Microbiology (1 996), 142, 1937-1 944 Printed in Great Britain Evidence of a role for NAD+-glycohydrolase and ADP-ribosyltransferase in growth and differentiation of Streptomyces griseus NRRL B-2682 : inhibition by m-aminophenylboronic acid Andras Penyige, Eleonora Deak, Attila Ka and Gyargy Barabas Author for correspondence: Andris Penygc. Tel/Fas: + 16 52 4 ma ncz h e I y i G 531. e-mail : p.andras(rr tot.dote.hu Institute of Biology, University Medical School Debrecen, Debrecen, Nagyerdei krt 98, H-4012 Hungary m-Aminophenylboronic acid (APBA) inhibited the germination, growth and sporulation of Streptomyces griseus NRRL B-2682 in an age- and concentration- dependent manner in submerged and solid cultures. When added to cells or cell extracts it irreversibly inhibited NAD'-glycohydrolase and ADP- ribosyltransferase activity. ADP-ribosyltransferase was more sensitive, but inhibition was not complete, even in the presence of 10 mM APBA. The in vivo effects of the inhibitor correlated with its in vitro effect on ADP-ribosylation and on the profile of ADP-ribosylated endogenous proteins. The physiological importance of ADP-ribosyltransferase was supported by the observation that APBA strongly inhibited the growth of a non-sporulating and NAD '- glycohydrolase-negative mutant of the parental strain. The resistance of 5. griseus NRRL B-2682 strains able to grow in the presence of APBA was due to permeability factors. A comparison of the ADP-ribosylated protein profiles of S. griseus NRRL B-2682 grown under various conditions showed similarities, but also specific differences. The results suggest that the ADP- ribosyltransferase of 5. griseus NRRL B-2682 is an indispensable enzyme for growth and differentiation of the strain. It may regulate the activity of key enzymes or developmental proteins by responding to intra- and extracellular conditions. ~ --- Keywords : S'treptoqices gri.ceu.r, h D P- r i bo s y 1 t ran s fe ra se , N ,\ D ' - g 1 y c o h y d c (I I a se , m-aminophenylboronic acid, sporulation INTRODUCTION Streptomycetes are filamentous Gram-positive soil bac- teria, well known for their complex morphological development and their ability to produce a wide variety of secondary metabolites. On solid media the spores ger- minate to produce a network of apically growing hyphae that form the substrate mycelium. This later develops into aerial mycelium, which eventually produces terminally differentiated spore cells. Certain species can sporulate in submerged cultures, although here the substrate my- celium develops into spores without forming aerial mycelium. The early events in sporulation often coincide with the initiation of secondary metabolite production. The genetics of the developmental process have been ex t en s i vel y an a 1 y s e d , e s p e c i a 11 y in .Strep to myces coelicolo r h3(2) and Strepfovyces grisetrs (Chater, 1984 ; Babcock & Kendrick, 1988 ; Ensign, 1988 ; Chater, 1993). In the latter organism cell differentiation and secondary metabolite production are initiated by A-factor, a hydrophobic y- butyrolactone (Khokhlo~ et d., 1973 ; hfiyake et a/., 1990 ; Horinouchi & Beppu, 1992). Some biochemical pathways are regulated post-trans- la t i o na1 1 y by c o ~7 a 1 en t mc ,d i fi c a t i o n of pro t e in s through phosphorylation, adenylylation, methylation or poly- or mono-ADP-ribosvlation (Streicher & Tyler, 1981 ; Nimmo, 1987; Fu et a/., 1990; 1,ouw-y & Ludden, 1990; Hang et a/., I991 ; Hoch, 993). rclono- ADP-ribosylation reactions are catalysed by mono- Abbreviations: ADPRTase, ADP-ribosyltransferase; APBA, m-amino- phenylboronic acid; NAD'ase, NAD--glycohydrolase. 993 ; bl(lrgan ef 0002-0584 0 1996 SGM 1937