Xenorhabdus innexi is a bacterial symbiont of Steinernema scapterisci nematodes, which is a cricket-specialist parasite and together the nematode and bacteria infect and kill crickets. Curiously, X. innexi expresses a potent extracellular mosquitocidal toxin activity in culture supernatants. We sequenced a draft genome of X. innexi and compared it to the genomes of related pathogens to elucidate the nature of specialization.

The ssgA gene of Streptomyces griseus B2682, when present in high copy number, results in both suppression of sporulation and fragmented growth of mycelia. Western analysis with polyclonal antibodies against the gene product (SsgA) revealed a close correlation between SsgA accumulation and the onset of sporulation in wild-type cells. The protein was only detected in the cytoplasm. Certain developmental mutants of S. griseus (afs, relC and brgA) which are defective in aerial mycelium formation in solid culture and submerged spore formation in liquid culture failed to accumulate SsgA. The SsgA protein appeared shortly (1 h) after nutritional shift-down of strain B2682 cells. afs mutant cells sporulated and expressed SsgA only when A-factor was present both before and after nutritional shift-down. Introduction of the ssgA gene in a low-copy-number vector into strain B2682 resulted in fivefold overexpression of SsgA, and was accompanied by fragmented growth of mycelia and suppression of submerged spore formation (in liquid culture) and aerial mycelium formation (in solid culture). Streptomycin production was not inhibited. In a control experiment, a nonfunctional ssgA gene possessing a frameshift mutation near its N-terminus had no effect on either growth or sporulation. It is proposed that the ssgA gene product plays a role in promoting the developmental process of S. griseus.

Flagella of some of the actinoplanete genera were purified and the molecular sizes of their flagellin subunits compared by SDS-PAGE analysis to flagellins of cells of other bacteria. Several species of Actinoplanes have a major flagellar protein of subunit sizes of 42-43 kDa and a lesser amount of a second protein, possibly a minor flagellin subunit, of 60 kDa, The flagellar protein sizes of other actinoplanetes ranged from 32-43 kDa (major) and 48-58 kDa (minor), Antibodies formed against the 42-kDa protein of A. rectilineatus showed cross-reactivity in Western blots against flagellar proteins of spores of other Actinoplanes species, two species of Dactylosporangium and an Ampullariella species, Cross-reactivity was also observed with motile cells of two other actinomycetes, Arthrobacter atrocyaneus and a Geodermatophilus species, and with Bacillus subtilis, No cross-reactivity was observed with Escherichia coli or Planomonospora parontospora flagellar proteins, The amino acid composition and partial N-terminal sequence of the 42-kDa flagellar protein of A. rectilineatus was compared to literature data for other bacterial flagellins and found to be most similar to B. subtilis 168.

An oxygen electrode inserted into a dead Manduca sexta larva infected with Heterorhabditis nematodes carrying the bacterium Photorhabdus luminescens showed barely detectable levels of oxygen in a 1 to 2 mm zone below the cuticle, and virtual anaerobiosis deeper in the carcass. This observation indicates that the bacteria in this habitat, where they are actively growing, are probably carrying out a fermentative metabolism. Therefore, the anaerobic metabolism of the primary and secondary form variants of P. luminescens Hm and NC1 was compared. Amino acids were not fermented by either strain, either singly or in mixtures. Glucose was fermented by both forms of both organisms, forming products typical of mixed acid fermentation by Enterobacteriaceae. The fermentation patterns were the same in the primary and secondary forms. Growth rates of the secondary form cells were higher in defined medium with glucose as energy and carbon source. Growth yields of the primary and secondary forms of strain Hm were nearly identical, whereas the growth yield of secondary form cells of strain NC1 was slightly higher than that of the primary form. The results of this study indicate that the observed predominance of primary form cells in infected insect larvae cannot be explained by an advantage over the secondary form cells related to the efficiency of anaerobic growth or fermentative metabolism.

Streptomyces griseus does not sporulate when grown in a nutrient rich medium. Two mutants that sporulate in such media were isolated and characterized. The mutants sporulate at the same time as the wildtype when grown in a nutrient poor medium. The mutants and wildtype exhibit the same commitment to sporulation following a nutritional shiftdown. Six genomic DNA fragments of wildtype cells when transformed into the mutants caused these mutants to acquire the wildtype phenotype of not sporulating in rich growth media. Restriction analyses show that the six cloned DNA fragments are different. The six clones suppressed sporulation when transformed into Streptomyces coelicolor and Streptomyces lividans and secondary metabolite production was suppressed in S. coelicolor.

A mutant of Streptomyces griseus, NY5, differs from the parent in sporulating when grown in a nutrient rich growth medium. Cloned genes of the wildtype organism in the multicopy plasmid pIJ702 transformed into the NY5 mutant cells resulted in converting the mutant phenotype of sporulating in rich medium to the parental genotype of not sporulating in rich medium. A 1.5 kb DNA fragment was sequenced and found to contain two open reading frame gene regions. Restriction deletion mapping and subcloning revealed a gene which when transformed in high copy number into S. griseus wildtype, mutant NY5 and S. lividans resulted in suppression of sporulation and fragmented cell growth. The gene, named ssgA (DDJB/EMBL/GenBank accession no. is D50051), encodes a 145 amino acid protein with a calculated size of 15.8 kDa. The predicted protein has a strong negative charge and shows no significant sequence homology to known proteins. Southern analysis detected regions in S. coelicolor and S. lividans DNA homologous to ssgA.

Addition of NH4+ to STreptomyces griseus 2682 cells grown in NO3- containing medium resulted in a rapid decline in glutamine synthetase activity due to covalent modification of the enzyme. The NH4+ promoted inactivation of the enzyme was inhibited by the ADP-ribosyltransferase inhibitor 3-methoxybenzamide. In the presence of ADP-ribosyltransferase activity the purified glutamine synthetase was also inhibited by NAD+ in a concentration-dependent manner. ADP-ribosylation of glutamine synthetase was demonstrated in vitro by showing the incorporation of labeled ADP-ribose from [alpha-32P]NAD+ into glutamine synthetase subunits. Beside ADP-ribosylation, adenylylation of glutamine synthetase was also shown in S. griseus since phosphodiesterase I treatment reactivated the enzyme in crude extracts of NH(4+)-shocked cells. Glutamine synthetase was also inhibited and modified by ATP in crude cellular extracts. These results suggest that in S. griseus 2682 ADP-ribosylation of glutamine synthetase could be an alternative modification to adenylylation to regulate glutamine synthetase activity.

Actinomycetes are widespread in the environment and are mainly organotrophic. Studies of their ecology have been primarily focussed on their detection and isolation, with comparatively little attention to the control mechanisms that determine their occurrence and behaviour in their natural environments. This session provided some diverse examples of approaches to this problem. Several actinomycete genera produce motile spores. The significance of flagella proteins and factors influencing spore motility and germination are considered. The genus Frankia forms nitrogen-fixing associations with non-leguminous plants. Molecular techniques have been used to clarify the endophyte-host relationships. Micromonospora species are common in the environment. The growth and physiology of a gentamicin-producing strain are described. Thermophilic actinomycetes in the genus Thermoactinomyces are common in composts and other self-heating environments. Novel isolates from acid soil, which grow and produce enzymes active at high temperatures and in acidic conditions, are discussed.

The role of ADP ribosylation of proteins in the physiological regulation of sporulation in Streptomyces griseus was studied. We report here that both the activity of NAD+: arginine ADP-ribosyltransferase (ADPRT) and the pattern of ADP-ribosylated proteins showed characteristic changes during the life cycle in S. griseus 2682. Analysis off ADP-ribosylated proteins revealed that in a nonsporulating mutant of the parental wild-type (wt) strain (Bld7 mutant), both the activity of ADPRT and the pattern of ADP-ribosylated proteins were different from those of the parental strain. Addition of 3-aminobenzamide (3AB), the most potent inhibitor of ADPRT, inhibited sporulation of S. griseus 2682 and the A-factor (AF)-induced sporulation of S. griseus Bld7, but in both cases the inhibitory effect of 3AB was strictly age-dependent. Using [alpha-32P]GTP, we have demonstrated the presence of GTP-binding proteins in purified cell membranes of S. griseus 2682 and S. griseus Bld7. The same GTP-binding proteins were observed in Bld7 and the wt. AF stimulated the basal GTPase activity of cell membranes of S. griseus 2682 in a concentration-dependent manner, suggesting that GTP-binding proteins might be involved in the AF-induced sporulation process.

Conditions for microcycle sporogenesis in two streptomycete strains without shifting the culture were found. The sporulation in Streptomyces granaticolor took place after 24 h of cultivation. The dry mass was increasing till 32 h probably due to production of a hydrophobic substance resembling fibrous sheath of aerial hyphae and spores. Ultrathin section of microcycle spores are presented.

Membranes purified from cells of Streptomyces griseus strain 52-1 possess an ADP-ribosyltransferase activity. The enzyme transfers the ADP-ribose moiety of NAD to one major membrane protein of Mr 32,000 and 2-3 minor proteins of larger molecular weights. The effects of inhibitors on the ADP-ribosyltransferase activity proves that the reaction is enzymatic and suggests that the enzyme ADP-ribosylates the guanidine group of arginine. The kinetics of liberation of ADP-ribose during alkaline hydrolysis of the modified proteins is consistent with the arginine-ADP-ribose bond. This is the first report of ADP-ribosylation of proteins in a Gram-positive bacterium.