Eight proteins, including CheAY, FliC, GlnA, InfB, Mfd, OsmY and PyrG (spot no. 42), were present only in the ada mutant strain, while AnsB,
GrcA (two spots), OppA and PyrG (spot no. 4) were detected only in the wild-type strain. Interestingly, the ada mutant cell showed a different isoform distribution for CTP synthase (PyrG) compared with that of the wild-type. This finding suggests that the ada mutation alters this protein by posttranslational modification. Tucidinostat chemical structure Consistent with the transcriptome data, the main differences between the two strains were identified as the flagellar biosynthesis protein (FliC) and chemotaxis proteins (CheAY). These results indicate that Ada might be a negative regulator of bacterial chemotaxis under normal growth condition. In addition, the small differences between the strains suggest the limited role that Ada plays under normal growth condition. In fact, there have been no reports on any other functions of Ada except its adaptive response to protect cells from DNA damage by alkylating agents . However, our study indicates that Ada plays an additional role as a transcriptional regulator under normal growth condition and this can be a reason why the final concentration of the ada mutant strain was lower than that of the wild-type strain, as shown in Selonsertib solubility dmso Figure 1. Expression levels of the genes in the Ada regulon As mentioned
previously, the adaptive response set of genes is comprised of the ada, alkA, alkB and aidB genes TEW-7197 in vivo . Expression of these genes is regulated by Ada, and their induction provides protection against alkylation damage to DNA. To validate the expression levels of these genes in response to alkylation damage, we examined their transcriptional levels in both E. coli W3110 and the ada mutant strains at three different time points after MMS treatment, by both DNA microarray and real-time PCR analyses (Figure
5). As expected, the results obtained from real-time PCR analysis strongly correlated with those from DNA microarray analysis (r2 = 0.90). The expression levels of the genes in wild-type and mutant strains HAS1 were obviously different in MMS-treated condition. However, they were not significantly changed over time in the ada mutant strain, compared with those of its parent strain. On the other hand, the alkA and related genes showed gradually increased or decreased expression levels over the time in the wild-type and mutant strains, respectively. This implies that the adaptive response resulting from the up-regulation of these genes was not induced in the absence of the ada gene. This finding is in good agreement with previous reports showing that the expression of these four genes is positively controlled by Ada only after it interacts with methylated DNA [11–14].