The relationship of valine resistance .to the expression of the ilvGEDA operon of Escherichia cob K-12 has been determined. DNA sequence and in vivo protein analyses indicate that in wild-type E. coli K-12 there is a frameshift site within the gene (ilvG) for valine resistance. The ilvG+2096 (formerly designated ilv02096) mutation displaces this frameshift site, resulting in the~expression of ilvG and the relief of transcriptional-polarity on the distal genes of this operon. Thus, the "ilvO" mutation, which concomitantly confers valine resistance and increased expression of the ilvEDA genes, is, in fact, the "reversion" of a polar site within the first structural gene-of the.ilvGEDA operon.

Galactose utilization in Streptomyces lividans was shown to be controlled by an operon that is induced in the presence of galactose and repressed by glucose. Two promoters, gaIPi and galP2, which direct transcription of two distinct polycistronic transcripts, have been identified. galPI is located immediately upstream of the operon and is induced in the presence of galactose. This promoter directs transcription of the gaiT, gaIE, and galK genes. The second promoter, gaLP2, is located within the operon just upstream of the gaIE gene. This promoter is responsible for constitutive transcription of the galE and galK genes. Comparison of the S. lividans gal operon to the Escherichia coli gal operon indicates the presence of a constitutive promoter positioned upstream of galE in both operons. We suggest that coupling the operon's constitutive promoter to the galE gene fulfills a physiological requirement for constitutive UDPgalactose 4-epimerase expression in Streptomyces.The coordinate activation of sets of genes often involves complex combinations of regulatory signals. Many basic concepts about the mechanisms of gene expression in bacteria were formulated from work with the carbon catabolic and amino acid biosynthetic operons of Escherichia coli (1, 2). There are, however, many organisms that utilize different regulatory mechanisms to accomplish the same metabolic events. For example, the genes responsible for galactose utilization in E. coli are organized within a polycistronic operon. The operon is transcribed from two overlapping promoters, PI and P2. The PI promoter is positively activated by a cAMP-receptor protein, whereas P2 is repressed upon PI activation. Both promoters are negatively regulated by the gal repressor (3). The galactose utilization genes of Saccharomyces cerevisiae are also clustered and coordinately expressed. However, each gene is transcribed from its own promoter. The promoters are negatively controlled by the gal80 gene product and positively activated by the gal4 gene product (4). As in E. coli, galactose utilization in Sa. cerevisiae is subject to catabolite repression. Catabolite repression in Sa. cerevisiae, however, is not mediated by cAMP but by the hexokinase isoenzyme PII (5, 6). Thus, the processes that control gene activation reflect both the unique biology of individual organisms and universal principles of gene expression.We are interested in gene regulation in the Gram positive, differentiating bacterium Streptomyces. Members of this genus express a variety of interesting gene sets including those responsible for its morphological differentiation (7) and the biosynthesis of antibiotics (8, 9). We have chosen to study the galactose utilization operon as an example of a regulated set of genes in Streptomyces lividans because it is subject to glucose repression (10) and galactose induction (11). This paper describes the isolation and characterization of the promoters that direct transcription of the St. lividans gal operon. We demonstrate that the St. lividans galactose ...

Much of our present knowledge about gene regulation in bacteria is the result of work done on the gram-negative bacterium Escherichia coli and its various bacteriophages. More recently, gene regulation studies have expanded to include the differentiating Streptomyces species (22). These studies have focused on the isolation and characterization of genes involved in diverse cellular functions such as antibiotic resistance (7,52,53) and biosynthesis (10,16,18,28,36,37,48), carbon catabolism (19,22,45), and morphological differentiation (38). For example, some of the best-characterized of these genes are those which confer antibiotic resistance to neomycin (aph) and erythromycin (ermE). One notable feature of the aph and ermE genes is that each gene is controlled by tandem promoters which may act to differentially control gene expression (7,23). Among the most complex of the gene systems under study are those responsible for actinorhodin (28, 29) and methylenomycin (10) biosynthesis. Each gene system consists of clustered, multiple genes which are organized within polycistronic operons (10, 29). Our understanding of these operons is limited, however, since the molecular details of operon organization and the strategies used to temporally regulate their expression have yet to be revealed.One example of a multiple gene system whose regulatory strategies have been described is the Streptomyces lividans galactose utilization operon. We have recently demonstrated that expression of this operon is directed by two independently regulated promoters: galPi, which is responsible for galactose-dependent transcription of the operon, and galP2, which is an internal, constitutive promoter (19). As observed for the E. coli gal operon, glucose represses galactose induction of the S. lividans operon (19). Glucose repression in Streptomyces species, however, does not appear to be mediated by cyclic AMP (14, 35) but is controlled, at least in part, by glucose kinase (21). Thus, studies of the S. lividans gal operon should elucidate general features of Streptomyces operon organization and novel regulatory mechanisms used by this bacterium.In this report, the isolation, gene organization, and structure of the S. lividans gal operon are described. We demon-* Corresponding author. t Present address: Beckman Instruments, Inc., Brea, CA 92621. strated by complementation of E. coli gal mutants and DNA sequence analysis that the S. lividans gal genes are organized in the order galT, galE, and galK. The DNA sequence of the S. lividans gal genes was also used to predict the protein sequences for the Streptomyces gal enzymes. By comparing these protein sequences with the sequences of the corresponding E. coli (13,25) and Saccharomyces carlbergensis (11) gal enzymes, we found regions of structural homology within each of the galactose utilization enzymes. Finally, the relationship between the gene organization of the operon and the potential role played by the gal enzymes and their metabolites in Streptomyces physiology is discussed below. MATER...

Six ilvG (IlvG+) mutations of Escherichia coli K-12 were transferred to recombinant plasmids, and the DNA sequence of each mutation was determined. This analysis confirmed that expression of the ilvG gene product (acetohydroxy acid synthase II) requires the deletion of a single base pair or the addition of two base pairs within ilvG to displace a frameshift site present in wild-type E. coli K-12. This system should be useful in the analysis of potential frameshift mutagens.

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