1. MARCO REFERENCIAL
1.3 OBJETIVOS
2.2.2 Principio de celeridad y procedimiento directo: la simplificación del
2.2.2.3 Carga de la Prueba
This study was done to compare the kinetics of induction of the two recA promoters over an
induction period of eight hours in M. smegmatis and twenty-four hours in M. tuberculosis.
Whilst a similar analysis had previously been done with the entire upstream region of recA
in both mycobacterial species (Papavinasasundaram et a l, 2001) this study would give an
indication of how the individual promoters behaved.
In M. smegmatis, after an initial lag, both promoters reached a maximum rate of induction
between one and three hours after the addition of mitomycin C (fig 4.5a), although the rate of induction is slightly higher for recAV2 than recAVl. However, after four hours post
induction, there is no further induction of recAVl and a plateau is reached, whilst recAVl
activity continues to increase through the entire time period of this study (fig. 4.5a).
The region upstream up the recA gene, and in particular the two promoter elements, has
been shown to be highly conserved in both M. smegmatis and M. tuberculosis. It, therefore,
might be assumed that the kinetics of induction of both recAVl and recAVl would be the
same in both saprophyte and pathogen. However, when the same study was undertaken in
M. tuberculosis it was shown that the kinetics of both recAPI and recAP2 induction in the
pathogen were quite different to that seen in M. smegmatis. In the pathogen, the initial
post-induction activity of recAP2 is higher that that seen for recAPI as is seen in the
saprophyte (Figs. 4.5a and 4.5b) although the increase in the rate of activity seen from
recAP2 in mitomycin C induced M. tuberculosis was more gradual that seen in the
6 hours post induction the rate of induction of recAPI increases dramatically over a period
of 12 hours resulting in the activity of recAPI becoming greater than that of recAP2 using
these constructs. However, when the more active recAP2 construct pKKG25 was tested in
the pathogen following induction for 24 hours, the induced level of expression was comparable to that of recAPI at the same time point (Fig. 3.8).
_ # - - - recAP^ a c t i v i t y recAP2 a c t i v i t y 5 0 0 -, c 3 4 0 0 - 0> 3 0 0 - > u <0 200 -
I
o 100 - M. smegmatis 0 2 4 6 8 10 Tim e (hours) B 1 2 0 0 -I E 3 1000 - im. 0) 8 0 0 - 6 0 0 - > Z <0 4 0 0 - o E 2 Q. M. tuberculosis 0 5 10 1 5 20 2 5 3 0 Tim e (hours)Figure 4.5. This figure illustrates the kinetics of induction study done for both recA
promoters in M. smegmatis (A) and M. tuberculosis (B). Aliquots of identical volume were taken from one culture of either M. smegmatis or M., tuberculosis and induced with mitomycin C for a variety of incubation times ranging from 0 - 8 hours for the M.
smegmatis study and 0-24 hours in the M. tuberculosis study. These samples were then assayed. The error bars represent standard errors between three independent experiments per promoter construct. From the results it can be seen that there is a difference both in the way either promoter acts in the same host and also in, the way the same promoter acts in the two different mycobacteria.
4.4. Discussion
It has been shown that in the region upstream of the recA gene in both M. tuberculosis and
M. smegmatis (Movahedzadeh et al, 1997; Papavinasasundaram et a l, 1998), there are two
promoter elements each of which share a common sequence in the two species. The presence of these two promoters could be seen as an indication that two modes of induction could be involved in activating transcription of recA in both species. The aim of the studies
mentioned in this chapter was to obtain a better understanding of why M. tuberculosis has
two DNA damage inducible promoter elements present upstream of its recA gene. It was
thought that seeing how the induction of both promoters was regulated could provide insight into this.
4.4.1. Is there a link between growth phase and uninduced promoter
activity?
Although genes that are associated with the SOS response become activated in conditions of DNA damage, it has been shown in E. coli that there are other conditions whereby SOS
genes are expressed. One such condition is that of growth phase and it has been shown in
E. coli that recA can be induced in stationary phase without the presence of DNA damage
(Taddei et a l, 1995). In addition, another gene regulated by LexA in E. coli, sbmC, is
induced upon entering stationary phase in a DNA damage free environment (Baquero et al,
1995). It has been shown in both cases that the presence of cyclic AMP (cAMP) can stimulate the SOS response, with cleavage of the LexA repressor and activation of these genes (Baquero et a l, 1995; Taddei et a l, 1995). Conversely, the E. coli sfiA gene, which
also is regulated through the presence of a LexA binding site, is active in early exponential phase without the presence of DNA damage (Dri and Moreau, 1993).
In the case of both recAV 1 and recAVl in M. smegmatis, it was found that uninduced
promoter activity remained constant in all phases indicating that there was no link between uninduced activity and growth phase for either promoter in this species (fig. 4.1). This in turn suggests that the induction of both promoters in this species is dependent on DNA damage. It was also shown that the activity of recAPI in M. tuberculosis was independent
of growth phase (fig. 4.2). In the case of uninduced recAPI activity, whilst it could be
argued that there may be a slight decrease in the activity of the LexA regulated promoter, the overlapping error bars seen can also be interpreted as showing no effect linked with the progression of growth.
Another finding of the growth phase study in M. smegmatis is that the activity of the
constitutive recAPI (with mutations in the SOS box) remained constant in the different
growth phases. As stated earlier, gearbox promoters in a stress-free environment, are associated with increased activity in stationary phase (Aldea et al, 1990; Ballesteros et al,
1998). Therefore, what this finding indicates is that recAPI is not a gearbox promoter.
However, this contradicts the information from the point mutations done in the recAPI -10
region, which showed that the -12A base (conserved in the gearbox consensus but not in the heat shock promoter consensus) is vital to the activity of the mycobacterial promoter. With these contradictions, it is obvious that it has become difficult to label this mycobacterial promoter based on the characteristics of promoters from other bacterial species. Therefore, another means of "grouping" this mycobacterial promoter would be to determine which
sigma factor is involved in it recognition. One way of achieving this aim would be to create
M. tuberculosis sigma factor mutants and screening these strains with recAPI and recAPI
constructs to see in which mutants promoter activity was lost.
The studies involving the constitutive rccAP2 construct in M. smegmatis show that
unrepressed promoter activity is constant over the whole growth process of the saprophyte. Furthermore, it was found that the activities of the constitutive and induced wild type
recAVl in the saprophyte were similar. This in itself does not indicate anything about the
sigma factor that recognises this promoter. However, it does show that induction of this promoter element in M. smegmatis is dependent on the cleavage of the repressor and not on
any associated factors that are produced in DNA damage conditions. The dependence on RecA* mediated cleavage of the LexA repressor has also been shown by inability of a wild type recAVl construct to have enhanced expressed in a M. tuberculosis tsrecA strain upon
induction with mitomycin C (E. O. Davis, personal communication).