Glucose served as an exogenous energy source for sporulation when a limited amount was fed continuously. Excess glucose repressed sporulation.

Cultures of Clostridium thermosaccharolyticum, under conditions of restricted growth achieved by slow feeding of glucose, showed a high degree of sporulation. Analysis of the end products showed an accumulation of ethyl alcohol in addition to butyrate and acetate, whereas, in the nonsporulating cultures, acetate and butyrate were the principal products. Incorporation of uniformly labeled "4C-glucose by sporulating cells was three to four times higher than by nonsporulating cells. The efficiency of acetate assimilation into the lipid fraction of sporulating cells was at least two times higher than that of glucose. When starch was used as the carbon source, the growth rate was reduced; sporulation occurred, and the end products and carbon distribution were similar. Alcohol dehydrogenase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase were preferentially formed by sporulating cells. In vegetative cells, the formation of these enzymes was repressed if the glucose concentration in the medium was increased. The change in enzyme activity appeared to be related to a morphological change in the cells and indicated an altered metabolic pattern for sporulating cells.

Good sporulation was induced in batch culture using carbon sources that have in common the effect of reducing growth rate. We have recently demonstrated that sporulation of Clostridiwn thermosaccharolyticum does not occur endotrophically but requires an exogenous source of carbon and energy (2). It was also demonstrated that sporulation occurred under conditions of restricted growth. Pheil and Ordal (3) had previously demonstrated that a medium containing L-arabinose, as the carbon and energy source, supported sporulation. A reexamination of growth and sporulation in this medium demonstrated that growth was restricted because arabinose was utilized slowly. The specific growth rates in arabinose-and glucosecontaining media were 0.105 and 0.578, respectively. This then raised the question of whether other carbon sources, which were utilized less

Selected gram-negative bacteria were differentiated by comparing profiles of their intracellular aminopeptidase activities. Aeromonas liquefaciens plus 14 species and serotypes of Enterobacteriaceae were used in the study. Substrates for the bacterial enzymes were 19 L-amino acid beta-naphthylamides. The beta-naphthylamines released by enzyme hydrolysis were determined for each amino acid by fluorometric analysis, and a profile for each culture was obtained in 4-6 hr. The technique appears to be useful for differentiation of bacteria and for demonstrating relatedness.

Production of polygalacturonic acid (PGA) trans-eliminase was greatly stimulated under conditions of restricted growth of Aeromonas liquejaciens. This was accomplished either by substrate restriction in a continuous-feeding culture or by restricting divalent cations in a batch culture, with the use of PGA as the sole source of carbon in a chemically defined medium containing inorganic nitrogen. Slow feeding of glucose, glycerol, or PGA to carbon-limited cultures allowed PGA trans-eliminase to be formed at a maximum differential rate 500 times greater than in batch cultures with excess substrate present. The differential rate of enzyme formation obtained by slow feeding of these three substrances or of a mixture of PGA plus glucose was observed to be the same. Therefore, PGA trans-eliminase produced by A. liquefaciens, contrary to the current view, appears to be constitutive. These observations also indicate that production of PGA trans-eliminase is subject to catabolite repression and that limiting the substrate reverses this repression. It was also found that, under conditions of unrestricted growth, any compound which the bacteria can use as a source of carbon and energy repressed constitutive PGA trans-eliminase production. The heritable reversal of catabolite repression of PGA trans-eliminase synthesis was demonstrated by isolation of mutant strain Gc-6 which can readily synthesize the constitutive catabolic enzyme PGA trans-eliminase while growing in the presence of excess substrate.