The xylose isomerase (xylA) and the xylulose kinase (xylB) genes from Streptomyces rubiginosus were isolated, and their nucleotide sequences were determined. The xylA and xylB genes encode proteins of 388 and 481 amino acids, respectively. These two genes are transcribed divergently from within a 114-nucleotide sequence separating the coding regions. Regulation of the xyl genes in S. rubiginosus was examined by fusing their promoters to the Pseudomonas putida catechol dioxygenase gene and integrating the fusions into the minicircle integration site on the S. rubiginosus chromosome. The expression of catechol dioxygenase was then measured under a variety of conditions. The results indicated that transcription of the xyl genes was induced by D-xylose and repressed by glucose. Data from quantitative S1 mapping were consistent with this conclusion and suggested that xylA had one and xylB had two transcription initiation sites. The transcription initiation site of xylA was 40 bp upstream of the coding region. The two transcription initiation sites of xylB were 20 and 41 bp 5' of its translation initiation codon. Under control of appropriate regulatory elements, the cloned xyl genes are capable of complementing either Escherichia coli xylose isomerase- or xylulose kinase-deficient strains. The deduced amino acid sequence of the S. rubiginosus xylA protein is highly homologous to sequences of other microbial xylose isomerases.

The d gene from the Bacillus subtilis temperate bacteriophage SP,3 was isolated. When introduced into an SP,l-sensitive strain of B. subtilis, the cloned d gene directed the synthesis of a 22-kilodalton protein and conferred on the host immunity to SP,i phage. A frameshift mutation, designated d2, was introduced into the cloned d gene, and it was subsequently crossed back into the SPI8 phage genome. The ml when needed, was used as a plating medium for phage and bacteria.Phage growth and DNA preparation. Phage SPI c2 was prepared from strain CU1147 by the heat induction method essentially as described by Rosenthal et al. (13). Wild-type SP,B phage was prepared either by mitomycin C (0.8 ,ug/ml) induction or, as for SP3 clO (described below), by growing phages lytically by the method of Warner et al. (16) with the following modifications: (i) the adsorption period at 37°C was 15 to 20 min; (ii) the centrifugation step to remove unadsorbed phage was omitted; and (iii) infected cells were diluted 30-to 100-fold into prewarmed (37°C) modified M medium.Purified phage particles were prepared by the method of Fink et al. (3) with several modifications. Polyethylene glycol 6000 was added to 10% (wt/vol) to the phage suspension along with NaCl to 0.5 M to precipitate most of the phage particles. Polyethylene glycol-precipitated phage pellets were suspended in CsCl (1.497 g/ml, in a 10 mM trischloride [pH 7.4]-2 mM MgSO4-10 mM CaC12 buffer) without attempting to remove the trace amount of polyethylene glycol and centrifuged to equilibrium with a Beckman Ti 70.1 rotor at 24,500 rpm for 28 h or with a VTi8O rotor at 60,000 rpm for 6.5 h at 18°C. After collection and dialysis of the phage band, phage suspensions were concentrated, and the phage DNA was extracted with TE (10 mM Trischloride [pH 8.0], 1 mM EDTA) buffer-saturated phenol followed by two chloroform extractions and ethanol precipitation.Isolation of clear-plaque SP13 c1O. During the preparation of SPI c+ phage from the SU+3 (SP1) strain, a spontaneous clear-plaque phage (designated SP, c1O) was fortuitously identified. In all the tests performed, SP clO was indistinguishable from SP, cl; they were both used in these studies.Construction of SPO c2 phage DNA library and other molecular cloning methods. Plasmid pLP1201 (11) replicates in both Escherichia coli and B. subtilis, and it was used for the construction of the phage gene library. BamHI and BglII doubly digested SP3 c2 DNA (2.8 ,ug) was ligated to BamHIdigested pLP1201 (1 ,ug) DNA at a final DNA concentration of about 300 xug/ml. E. coli MM294 (9)