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 pdx4, 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 pdrA, pdxB, and pdxH but not in pdr. This observation suggested that pd4, like pdx4, 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 ofpdrJ::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.

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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.

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.

Single and tandem insertions of prophage X into R100 have been isolated. Insertions into the transfer genes, insertions into the transfer control gene finO, and insertions into regions that result in no detectable phenotypic change were found. From the last type, deletion mutants were isolated which established the sequence of antibiotic resistance genes as tet-cml-fus-str-sul-mer in R100. Highfrequency transducing phage preparations Xmer, Xsul str, and Xsul str cml were also isolated from this type. R factor R100 is a conjugative plasmid existing as a covalently closed circular deoxyribonucleic acid (DNA) molecule of 60 x 10 to 70 x 106 molecular weight in Escherichia coli K-12. The functions that this plasmid determines include: (i) the ability to replicate in a controlled fashion in the host cell, together with the incompatibility of this replication with that of related plasmids in the same cell; (ii) resistances to tetracycline, chloramphenicol, streptomycin (and spectinomycin, using the same adenylase [3,9]), sulfonamides, mercuric ions, and fusidic acid (5); (iii) a transfer system that allows it to transfer its DNA by conjugation to a suitable recipient cell; and (iv) a two-stage system that controls expression of the transfer genes (7).As a long-term goal, we are interested in elucidating the detailed genetic and physical structure of R100, including in particular the mechanisms whereby expression of the transfer genes is controlled. As a step towards that end, we first isolated a number of "co-integrates" of R100 and X by using the techniques for X insertion into "unusual" attachment sites pioneered by Shimada et al. (16). These X insertions are frequently strongly polar and therefore help to identify operons. Second, from insertions of a XcI857 phage, deletion mutants can be selected as survivors to high temperatures. These can be used to locate the genes responsible for the various R factor functions, and. they are also useful in the characterization of operons and the genes controlling them. Third, it is possible to isolate transducing phages from such insertions, which should allow gene amplification where it is required to purify a gene product, and serve as a source of DNA for measuring transcription of discrete regions of R100 using hybridization techniques. This report presents the methods used to isolate R100 (X) co-integrates and summarizes results showing the approximate locations of the prophage insertions. Deletion mutants from two integrates are described that have allowed a genetic map of the antibiotic resistance genes of R100 to be constructed. MATERIALS AND METHODSBacterial strains. The host strain for R100 or R100-1 in which the X insertions were generated was ED2149. This is a derivative of ED395 that is T66 and carries a deletion covering nia, gal, att,, and bio (ED395 is a LacAu,24-derivative of W3110). The att, deletion was transduced with P1 grown on CT1O (obtained from C. Town) into a (XcI857 susJ6 xis-i) + lysogen ofthe parental attx+ strain, since all spontaneous gal att, bio ...