Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning.

A novel plasmid vector that is able to replicate both in Escherichia coli and in Streptococcus sanguis is described. This 9.2-kb plasmid, designated pVA856, carries Cmr, Tcr, and Emr determinants that are expressed in E. coli. Only the Emr determinant is expressed in S. sanguis. Both the Cmr and the Tcr of pVA856 may be insertionally inactivated. This plasmid affords several different cleavage-ligation strategies for cloning in E. coli followed by subsequent introduction of chimeras into S. sanguis. In addition, we have modified a previously described E. coli-S. sanguis shuttle plasmid [pVA838; Macrina et al., Gene 19 (1982) 345-353], so that it is unable to replicate in S. sanguis. The utility of such a plasmid for cloning and selecting sequences enabling autonomous replication in S. sanguis is demonstrated.

Demonstration of a metabolic grid at an early step in the streptonigrin biosynthetic pathway in Streptomyces flocculus.

The enzyme activities which catalyze the conversion of tryptophan to beta-methyltryptophan by two different routes have been demonstrated in cell-free extracts of streptonigrin-producing Streptomyces flocculus. The first route involves direct methylation of tryptophan by a C-methyltransferase. The second involves transamination of tryptophan to indolepyruvate, methylation of indolepyruvate to beta-methylindolepyruvate, followed by a reverse transamination reaction to yield beta-methyltryptophan. The direct methylation route was confirmed by the fact that the methyltransferase activity is still present after the transaminase has been inactivated by hydroxylamine treatment. The L-tryptophan C-methyltransferase has been purified 30-fold by ammonium sulfate precipitation and a Sephadex G-150 column. The indolepyruvate C-methyltransferase activity copurified with the tryptophan C-methyltransferase activity, but the transaminase did not. These results show that a metabolic grid exists for the first antibiotic-committed step of the streptonigrin biosynthetic pathway.

Focused, multiple-pass cell for Raman scattering.

A simple optical system is described that makes use of a unique property of ellipsoidal mirrors, viz., light brought to one focus will be reflected alternately through the two foci and collapse to the major axis. This system consists of an on-axis ellipsoidal mirror facing a coaxial flat-spherical mirror assembly that is positioned at the minor axis. Calculations indicate that gains of the order of 500 in the light flux at the point of observation should be attainable with low-eccentricity ellipsoids. Raman-scattered light from atmospheric N(2) was obtained with a system employing a 0.2 eccentricity ellipsoid. An experimental gain of 93 was determined by the ratio of the scattering with the system to the scattering obtained with one beam. This result is in good agreement with the theory.

A tryptophan C-methyltransferase involved in streptonigrin biosynthesis in Streptomyces flocculus.

A C-methyltransferase that catalyses the transfer of a methyl group from S-adenosylmethionine to C-3 of tryptophan, resulting in beta-methyltryptophan, has been identified in cell-free extracts of streptonigrin-producing Streptomyces flocculus. The absolute configuration of the product was shown to be (2S,3R)-beta-methyltryptophan by high-pressure liquid chromatography and reactivity with D- and L-amino acid oxidases. In shake culture, maximum specific activity occurs after S. flocculus enters stationary phase, but before significant streptonigrin accumulates.

Purification, properties, and sequence specificity of SslI, a new type II restriction endonuclease from Streptococcus salivarius subsp. thermophilus.

SslI, a type II restriction endonuclease, was purified from Streptococcus salivarius subsp. thermophilus strain BSN 45. SslI is an isoschizomer of BstNI. SslI activity was maximum at pH 8.8, 0 to 50 mM NaCl, 2 to 8 mM Mg2+, and 42 degrees C. Activity against phage DNA in vitro was demonstrated.

Disseminated tetracycline resistance in oral streptococci: implication of a conjugative transposon.

A DNA sequence specifying tetracycline resistance (Tcr) has been previously cloned from a clinical isolate of Streptococcus mutans designated U202 (J. A. Tobian and F. L. Macrina, J. Bacteriol. 152:215-222, 1982). We used this sequence as a molecular probe in studying the dissemination of Tcr among oral streptococcal species isolated from patients treated with tetracycline. Eleven strains (including S. sanguis I, S. sanguis II, S. mitis, and S. salivarius) from seven patients were examined by Southern blot analysis. Seven strains showed strong hybridization to the Tcr probe, two showed weak hybridization, and two did not display detectable hybridization. Based on previous characterization of the cloned sequence, our data suggest the dissemination of the tetM class of resistance determinants among these oral streptococci. One of the clinical S. sanguis I isolates studied was able to transfer its Tcr phenotype to other oral streptococci and to enteric streptococci in the absence of plasmid DNA. This transfer appeared to be conjugation-like on the basis of its insensitivity to DNase and its dependence on intimate cell-to-cell contact. Using the cloned Tcr sequence, we were able to study the progeny of the matings. Our data suggest that this resistance transfer element occupies a chromosomal location in streptococcal cells and that it strongly resembles the conjugative transposon Tn916 in its behavior.