Protein turnover in nitrogen starving and shift down cells of Bacillus megaterium

Abstract

I n the last decade turnover of protein in growing and non-growing cells of some gramnegative and grampositive bacteria was extensively studied. I n a starving population of E. coli the balanced degradation and resynthesis of protein was found to be about, b%/hr. ; while in growing cells the protein turnover was nearly negligible (MANDELSTAM 1951). The turnover of protein in different subcellular components of E. coli was analyzed by MANDELSTAM and HALVOR-SON (1960) and PINE (1965). I n B. cereu8 the rates of protein turnover in growing and non-growing cell sare 1.4% and 7%/hr. respectively (URBX 1959). More recently WILLETS (1965) employing membrane filter technique, found, that prot>ein degradation during lag phase of diauxic growth of E. coli amounts about 6%/hr.In the following experiments the rate of protein turnover in non-growing cells of non sporogenic B. megaterium KM has been determined. The strain was chosen because according to data of AUBERT and MILLET (1963) and BALASSA (1964) the capability of turnover of protein in bacilli is related to their capability to sporulate and to form or activate an intracellular proteinase. B. megaterium KM forms extracellular and intracellular proteinase ( CHALOUPRA and Kft~6~0v.4 1962) the synthesis of the former is repressed by amino acids. The pool of free amino acids in this organisms is relatively high amounting to about 10% of protein. I n the present study the turnover of protein was determined by the following two methods: 1) by measuring decrease in specific radioactivity of the prelabelled protein 2) by the measurement of decrease in original radioactivity of a constant volume of starving cells, using membrane filter technique. The sample was precipitated with cold TCA (final concentration 5%) and succesively washed on HUFS membranes with TCA and water.The niethionine requiring strain of B. megaterium KM was grown on a synthetic C/G medium containing ammonia, mineral salts and glucose (MCQUILLEN 1955) supplemented with DL-methionine (5 X M). The cells were prelabelled with C'* lysine or 14C leucine (0.1 pc/ml).In the shift down experiments the cells were growing logarithmically for 4 hours and then transferred after washing into either C/G medium without nitrogen or C/G nitrate+methionine medium. DL-lysinehydrochloride or DL-leucine (5 x M) were added t o "trap", labelled amino acid released by protein breakdown. l ) Unesco doctoral fellow. Permanent address : West Regional Laboratories, Lahore-16, Pakistan