Salt-induced genes under the control of DegU~P

To determine whether certain gene is up-regulated from the DegU-phosphate, a similar procedure as for the degSU operon was followed. Initially, B. subtilis wild type, hyper mutant and deletion mutant strains were grown as described in Helmann medium and in Helmann medium supplemented with 1.2M NaCl, respectively. The exponentially grown cells (OD578

of 1.0) were harvested and the cell pellets were subsequently used for isolation of total RNA.

Finally, the expression pattern of the genes was explored through hybridization of the isolated RNA with a single-stranded DIG-labelled RNA probe specific for the particular gene.

In Figure 27 is shown the Northern analysis of the yddT gene. It codes for a 228 amino acid protein with unknown function. The mRNA transcripts could be detected only from cultures grown under high salt conditions perhaps because the induction in the non-salt environment is too low. It became also clear that the yddT gene is up-regulated from the DegU-phosphate since the amount of the mRNA transcript was higher in the hyper mutant than that detected in the wild type strain. Additionally, the gene was expressed also in the absence of the degS and degU products although not to a very high extent. This observation implies that an additional factor is involved in the regulation of the yddT gene. From the size of the detected bands (approximately 1.3 kb) could be concluded that the gene is transcribed alone because the other two neighbour genes were located quite far from its coding region (Fig. 27).

1308 bp 684 bp 591 bp

yddS yddT ydeA

786 bp 962 bp

500 bp 1000 bp 1500 bp 2000 bp

probe

0M NaCl 1.2M NaCl

1 2 3 4 5 6

Fig. 27. Northern blot analysis of yddT

Shown is the genetic region of the yddT gene. Position of the RNA probe, the size of the corresponding genes and intergenic regions are indicated (upper part). Total RNA was isolated from the wild type JH642 (lane 1 and 3), hyper mutant (THB300) (lane 2 and 4) and deletion mutant (THB282) (lane 3 and 6) grown in Helmann medium with 0M and 1.2M NaCl, and hybridized with an yddT-specific probe (lower part).

Another gene that was found to have very similar transcriptional profile was yomL (Fig. 28).

It also codes for a protein with unknown function and size of 228 amino acids. RNA transcripts could be seen also only when the cells were subjected to high salt concentration.

The mRNA bands were visible in the three explored strains whereas the amount was different.

Again a higher expression level was detected when higher amount of DegU-phosphate were present and on the other hand the transcription was not completely abolished in its absence. It appears that even the yomL gene is obviously regulated from the DegS-DegU two-component system it is not the only factor involved in that process. Concerning the size of the transcript, it was not entirely clear if this gene is transcribed alone or together with the downstream situated yozP (Fig. 28). The calculated size from the Northern analysis corresponds to 1.2 kb and the size of the yozP gene is only 327 bp which makes the estimation difficult, moreover the transcription start point of the yomL is not known. For that reason another Northern blot analysis was carried out by using an RNA probe specific to the yozP internal region. There could not be detected any signals after the hybridization (data not shown) and consequently the yomL gene was postulated to be transcribed alone.

yozP yomL yomK

327 bp 684 bp

178 bp 546 bp

probe

500 bp 1000 bp 1500 bp 2000 bp

1 2 3 4 5 6

0M NaCl 1.2M NaCl

Fig. 28. Northern blot analysis of yomL

Shown is the genetic region of the yomL gene. Position of the RNA probe, the size of the corresponding genes and intergenic regions are indicated (upper part). Total RNA was isolated from the wild type JH642 (lane 1 and 3), hyper mutant (THB300) (lane 2 and 4) and deletion mutant (THB282) (lane 3 and 6) grown in Helmann medium with 0M and 1.2M NaCl, and hybridized to a yomL-specific probe (lower part).

Another gene which expression was followed under the adopted conditions was yitM. The function of the gene product is also unknown and it contains 194 amino acids. The Northern analysis revealed clearly that the yitM belongs to the DegS-DegU regulon since higher amount of mRNA could be detected in the hyper mutants strain in both cultivating conditions – with and without salt (Fig. 29A). At the same time, the amount of the transcript was slightly induced when the cells were subjected to high salt which indicated that the gene fulfils both

requirements mentioned earlier. Taking into account the size of the corresponding transcript of about 900 bp, it was not entirely clear if the neighbour gene yitN is co-transcribed (Fig. 29).

In parallel to these observations, another very weak band was noticed on the same membrane with size of approximately 2.3 kb. To elucidate the transcription pattern more precisely another Northern blot was carried out with RNA probe specific to the yitN gene. In this case only one band with size of about 2.3 kb could be seen. Although difficult to detect, the amount of the mRNA was slightly higher in the hyper mutant in comparison with the wild type strain in the cultures grown under high salt. From size of the observed band it was evident that these genes are transcribes as an operon which includes all four genes from the region (Fig. 29B). At the same time most probably there is a second promoter within that operon upstream from the yitM and in the coding sequence of the yitN gene. In the deletion mutant background on the other hand, no transcript was detectable which shows that most likely these genes are controlled solely from DegS-DegU two-component system.

yitL yitN

582 bp

894 bp 366 bp

yitM yitO

210 bp

yitP

534 bp

58 bp 134 bp 203 bp 372 bp

yitQ

735 bp

1 2 3 4 5 6 1 2 3 4 5 6

A B

500 bp 1000 bp 1500 bp 2000 bp 3000 bp

0M NaCl 1.2M NaCl 0M NaCl 1.2M NaCl

probe probe

Fig. 29. Northern blot analysis of yitMNOP operon

Shown is the genetic region of the yitM gene. Position of the RNA probes, the size of the corresponding genes and intergenic regions are indicated (upper part). Total RNA was isolated from the wild type JH642 (lane 1 and 3), hyper mutant (THB300) (lane 2 and 4) and deletion mutant (THB282) (lane 3 and 6) grown in Helmann medium with 0M and 1.2M NaCl, and hybridized to a yitM- (A) and yitN-specific (B) probes (lower part).

The next gene that was determined to be a subject of salt induction and was regulated from the DegU-phosphate is yoaJ. The gene product constitutes a 232 amino acid protein and has an unknown function but it is similar to extracellular endoglucanase precursor. The expression pattern of the yoaJ revealed a higher amount of mRNA in the hyper mutant background on one hand and an additional induction when the cells were cultivated in a high salt environment (Fig. 30). It is also probable that the gene is controlled only from the

DegS-DegU system since no signal could be detected form cells with disrupted degS and degU genes. Nevertheless, one could claim that the expression pattern in the deletion mutants is ambiguous since there was also no product in the wild type strain. To clarify this, further experiments were performed (see section 6.2.). As regards the size of the detected band (about 900 bp), an operon structure with the upstream yoaK is excluded (Fig. 30).

yoaI yoaJ yoaK

1125 bp 696 bp 675 bp

42 bp probe 272 bp

1000 bp 1500 bp

1 2 3 4 5 6

0M NaCl 1.2M NaCl

Fig. 30. Northern blot analysis of yoaJ

Shown is the genetic region of the yoaJ gene. Position of the RNA probe, the size of the corresponding genes and intergenic regions are indicated (upper part). Total RNA was isolated from the wild type JH642 (lane 1 and 3), hyper mutant (THB300) (lane 2 and 4) and deletion mutant (THB282) (lane 3 and 6) grown in Helmann medium with 0M and 1.2M NaCl, and hybridized to a yoaJ-specific probe (lower part).

One further gene with comparable transcription profile is yqxI. It encodes a small protein with 159 amino acids and unknown function as well. The Northern analysis that was carried out with specific RNA probe against yqxI gene revealed the presence of two bands (Fig. 31).

Despite of the different RNA preparations that were applied, these two bands appeared in all experiments. Although very distinct, the upper band was considered as unspecific because its expression profile could not be attributed to the overall model exhibited by the investigated genes. The profile of the smaller band on the other hand, corresponded to a pattern where the expression level is higher in the hyper mutant strain, and even more increased in the high salt conditions. Nevertheless, no transcript was detected in the deletion mutant background from both bands which made the interpretation of the data complicated. That is why another experiment was carried out to clarify this issue (see section 6.2.). The size of the indicated transcripts was estimated to be 1 and 1.2 kb, respectively. The analysis of the corresponding gene region revealed that it is likely yqxI and yqxJ to be transcribed as an operon (Fig. 31).

The upstream located cwlA gene is not part of that operon as it can be calculated from its size.

yqcF yqxJ yqxI cwlA

576 bp 360 bp 477 bp 816 bp

266 bp 18 bp _probe 167 bp

500 bp 1000 bp 1500 bp 2000 bp

1 2 3 4 5 6

*

0M NaCl 1.2M NaCl

Fig. 31. Northern blot analysis of yqxIJ operon

Shown is the genetic region of the yqxIJ. Position of the RNA probe, the size of the corresponding genes and intergenic regions are indicated (upper part). Total RNA was isolated from the wild type JH642 (lane 1 and 3), hyper mutant (THB300) (lane 2 and 4) and deletion mutant (THB282) (lane 3 and 6) grown in Helmann medium with 0M and 1.2M NaCl, and hybridized to a yqxI-specific probe (lower part). * indicates a non specific band