- Volume 143, Issue 11, 1997

This paper reports a novel use of cluster analysis for the identification of intermediary metabolites that are produced at rates closely correlated with those of antibiotic biosynthesis. This information was used to devise culture feeds resulting in enhanced production of clavulanic acid, an antibiotic of current worldwide commercial interest. The feeding strategies apparently alleviated a rate-limiting supply of the C3 precursor of clavulanic acid. C3 limitation may be a consequence of unusual nitrogen and carbon metabolism in Streptomyces clavuligerus. This approach has potential as a generic method for influencing biosynthetic pathway fluxes using feeds without knowledge of the biosynthetic pathway.

The life cycle of Streptomyces coelicolor during development on solid medium has been studied from a physiological perspective. A biphasic growth pattern was demonstrated, evidenced by a continuous transition from an initial exponential growth period into a slower phase of biomass accretion. The switch between the two phases coincided with the exhaustion of nitrate from the medium. The depletion of nitrate from the medium coincided with the initiation of aerial mycelium formation within the cultures and the development of hydrophobic surface properties. During secondary growth, cultures remained metabolically active, continuing to accumulate DNA, despite a cessation in the levels of RMA and cell protein accretion. In addition, the accumulation of glycogen and lipid contributed to the observed accretion of biomass in this phase. The depletion of nitrate also marked an increase in the production of α-ketoglutarate by the culture and a coincident decrease in medium pH. Latter stages of the secondary growth phase saw the development of spores within the culture, this in turn was associated with a decrease in cellular glycogen. This supported previous observations that glycogen degradation and spore maturation were intimately associated.

The constitutive and the heat-shock-induced expression of members of heat-shock protein families changed during vegetative development and conidiation of Neurospora crassa as determined by two-dimensional gel electrophoresis. Western blot, and ELISA analyses revealed the highest amounts of the constitutive heat-shock protein 70 (HSC70) in conidiating aerial hyphae and dormant conidia. During conidial germination the amount of HSC70 decreased and subsequently increased during vegetative growth. Stationary mycelia and young aerial hyphae exhibited the lowest HSC70 level. The stationary-phase-dependent decrease in HSC70 was accompanied by a concomitant increase in its nuclear localization, whereas no significant changes in the amount of nuclear HSC70 were found during aerial hyphae development. The cAMP content during aerial hyphae development was inversely correlated with that of HSC70. To examine possible causal relations between HSC70 expression and cAMP content, the adenylate-cyclase-deficient mutant crisp (cr-1) was analysed, which exhibits low concentrations of endogenous cAMP. This mutant, however, showed a lower constitutive HSC70 level, compared to the bdA strain. Treatment of the bd strain and cr-1 mutant with 20 μM 8-bromo-cAMP did not result in significant changes of the constitutive HSC70 level, but in the level of heat-induced HSC/HSP70. In a developmental mutant (acon-2) which is defective in a differentiation step toward conidiation, the expression of HSC70 in aerial hyphae was delayed until the first proconidial chains were observed. It is concluded that the differential expression of HSC/HSP70 does not depend on different nuclear levels of HSC70 or on changes in cAMP concentrations, but rather on developmental genes controlling conidiation.

The 200 kb region of the Bacillus subtilis chromosome spanning from 255 to 275° on the genetic map was sequenced. The strategy applied, based on use of yeast artificial chromosomes and multiplex Long Accurate PCR, proved to be very efficient for sequencing a large bacterial chromosome area. A total of 193 genes of this part of the chromosome was classified by level of knowledge and biological category of their functions. Five levels of gene function understanding are defined. These are: (i) experimental evidence is available of gene product or biological function; (ii) strong homology exists for the putative gene product with proteins from other organisms; (Hi) some indication of the function can be derived from homologies with known proteins; (iv) the gene product can be clustered with hypothetical proteins; (v) no indication on the gene function exists. The percentage of detected genes in each category was: 20, 28, 20, 15 and 17, respectively. In the sequenced region, a high percentage of genes are implicated in transport and metabolic linking of glycolysis and the citric acid cycle. A functional connection of several genes from this region and the genes close to 140° in the chromosome was also observed.

A 15 kb DNA fragment from the Bacillus subtilis chromosome between citB and ppsC has been sequenced, and new ORFs encoding putative enzymes involved in lipopolypeptide synthesis, which complete a partial operon previously reported, and a new set of enzymes responsible for lipid metabolism have been identified. From the analysis of DNA sequence homology of the fragment it was deduced that these new peptide synthetase genes are part of an operon for the biosynthesis of the fungicide fengycin.