We constructed a series of recombinant plasmids containing a kanamycin resistance (Kmr) cassette upstream from, within, and downstream from hisT, which encodes the tRNA modification enzyme pseudouridine synthase I. These Kmr insertions were then crossed directly into the bacterial chromosome. We determined growth characteristics, assayed in vivo hisT expression, and mapped in vivo hisT operon transcripts for the Kmr insertion mutants. We also analyzed polypeptides synthesized in minicells from plasmids containing Kmr cassettes. The combined results from these experiments demonstrate new features concerning the structure and expression of the complex operon that contains hisT. We show that the minimum size of the operon is approximately 3,500 base pairs and that it contains at least four genes, which are arranged in the order usg-2 (pdxB), usg-1, hisT, and dsg-l and encode polypeptides with apparent molecular masses of 42,000, 45,000, 31,000, and 17,000 daltons, respectively. Of these genes, only the functions of usg-2 (pdrB) and hisT are known, and genetic evidence suggests that these two genes do not require usg-l or dsg-l for function. usg-2 (pdxB) is required for growth of bacteria on minimal medium at 37°C. In contrast, the three genes at the end of the hisT operon are dispensable and form a transcription unit that is expressed from a relatively strong internal promoter. The phenotypes of the Kmr insertion mutants and results from gene expression experiments further confirm the position of the internal promoter and locate additional genetic signals in the DNA sequence around hisT. The experiments reported here also indicate several interesting properties of the Kmr cassette as a tool for probing complex operons.

We show that hisT function is required for normal growth of Escherichia coli K-12, since a lack of hisT-mediated pseudouridine tRNA modification causes a uracil requirement that interferes with cell division. We also show that hisT transcription is positively growth rate regulated in exponentially growing bacteria and is induced during the transition from exponential to stationary growth phase.

The DNA sequence of a 2.3-kilobase segment of the E. coli hisT operon was determined. Analysis of the sequence indicated that the upstream gene in the operon encodes a 36,364-dalton polypeptide, which runs aberrantly on SDS-polyacryl ami de gel s. The distal hisT gene encodes the tRNA modification enzyme, pseudouridine synthase I, whichwias shown to have a polypeptide molecular mass of 30,399 daltons. The DNA sequence was consistent with the phenotypes and hisT expression of mutant operons. Analysis of the sequence and genetic complementation experiments demonstrated that the upstream and hisT genes are evolutionarily, structurally, and functionally unrelated; however, translation signals for the two genes overlap, which is consistent with genetic evidence suggesting translational coupling. Codon usage in the upstream gene is radically different from the hisT gene and may underlie the differential expression observed from the operon. Gene-inactivation experiments and Sl-mapping of in vivo transcripts indicated that the operon contains an additional upstream gene. S1-mapping experiments al so confirmed the presence of an internal promoter, which might be stringently controlled. Taken together, these results show that the structure of the hisT operon is complex and suggest that the operon might be regulated at sever levels.INTRODUCTI ON Maturation of stable RNA molecules occurs by multistep processing and modification of primary transcripts. A considerable number of biochemical and genetic studies have outlined the organization of genes that specify stable RNA molecules and the enzymatic cleavage steps involved in processing (reviewed in 1). In contrast, comparatively little is known about modification, even though this process is a dynamic, integral part of stable RNA biosynthesis in both prokaryotes and eukaryotes. In Escherichia coli, greater than 1% of the chromosome encodes the enzymes that modify tRNA and rRNA molecules (2). With the exception of the work of Bjork and his associates on tRNA-methyl transferase genes (sumimarized in 2), the structure and regulation of these genes are largely unknown. In addition, there is currently inadequate knowledge of the functions played by RNA modifications. Modifications undoubtedly play structural roles in stable RNA molecules, and they may influence the interac-© I R L Press Limited, Oxford, England.

The sections in this article are Biofilm Formation Corrosion Promoting Mechanisms Sulfur, Sulfate and Thiosulfate Reduction Practical Aspects Sulfur/Sulfide Oxidation Metal‐oxidizing Bacteria Metal‐reducing Bacteria Acid‐producing Organisms Slime‐producing Bacteria Corrosion Inhibiting Mechanisms

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