Statistical Estimate of the Total Number of Operons Specific for Bacillus subtilis Sporulation

D. Hranueli, P. J. Piggot, J. Mandelstam
1974 Journal of Bacteriology  
As an alternative to exhaustive mapping, an attempt has been made to obtain a rough estimate of the total number of sporulation operons by statistical treatment. Sixteen sporulation mutants taken at random were characterized biochemically and morphologically. The mutations they contained were mapped to determine whether they fell into any one of 23 known operons. From the proportion that do so ('0A6), it is calculated that the most probable number of sporulation operons is 37 (68% confidence
more » ... its of 31 and 46). If allowance is made for the fact that two of the operons apparently contain mutagenic "hot spots" and the calculation is amended accordingly, the most probable numbers of operons becomes 42 (limits 33 and 59). In considering the regulation of bacterial spore formation, it is obviously important to have some idea of the complexity of the process as indicated by the number of operons specifically concerned with sporulation. An attempt to obtain a minimal estimate of this number was made by Piggot (19) , who mapped 37 characterized sporulation mutations in Bacillus subtilis 168. Sporulation-specific mutations do not affect vegetative growth and are presumed to be in genes that are only expressed during sporulation. Thus, each gene identified by a sporulation mutation must have a "switch-on" mechanism that activates it during spore formation. Several adjacent genes may share the same mechanism. If they are closely linked and concerned with the same stage, we have assumed, to obtain a minimal estimate, that they do share the same mechanism. Each cluster of genes with its own switch-on mechanism is called a sporulation operon (23). To make a minimal estimate of the number of operons, the following three criteria were adopted as indications that two Spo mutations were in separate operons: (i) if they were separated by an auxotrophic marker, (ii) if they were unlinked by transformation, (iii) if they were concerned with different stages of sporulation and, thus, expressed at different times. On the basis of these criteria, the 37 mutations were found to fall into 16 separate operons (19). In addition, the mapping data published by others (4, 8, 11, 13, 20, 25, 26) indicate 12 additional operons which were almost certainly different, making a total of 28 in all. At this stage, there seemed to be two ways of following up the work. The first was to continue to isolate and characterize mutants and map the Spo genes until the map was saturated. The alternative approach, which we adopted, was to map a limited number of randomly isolated mutations, ascertain what proportion of them fell into known operons, and hence calculate the probable number of operons that would be discovered by further mapping. It should also be possible from the results of such a limited investigation to calculate how many more mutations would need to be mapped to provide a value for the probable number of operons to a specified degree of accuracy. For this work, we had to disregard the additional operons indicated by the work from other laboratories because the relevant mutants were not immediately available to us. However, those mutants which were isolated by Coote (4) in this laboratory and which lie in distinct operons have been included in the study. This gives a total of 23 "known" operons. Sixteen new mutants were characterized and the Spo mutations were mapped. Of these, 10 fell into the 23 known operons. From the raw data, it can be calculated that the number of operons is 37 (68% confidence limit of 31 and 46). However, account needs to be taken of the fact that some operons are likely to be more 684 on May 9, 2020 by guest
doi:10.1128/jb.119.3.684-690.1974 fatcat:czd5zkthgzccxcr2m6vi4dyuza