No abstract

The Haemophilus influenzae rec-1+ protein plays a central role in DNA metabolism, participating in general homologous recombination, recombinational (postreplication) DNA repair, and prophage induction. Although many H. influenzae rec-1 mutants have been phenotypically characterized, little is known about the rec-1+ gene at the molecular level. In this study, we present the genetic organization of the rec-1+ locus, the DNA sequence of rec-1+, and studies of the transcriptional regulation of rec-1+ during cellular assault by DNA-damaging agents and during the induction of competence for genetic transformation. Although little is known about promoter structure in H. influenzae, we identified a potential rec-1+ promoter that is identical in 11 of 12 positions to the bacterial sigma 70-dependent promoter consensus sequence. Results from a primer extension analysis revealed that the start site of rec-1+ transcription is centered 6 nucleotides downstream of this promoter. We identified potential DNA binding sites in the rec-1+ gene for LexA, integration host factor, and cyclic AMP receptor protein. We obtained evidence that at least one of the proposed cyclic AMP receptor protein binding sites is active in modulating rec-1+ transcription. This finding makes rec-1+ control circuitry novel among recA+ homologs. Two H. influenzae DNA uptake sequences that may function as a transcription termination signal were identified in inverted orientations at the end of the rec-1+ coding sequence. In addition, we report the first use of the Escherichia coli lacZ operon fusion technique in H. influenzae to study the transcriptional control of rec-1+. Our results indicate that rec-1+ is transcriptionally induced about threefold during DNA-damaging events. Furthermore, we show that rec-1+ can substitute for recA+ in E. coli to modulate SOS induction of dinB1 expression. Surprisingly, although 5% of the H. influenzae genome is in the form of single-stranded DNA during competence for genetic transformation, an event that could be a potent SOS-inducing signal, we failed to detect significant changes in rec-1+ transcription during the induction of genetic competence.

S-Adenosylhomocysteine metabolism was studied in cell extracts of streptonigrin-producing Streptomyces flocculus. The major route of metabolism was found to be deamination to form S-inosylhomocysteine. The metabolite was purified by high-performance liquid chromatography and identified by its UV and nuclear magnetic resonance spectra and by its chemical degradation to hypoxanthine.Streptomyces flocculus (ATCC 13257) produces the antitumor antibiotic streptonigrin (9). The biosynthetic pathway which yields streptonigrin involves an S-adenosylmethionine-mediated C-methylation of the I position of the side chain of tryptophan to form P-methyltryptophan (7), as well as two 0-methylations later in the pathway (6). The tryptophan C-methyltransferase is the first enzyme in the pathway which commits a primary metabolite to antibiotic biosynthesis and therefore is a likely site of regulation for the pathway. One product of the reaction, S-adenosyl-L-homocysteine (AdoHcy), is known to be a potent inhibitor of S-adenosylmethionine-dependent methyltransferases (2, 4), and inhibition of the tryptophan C-methyltransferase by AdoHcy has been demonstrated in partially purified enzyme preparations (M. K. Speedie and B. Fox, unpublished data).Cellular levels of AdoHcy are known to be regulated in eucaryotic cells by AdoHcy hydrolase (EC 3.3.1.1), which hydrolyzes AdoHcy to adenosine and homocysteine (12). In procaryotic organisms, the predominant route of metabolism is catalyzed by AdoHcy nucleosidase (EC 3.2.2.9), which generates adenine and S-ribosylhomocysteine (14), although recently Shimizu et al. (12) demonstrated the occurrence of AdoHcy hydrolase in some procaryotic organisms, including one streptomycete. In order to examine AdoHcy metabolism in S. flocculus, we incubated cell extract with AdoHcy and discovered an unexpected metabolite, S-inosylhomocysteine (InoHcy), as the major product. The identification of this metabolite is the subject of this report.Cultures of S. flocculus (ATCC 13257) were inoculated with a 5% inoculum of seed culture which had been grown for 48 h from a spore suspension in 100 ml of Emerson broth (1.0% glucose, 0.4% beef extract, 0.4% Gelysate peptone, 0.1% yeast extract, 0.25% NaCl in 1 liter of distilled water, adjusted to pH 7.0) in a 1-liter Erlenmeyer flask at 28°C and 180 rpm. Cultures (250 ml of Emerson broth in 2-liter Erlenmeyer flasks) were grown for 24 h at 28°C and 180 rpm (New Brunswick model M52 Controlled Environment Incubator Shaker) and then harvested by filtration. A cell slurry in 10 mM sodium phosphate buffer (pH 8.0) with 1 mM dithiothreitol was sonified at setting 5 on an Ultrasonics sonifier in an ice bath and centrifuged for 20 min at 30,000 x g to remove cell debris. The crude cell extract was precipitated with 70% ammonium sulfate and centrifuged at 30,000 x g for 20 min at 6°C. The precipitate was redissolved in 20 mM potassium phosphate buffer, pH 7.0, and dialyzed for 24 * Corresponding author. h against two changes (2.5 liters) of the same buffer at 4°C. This prepar...

A heterologous phosphotriesterase (parathion hydrolase) containing the native Flavobacterium species signal sequence was previously shown to be secreted by Streptomyces lividans. Western blot analysis of the recombinant phosphotriesterase produced by S. lividans demonstrated only the mature form extracellularly but both processed and unprocessed forms in cell-associated samples. To investigate the efficiency of secretion in Streptomyces, a construction was made that substituted a native Streptomyces beta-galactosidase signal sequence for the Flavobacterium signal sequence. This resulted in a higher proportion of hydrolase in the extracellular fluid and a lower proportion of parathion hydrolase remaining cell-associated. These results suggest that use of a native Streptomyces signal sequence may result in more efficient secretion of heterologous proteins.

An S-adenosylhomocysteine deaminase has been isolated and purified from streptonigrin-producing Streptomycesflocculus ATCC 13257. Deamination represents the major metabolic route of S-adenosylhomocysteine in this organism. The protein was found to be monomeric with a molecular weight of 56,100 ± 1,600. The activity was optimal at pH 7.0 and 37°C, and the deaminase was inactivated by p-chloromercuribenzoate but not by metal chelators. The Km for S-adenosylhomocysteine is 2.5 mM, and the Ki for inhibition by deoxycoformycin is 1.6 nM.