The Global Transcriptional Response of Escherichia coli to Induced σ32 Protein Involves σ32 Regulon Activation Followed by Inactivation and Degradation of σ32 in Vivo

Abstract

Escherichia coli core DNA-dependent RNA polymerase consists of four different subunits and has the composition ␣ 2 ␤␤Ј. Core RNA polymerase (E) together with a factor constitutes a holoenzyme complex (E) (1, 2). The holoenzyme complex is able to initiate transcription at specific DNA sequences termed promoters. Since the discovery of 70 36 years ago, numerous factors have been described in E. coli and other prokaryotic organisms (3-7). The seven known E. coli factors are 70 , 54 , 32 , S , F , E , and fecI .The major factor, 70 , is involved in the transcription of the majority of genes in the cell. The other factors are alternative factors that enable RNA polymerase to transcribe genes required for cellular adaptation to changes in the external environment. Each factor recognizes and directs RNA polymerase to a different set of promoters.Among the six known alternative factors, 32 , which is encoded by rpoH (htpR, hin, and fam), was the first minor factor to be discovered in E. coli. For heat shock and some other general stress responses (such as sublethal concentrations of ethanol, viral infection, etc.), transcription initiation is regulated largely by 32 . The first step of the transcription initiation pathway is the binding of 32 to core RNA polymerase to form an E 32 holoenzyme complex. This binding results in the expression of many heat shock proteins (HSPs) 1 that play important roles in protein folding, repair, and degradation under normal and stress conditions. Because 32 plays an important role in heat shock stress, early work on 32 -dependent genes was focused on the induction of a group of genes upon heat shock stress. Most known 32 -dependent genes were identified either by monitoring synthesis rates of individual proteins before and after heat shock on two-dimensional gels (8) or by hybridizing cDNA (generated mRNA from heat-shocked cells) with membrane filters containing an ordered E. coli genomic library (9). However, in response to temperature upshift, the induction of S was shown in a Western blot experiment, and the induction of S -dependent genes was confirmed by using a S -dependent promoter-lacZ fusion approach (10). Also, Taylor and co-workers (11) found 54 -controlled genes are another group of genes that can be induced by heat shock in addition to 32 and E regulons (12). Therefore, although the heat shock response is mainly mediated by 32 , there are some other global gene regulators that increase and turn on genes during the heat shock response. This makes the heat shock stimulon a complicated group of different regulons.Here we report the results of using transcription profiling experiments to identify the 32 regulon in E. coli. Our basic strategy is to minimally perturb steady-state growth (E. coli MG1655 growing exponentially in minimum medium at 37°C) by moderate induction of 32 . We then monitor global RNA transcript abundance changes as a function of time using Affymetrix GeneChip R E. coli antisense genome arrays. This approach allows us to reduce the possibility of induction ...

Pivotal role of the Francisella tularensis heat-shock sigma factor RpoH

“…These genes encode the heatshock proteins Hsp40, HptG, DnaK, DnaJ, GroES and GroEL, which were all induced after heat stress as well. The previous studies of the s 32 regulon in E. coli, V. cholerae and N. gonorrhoeae identified these genes also (Gunesekere et al, 2006;Nonaka et al, 2006;Slamti et al, 2007;Zhao et al, 2005), demonstrating common features of RpoH regulation in different species. However, the number of genes identified in F. tularensis was unexpectedly low when compared with other bacterial species.…”

Exploring the antimicrobial action of a carbon monoxide-releasing compound through whole-genome transcription profiling of Escherichia coli

“…Accordingly, we verified that together with the induction of cpxP occurred a high number of transcriptionally modified genes involved in cell wall biogenesis and in protein folding, including genes encoding heat-shock proteins (ibpAB, hslJ, htpX), chaperones (dnaJ, htpG, clpB) and proteases (ftsH) ( Supplementary Table S1), mainly under anaerobic conditions. These genes are under the control of rpoH (s 32 ) (Zhao et al, 2005), a gene that was also found to be upregulated by CORM-2 under anaerobic conditions. The study of the growth behaviour of the DcpxP mutant revealed that this strain is approximately 35 % more sensitive to CORM-2 than the parental strain, but only under anaerobic conditions (Fig.…”

Regulon and promoter analysis of theE. coliheat-shock factor, σ32, reveals a multifaceted cellular response to heat stress

“…Several global approaches have been utilized in E. coli to identify genes under 32 control. Both two-dimensional protein gels (Lemaux et al 1978) and global transcriptional approaches have identified genes induced after transfer to high temperature (Chuang et al 1993b;Richmond et al 1999) or after overexpression of 32 (Zhao et al 2005); however, there has been no systematic determination of whether these induced genes are directly expressed from 32 -dependent promoters. The few cases where the functions of 32 -dependent genes were identified indicates the importance of this determination: Hsp33, a widely conserved hsp, is a redox activated chaperone (Jakob et al 1999), thereby providing the first identification of a chaperone of this type, and FtsJ was shown to be a methyltransferase whose substrate is 23S RNA, revealing an unexpected link between the HSR and RNA (Bugl et al 2000).…”

“…We also tested seven previously proposed 32 promoters located upstream of genes that were not significantly induced on our arrays and detected no 32 -dependent transcripts (Table 1C). Finally, neither 5ЈRACE nor in vitro transcription identified functional promoters for three of the 32 promoters proposed by Zhao et al (2005) (ldhA, macB, and ybbN; Table 1B) based on electrophoretic mobility gel shift by stream of sequences not present on our arrays (Table 1C) comprise a total of 51 promoters that drive the expression of 49 TUs. The sequence logos of the conserved sequence motifs upstream of the 50 chromosomal 32 promoters (note repE is on the F factor) together with their information content are displayed in Figure 4A.…”