Bichemical genetics of bacterial sporulation

Summary Pleitropic interactions among genes controlling the formation of bacterial spores and of sporulation-associated products are studied. In order to obtain sporulation mutants, spores have been germinated in the presence of chloramphenicol and then treated with nitrosoguanidine. In the most favorable conditions 25% of sporulation mutants have been found among the 40% surviving bacteria. This number is at least four times higher than the number of auxotrophic mutants, therefore a rough estimate of the number of genes involved in sporulation is 800.Rapid plate-tests have been developed for the oxidation of terrazolium salts, the formation of various proteolytic enzymes and the production of antibiotics. Although the exact biochemical nature of the products is not yet known, the results suggest that distinct factors, probably various enzymes (including several proteases) are detected by these tests. All of them are associated with spore formation and absent from a large number of sporulation mutants. Using these tests, the phenotypes of 500 randomly selected sporulation mutants were determined. No important differences were found between asporogenous and oligosporogenous mutants. The number of mutants deficient for several sporulation-associated characters is large, pleiotropic interactions following a defined pattern are observed. Statistical analysis indicates the existence of a unidirectional pleiotropic system. All the results agree with the hypothesis of sequential gene activation. Consequently, the sporulation-associated characters can be ordered into a linear sequence, presumably reflecting the consecutive steps in spore formation. The order obtained is the following: gelatinase, proteases acting on casein and on denatured albumin, oxidation of tetrazolium No 7, digestion of protamine, production of antibiotics (against a Staphylococcus and a Bacillus), hydrolysis of hemoglobin, oxidation of tetrazolium No 2, digestion of native albumin, synthesis of elastase. Another category of mutants, blocked in a late step of sporulation and apparently derepressed for the formation of elastase, is also described.In conclusion, arguments are put forward in favor of sequential gene activation. Sporulation genes, related by unidirectional pleiotropic interactions, form a sporulon. Generalization of this concept to other differentiating systems (a differon), its predictions and possible experimental confirmation are considered.The author was a Gosney Research Fellow in 1966/67, on leave of absence from the Centre National de la Recherche Scientifique, Paris. Present adress: see end of paper.     

Genetic instability of sporulation-associated characters in a Bacillus subtilis mutant: relationship between sporulation, segregation and the synthesis of extracellular enzymes (kinetic studies).

In the genetically unstable, protease-overproducing 'medusa (M) strains of Bacillus subtilis, segregation of stable, wild-type-like B cells occurred mainly during sporulation. After the end of the exponential growth phase, a small fraction of M cells sporulated quickly and formed M spores, while the majority of the cells, after a 'critical period', gave rise to B segregants which sporulated after a delay. Segregation occurred without cell division. Delayed sporulation, segregation and protease overproduction are related. Similar but more complex results were obtained with the highly unstable TD strains. Sporulation and the kinetics of protease overproduction were also followed in several stable segregants. Depending on the strain, either the rate of protease production or both the rate and time course were affected. The results are interpreted in terms of sequential activation and de-activation of sporulation genes. The production of the alkaline and the neutral proteases was, in general, under common genetic control. In some strains alpha-amylase was also overproduced.     

Physiological and Molecular Background of Maize Cold-Tolerance Enhancement with S-methylmethionine Salicylate

Low temperature is amongst the most influential abiotic stress factors, having deep impact on plant growth, yield and productivity. Studies on beneficial effects of certain biologically active substances, S-methylmethionine (SMM) and salicylic acid (SA) have provided a lot of valuable information regarding their role to counteract harmful effects of environmental stresses such as chilling. To obtain a more complex and stable defence compound with an extended range of stress-protective effect, the new derivative S-methylmethionine salicylate (MMS) was synthesised from the natural, biologically active substances SMM and SA. Since both original materials have complex stress-protective roles, the new compound was expected to combine the effects of original substances and to stabilise the unstable SMM in the new compound, thus providing an extended stress tolerance. Photosynthetic efficiency and accumulation of stress-related metabolites (polyamines and flavonoids) were measured in chilled and control plants, with and without MMS pretreatment, and expression changes of several genes involved in the cold stress response were analysed by quantitative real-time PCR (RT-qPCR) and a detailed microarray study. Our data show how the MMS combines the effect of SMM and SA on molecular level, causing numerous changes in the gene expression pattern and metabolite content. MMS gives rise to a better physiological condition, thus it could provide an alternative, environmental friendly way to enhance the plants defence mechanisms against stressors. As MMS is more stable than SMM, it promises easier, more long-lasting and more cost-effective usage in agriculture, with a complementing effect of SA.

     

Biochemical genetics of bacterial sporulation

Summary The development of membrane structures during the early steps of spore formation was reexamined. These steps include the formation of a ringshaped membrane invagination in contact with a mesosome, the growth of which leads to a straight closed double-membrane diaphragm with a cell wall spike (ringshaped thickening) at its base. Later the diaphragm protrudes into the sporangial cytoplasm in the form of a half-blown balloon. The last step toward completion of the prespore consists of the detachment of the diaphragm from the cell wall spike and its growth (probably from a new growing region) into a completely closed spherical structure.Some of the sporulation mutants are blocked at one of these steps, others present abnormal structures. Bilateral development, the occurrence of multiple membrane invaginations and derepression of cell wall growth are at the origin of certain abnormal mutants. In other mutants the diaphragm is not detached from the cell wall spike, perhaps because of the lack of initiation of a new membrane growth site. This leads to abnormal hemisphere prespores. Satellite prespores have been also observed and attributed to derepressed growth of the cell wall spike.The author was on leave of absence from the Centre National de la Recherche Scientifique, Paris. Present adress: see end of paper.     

Lytic enzymes in sporulating Bacillus subtilis

Two specific lytic enzymes were found in sporulating $\text{B. subtilis}$ cells: a N-acetyl muramyl L-alanine amidase and a $\text{γ-D-glutamyl-(L)}\text{meso}$ diaminopimelyl endopeptidase. Both enzyme activities were measured using radioactive synthetic substrates. They are low in vegetative cells and increase during sporulation. The highest rates of increase are concomitant with cortex formation. In a mutant with delayed sporulation enzyme synthesis is also delayed. We suggest that both enzymes play a role in the synthesis of the specific cortex peptidoglycan.     

Genetic instability of sporulation-associated characters in a Bacillus subtilis mutant: analysis of the segregation pattern and genetic studies.

A Bacillus subtilis mutant which formed dark-brown 'medusa' (M) colonies was obtained. It sporulated at a high frequency, overproduced extracellular protease during sporulation and possessed a high genetic instability with a complex segregation pattern. Segregation was maintained after repeated re-isolation of single M colonies. The major wild-type-like class of segregants (B) was stable, sporulated normally and produced normal amounts of protease. Occasionally segregants were obtained which produced extremely high amounts of protease, sporulated poorly, formed transparent colonies and were either highly unstable (TD) or stable (TDst). Rarely B(D) (stable, normal sporulation and protease overproduction) and W and T (both stable and asporogenous) segregants were produced. The M phenotype was transmitted as a single factor by transformation but not by transduction. The results of transduction experiments suggest the presence of two mutations, ScoC and ScoD. It is proposed that this new segregating system in B. subtilis may result from tandem duplication of part of the bacterial chromosome.