Transcription Analysis of Central Metabolism Genes in Escherichia coli. Possible Roles of σ38 in Their Expression, as a Response to Carbon Limitation

Abstract

The phosphoenolpyruvate: carbohydrate transferase system (PTS) transports glucose in Escherichia coli. Previous work demonstrated that strains lacking PTS, such as PB11, grow slow on glucose. PB11 has a reduced expression of glycolytic, and upregulates poxB and acs genes as compared to the parental strain JM101, when growing on glucose. The products of the latter genes are involved in the production of AcetylCoA. Inactivation of rpoS that codes for the RNA polymerase σ38 subunit, reduces further (50%) growth of PB11, indicating that σ38 plays a central role in the expression of central metabolism genes in slowly growing cells. In fact, transcription levels of glycolytic genes is reduced in strain PB11rpoS − as compared to PB11. In this report we studied the role of σ70 and σ38 in the expression of the complete glycolytic pathway and poxB and acs genes in certain PTS− strains and their rpoS − derivatives. We determined the transcription start sites (TSSs) and the corresponding promoters, in strains JM101, PB11, its derivative PB12 that recovered its growth capacity, and in their rpoS− derivatives, by 5′RACE and pyrosequencing. In all these genes the presence of sequences resembling σ38 recognition sites allowed the proposition that they could be transcribed by both sigma factors, from overlapping putative promoters that initiate transcription at the same site. Fourteen new TSSs were identified in seventeen genes. Besides, more than 30 putative promoters were proposed and we confirmed ten previously reported. In vitro transcription experiments support the functionality of putative dual promoters. Alternatives that could also explain lower transcription levels of the rpoS − derivatives are discussed. We propose that the presence if real, of both σ70 and σ38 dependent promoters in all glycolytic genes and operons could allow a differential transcription of these central metabolism genes by both sigma subunits as an adaptation response to carbon limitation.

Sigma factors and promoters in Corynebacterium glutamicum

“…In M. tuberculosis, the sigB gene is transcribed from the same promoter by RNAP + H after heat shock and by RNAP + E after surface stress (Manganelli et al, 2002). Evidence for dual promoters recognized by two or more sigma factors was also obtained in Bacillus subtilis (Qiu and Helmann, 2001) and E. coli (Olvera et al, 2009).…”

“…However, even these data could be misleading, since e.g. E. coli promoters that are A -dependent in vivo can be recognized by B in vitro and vice versa (Olvera et al, 2009). Sequences of the selected presumed A -dependent promoters localized by mapping the TSPs are shown in Fig.…”

“…Due to the similarity of 70 -and 38 -dependent E. coli promoters, a recognition overlap of 70 -and 38 in some E. coli promoters was supposed. In a number of promoters (designated dual promoters) mostly involved in glucose utilization, alternative transcription initiation at the same nucleotide with RNAP + A or RNAP + B was proven (Olvera et al, 2009). Transcription initiates from these promoters predominantly by action of RNAP + A during the optimal exponential growth, whereas the same promoters are 38 -dependent during slow growth under glucose limitation.…”

“…These promoters may be recognized by RNAPs with the same or different sigma factors. In specific cases, transcription from a single promoter directed alternatively by two different sigma factors can initiate at the same nucleotide (Olvera et al, 2009). Two or more signals were observed in analyses of the transcriptional start points of a number of C. glutamicum genes (Pátek et al, 1996(Pátek et al, , 2003aTanaka et al, 2008).…”

Use of In Vitro Transcription System for Analysis of Corynebacterium glutamicum Promoters Recognized by Two Sigma Factors

“…In Escherichia coli, functional overlap between primary sigma factor r 70 and heat-shock sigma factor r 32 [26] or general stressresponding sigma factor r 38 [14] was observed. Overlapping specificity of Bacillus subtilis alternative stress-responding sigma factors r X and r M [11] or r X and r W [18] was also found, and the respective dual promoters (i.e., recognized alternatively by two sigma factors) were described.…”