

Three lambdamer (resistance to Hg2+ and mercurials) transducing phages were prepared from three independent cointegrate isolates of bacteriophage lambda and plasmid R100. DNA heteroduplex and restriction nuclease analyses of the lambdamer DNA showed that all three phages had resulted from lambda insertion at kilobase coordinate 8.6 of plasmid R100, followed by loss of different lengths of lambda DNA and replacement with different lengths of R100 DNA. Two of the lambdamer phages were defective, containing deletions from lambdaatt through the lambdaN gene and into the lambdarex gene; the third, VAlambda14, was an N+ Spi- plaque-forming phage. With VAlambda14, N-dependent transcription of R100 mer from the lambdapL promoter suggested that transcription of mer proceeded in the direction from IS1b toward the sulfonamide resistance determinant (i.e., from a plasmid promoter in restriction nuclease fragment EcoRI-H toward fragment EcoRI-I). Phage-directed protein synthesis in a UV-irradiated lambdaind- lysogen showed the Hg2+-inducible synthesis of three major polypeptides of molecular weights 68,000, 11,500, and 8,500 and three minor ones of molecular weights 54,000, 33,000, and 13,500. The largest of the major polypeptides is identified as the subunit of the mercuric reductase enzyme. The functions of the smaller polypeptides are not known. Hg2+ reductase enzyme assays confirmed the regulation of mer synthesis during phage infection.