Activity of Rhodobacter sphaeroides RpoH _II , a Second Member of the Heat Shock Sigma Factor Family

Abstract

We have identified a second RpoH homolog, RpoH II , in the ␣-proteobacterium Rhodobacter sphaeroides. Primary amino acid sequence comparisons demonstrate that R. sphaeroides RpoH II belongs to a phylogenetically distinct group with RpoH orthologs from ␣-proteobacteria that contain two rpoH genes. Like its previously identified paralog, RpoH I , RpoH II is able to complement the temperature-sensitive phenotype of an Escherichia coli 32 (rpoH) mutant. In addition, we show that recombinant RpoH I and RpoH II each transcribe two E. coli 32 -dependent promoters (rpoD P HS and dnaK P1) when reconstituted with E. coli core RNA polymerase. We observed differences, however, in the ability of each sigma factor to recognize six R. sphaeroides promoters (cycA P1, groESL 1 , rpoD P HS , dnaK P1, hslO, and ecfE), all of which resemble the E. coli 32 promoter consensus. While RpoH I reconstituted with R. sphaeroides core RNA polymerase transcribed all six promoters, RpoH II produced detectable transcripts from only four promoters (cycA P1, groESL 1 , hslO, and ecfE). These results, in combination with previous work demonstrating that an RpoH I mutant mounts a typical heat shock response, suggest that while RpoH I and RpoH II have redundant roles in response to heat, they may also have roles in response to other environmental stresses.The ability to regulate gene expression through the use of alternate sigma factors enables bacteria to respond quickly to changes in the environment. These secondary sigma factors guide RNA polymerase to promoter elements whose target sequences differ from those of the primary sigma factors, thereby directing the transcription of a specific set of genes (44). One such rapid response to environmental perturbations, known as the heat shock response, uses an alternate sigma factor to increase the expression of proteins needed to maintain an intracellular milieu conducive for protein folding. While these heat-inducible gene products have traditionally been referred to as the heat shock proteins, increased heat shock protein expression is also induced by other stress conditions (11, 33). We are interested in alternate sigma factors that play a role in the heat shock or other stress responses of the ␣-proteobacterium Rhodobacter sphaeroides.In the ␥-proteobacterium Escherichia coli, 32 (encoded by rpoH) is the alternate sigma factor responsible for recognizing heat shock gene promoters (12). As a member of the 70 family of eubacterial sigma factors, 32 recognizes unique promoter sequences centered at positions Ϫ10 and Ϫ35 relative to the transcriptional start site (44). All 32 -like proteins are defined by a conserved region known as the "RpoH box"; they also contain conserved sequences in regions 2.4 and 4.2 that recognize the Ϫ10 and Ϫ35 elements, respectively, of heat shock promoters (29). While E. coli, with its single rpoH gene, sets the stage for much of what we know about bacterial heat shock regulation, several ␣-proteobacteria are known to encode multiple RpoH homologs. Three rpoH-like genes in...

RpoH II Activates Oxidative-Stress Defense Systems and Is Controlled by RpoE in the Singlet Oxygen-Dependent Response in Rhodobacter sphaeroides

“…Those putative target sequences were similar to those of previously predicted RpoH II -dependent promoters (6) but exhibited differences in the Ϫ35 and Ϫ10 regions compared to target sequences located upstream of the start codon of several heat shock-triggered genes and recognized by both RpoH I and RpoH II (19). The Ϫ35 region putatively recognized by RpoH II during 1 O 2 exposure contains not only TTG but also TTT or TGG and is flanked by GC-rich positions (Fig.…”

“…Like RpoH I of R. sphaeroides, RpoH II can complement the temperature-sensitive phenotype of an E. coli rpoH mutant (19). We therefore compared the mRNA levels of rpoH I and rpoH II and also of rpoE after exposure to 1 O 2 and heat shock (Fig.…”

“…RpoH II -like sigma factors have so far been recognized only in the Alphaproteobacteria (19) and may control rather specific functions. In Brucella melitensis, the gene BMEI0280 (rpoH1) represents the R. sphaeroides rpoH II homolog.…”

“…The role of RpoH II -type sigma factors, which are widely distributed among organisms in the Alphaproteobacteria (19), is largely unknown with respect to environmental response and function of the controlled genes. The proposed function of genes under RpoH II control may explain the adaptive response to photooxidative stress observed for R. sphaeroides (17).…”

“…In the alignment, transcription start sites are underlined and putative Ϫ35 and Ϫ10 regions are printed in bold letters. The dnaK P1 promoter sequence is shown for comparison and is recognized only by RpoH I in vitro (19 (Fig. 1A).…”