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Following exponential growth, cultures of Bacillus subtilis are heterogeneous. In fact, cells within a culture differentiate with respect to the process of sporulation, the development of the competent state, and the development of motility, as well as the production of secondary metabolites, etc. (4-6, 8, 12).

Here, we report on a transient requirement for exogenous amino acids manifested during postexponential growth. We found that strains of B. subtilis exhibit a transient growth requirement for the nonessential amino acids glutamic acid and isoleucine shortly after the bacteria enter stationary growth. Essentially, the observations were made while we were testing mutation frequencies in strains of B. subtilis that had been made competent using the two-step procedure previously described (17, 18). The three strains utilized were BR151 (trpC2 metB10 lys-3) (18), YB886 (trpC2 metB5 xin-1 amyE sigB Spbeta^sens) (15), and a derivative of YB886 that was auxotrophic for Met, His, and Leu (YB955) (16).

Initially, strain YB955 was grown in the complex medium GM1 (18) at 37°C with aeration, and growth was determined using a Klett-Summerson colorimeter (filter no. 66). Ninety minutes after the cessation of exponential growth (T₉₀), the bacteria were diluted 1/10 into the complex GM2 medium (18). The competent and noncompetent subpopulations in this culture (3) were then separated on Renografin gradients (7), and the cells were plated on Spizizen minimal medium (SMM) (14) supplemented with known essential amino acids, and on the complex tryptose blood agar base medium (TBAB; Difco). Originally, we had been plating on SMM that lacked one essential amino acid in order to quantitate mutation frequencies among the competent and noncompetent bacteria within the culture. However, it appeared that the mutation frequencies for both populations were well below what had been previously observed (16). In this previous study, the bacteria were grown on a defined minimal medium before being plated onto a selective minimal medium. Thus, these cells were synthesizing all of the amino acids for which they were prototrophic before they had been plated onto the selective medium.

In the present study, when we compared the viable number of CFU on SMM (with all of the auxotrophic requirements added) to those on TBAB, we observed a difference of 3 to 4 orders of magnitude in survival (Table 1). This loss of viability was true for both the competent and noncompetent subpopulations. In order to determine the nature of this loss of viability following dilution of the cells into GM2, the bacteria were incubated at 37°C, with aeration, for various periods of time and aliquots were taken and plated on three different media: TBAB, SMM (supplemented with auxotrophic requirements), and SMM supplemented with tryptophan and casein hydrolysate (SMM+Trp+CH; the Trp was purchased from Fisher, and the CH and agar were purchased from Difco). Essentially, SMM+Trp+CH contained all 20 amino acids. The results shown in Table 2 demonstrate that the addition of all 20 amino acids to the SMM almost restores the colony-forming ability of the cells in the culture to that observed when the cells are plated on the rich TBAB medium.

It was also noted that the cells plated on the three media immediately before and following dilution into GM2 were equal in their ability to form viable colonies. However, 30 min following the dilution of the cells into GM2, there was a substantial loss of colony-forming ability (2 to 3 orders of magnitude) for cells plated on SMM as compared to the cells plated on SMM+Trp+CH or on TBAB. The ability of the cells plated on SMM to form viable colonies returned with continued incubation of the cells in GM2 such that by 2 h following the dilution, survival of the cells on the three media was very similar (Table 2). These results indicated that there was a transient requirement for an exogenous supply of some amino acid(s) for these postexponential cells. Without this amino acid(s) the bacteria were severely impaired in their ability to form viable colonies. This impaired ability was responsible for the apparent significant decrease in the mutation frequency that we had initially observed. To further investigate this phenomenon, we utilized different groupings of amino acids (2) in order to try and determine which supplements were needed for the cells not to lose their colony-forming ability (data not shown). The results of this analysis indicated that the presence of six amino acids (50 µg/ml each) in the SMM (in addition to the three auxotrophic requirements) significantly enhanced the colony-forming ability of these cells (Table 3). These six transient essential amino acids were Glu, Gln, Asn, Ile, Cys, and Trp. Interestingly, further analysis revealed that not only wasn't cysteine required but the presence of this amino acid in excess actually decreased the colony survival (Table 3). Finally, the systematic pairing of the five remaining amino acids lead to the definitive identification of glutamic acid and isoleucine as being the critical components necessary for colony survival.

Another aspect of this transient requirement for glutamic acid and isoleucine was to determine whether or not this phenomenon was universal among B. subtilis 168 and its derivatives. The data in Table 3 (as well as additional data not shown) demonstrated that this transient amino acid requirement was not specific for strain YB955, for strains lacking ^B (a general stress-related sigma factor for B. subtilis, or for strains carrying any specific amino acid auxotrophic requirements. On the other hand, the strains that we utilized were related (15, 16, 18), and therefore we cannot establish whether this phenomenon exists in all B. subtilis 168 derivatives.

In summary, we have demonstrated that during a particular type of stationary-phase transition B. subtilis strains have a transient requirement for glutamic acid and isoleucine. If the bacteria are forced to synthesize their own glutamic acid and isoleucine (by having them grow on a minimal medium) before they enter stationary phase, then these cells can divide and produce colonies following plating on a minimal medium (16). However, if these bacteria have exogenous supplies of glutamic acid and isoleucine throughout exponential growth (i.e., growth on GM1 or other complex media; data not shown) and are then again placed in a complex growth medium, an exogenous supply of these amino acids must continue to be provided in order for all of the cells to divide and produce colonies when they are finally plated onto a minimal medium. Thus, there is transient period of time in which the bacteria cannot synthesize glutamic acid and isoleucine and the lack of these amino acids results in an inability of the cells to form colonies on media lacking these amino acids. These results strongly suggest that during this transitional phase of growth B. subtilis may go through an imbalance with respect to the regulation of some global network such as the stringent response. Clearly, the stringent response of B. subtilis is a regulatory network that is involved in the control of a variety of functions and processes associated with the types of metabolic changes represented by entrance into stationary growth (9-11, 13). The use of appropriate mutants should allow for the testing of this hypothesis. In any case, the observations reported here again demonstrate the complexity of the parameters associated with the growth of B. subtilis. Because of this complexity, depending upon the media utilized and the time of sampling it is possible to grossly underestimate mutation frequency as well as the number of competent cells found in a culture of B. subtilis. Such underestimations could very well explain discrepancies in the literature related to mutagenesis and transformation frequencies of B. subtilis.