Regulation of Nitrogen Catabolic Enzymes in Streptomyces clavuligems

Abstract

The levels of several enzymes involved in assimilation of different nitrogen compounds were investigated in Streptomyces clavuligerus in relation to the nitrogen source supplied to the cultures. Threonine dehydratase, serine dehydratase, proline dehydrogenase, histidase and urocanase were not decreased in the presence of ammonium. The latter two enzymes were induced by histidine in the culture medium, while proline dehydrogenase was induced by proline. Glutamine synthetase, urease and ornithine aminotransferase levels were higher with poor nitrogen sources and were repressed by ammonium. Arginase was induced by arginine and repressed by ammonium. Glutamine synthetase was rapidly inactivated upon addition of ammonium to the culture, and could be reactivated in vitro by treatment with snake venom phosphodiesterase, which suggested that adenylylation is involved in the inactivation. Three previously isolated mutants with abnormal glutamine synthetase activities showed pleiotropic effects on urease formation. All these data point to a mechanism controlling preferential utilization of some nitrogen sources in this species.

Overexpression of a Streptomyces viridochromogenes gene (glnII) encoding a glutamine synthetase similar to those of eucaryotes confers resistance against the antibiotic phosphinothricyl-alanyl-alanine

“…Our hybridization data suggest that not only S. viridochromogenes but also other Streptomyces strains might possess two types of GS enzymes, GSI and GSII. Several reports on GS enzymes from a variety of Streptomyces strains identified GSI-like enzymes with regard to molecular mass, holoenzyme structure (4,42,63), and regulation via adenylylation (22,54). However, the possibility of the presence of multiple GS forms has never been considered.…”

Nitrogen metabolism in Streptomyces coelicolor A3(2): modification of glutamine synthetase I by an adenylyltransferase The EMBL accession number of the internal Streptomyces coelicolor glnE fragment is Y17736.

“…Adenylylation of GSI after administering an ammonium shock to cells growing in a nitrogen-limited environment has been detected in a number of Gram-negative prokaryotes (Merrick & Edwards, 1995). In Grampositive streptomycetes, GSI modification by adenylylation (Streicher & Tyler, 1981 ;Bascara! n et al, 1989 ;Fisher & Wray, 1989 ;Hillemann et al, 1993) as well as by ribosylylation (Penyige et al, 1994) had been proposed.…”

Stringent response and initiation of secondary metabolism in Streptomyces clavuligerus

“…This medium was buffered with MOPS and adjusted to pH 6.9 with ~M -K O H ; 25mM-MOPS was used for incubations stopped in mid-growth phase, and 100 mM-MOPS for incubations continued up to stationary phase. Trace elements were added as previously described (Bascaran et al, 1989) but the concentration was increased from 1 to 5 mg I-'. This modification increased protease titres three-to fourfold over those obtained in batch cultures by Bascaran et al (1990).…”

“…The suspensions were sonicated in an ice-cold ultrasonic bath for 5 min and kept at -70 "C. Unless otherwise indicated, cultures were inoculated with mycelium grown for 48 h in seed medium inoculated with spores (Aharonowitz & Demain, 1979). Culture conditions, growth determinations and strain maintenance were as described previously (Bascaran et al, 1989). In experiments requiring precise growth measurements, the flasks were siliconized with Sigmacote (Sigma), a treatment that prevented wall growth.…”

“…Extracellular protease was assayed with azocasein (Sigma) as the substrate (Ginther, 1979). One U of activity is defined as the amount of enzyme that produces an increase in absorbance of 1.0 in 1 h. Lysine E-aminotransferase (EC 2.6.1 .36) (LAT) and diamine aminotransferase (EC 2.6.1 .29) (DAT) were determined in crude extracts obtained by ultrasonic disruption in 10 mM-MOPS, pH 7.2, containing 1 mM-MnC12 (Bascaran et al, 1989). Activities were estimated by spectrophotometric determination of the reaction products derivatized with o-aminobenzaldehyde (Sigma), as described by Madduri et al (1989).…”