ABSTRACT

Singlet oxygen (1O2) is a reactive oxygen species generated by energy transfer from one or more excited donors to molecular oxygen. Many biomolecules are prone to oxidation by 1O2, and cells have evolved systems to protect themselves from damage caused by this compound. One way that the photosynthetic bacterium Rhodobacter sphaeroides protects itself from 1O2 is by inducing a transcriptional response controlled by ChrR, an anti-σ factor which releases an alternative sigma factor, σE, in the presence of 1O2. Here we report that induction of σE-dependent gene transcription is decreased in the presence of 1O2 when two conserved genes in the σE regulon are deleted, including one encoding a cyclopropane fatty acid synthase homologue (RSP2144) or one encoding a protein of unknown function (RSP1091). Thus, we conclude that RSP2144 and RSP1091 are each necessary to increase σE activity in the presence of 1O2. In addition, we found that unlike in wild-type cells, where ChrR is rapidly degraded when 1O2 is generated, turnover of this anti-σ factor is slowed when cells lacking RSP2144, RSP1091, or both of these proteins are exposed to 1O2. Further, we demonstrate that the organic hydroperoxide tert-butyl hydroperoxide promotes ChrR turnover in both wild-type cells and mutants lacking RSP2144 or RSP1091, suggesting differences in the ways different types of oxidants increase σE activity.

IMPORTANCE

Oxygen serves many crucial functions on Earth; it is produced during photosynthesis and needed for other pathways. While oxygen is relatively inert, it can be converted to reactive oxygen species (ROS) that destroy biomolecules, cause disease, or kill cells. When energy is transferred to oxygen, the ROS singlet oxygen is generated. To understand how singlet oxygen impacts cells, we study the stress response to this ROS in Rhodobacter sphaeroides, a bacterium that, like plants, generates this compound as a consequence of photosynthesis. This paper identifies proteins that activate a stress response to singlet oxygen and shows that they act in a specific response to this ROS. The identified proteins are found in many free-living, symbiotic, or pathogenic bacteria that can encounter singlet oxygen in nature. Thus, our findings provide new information about a stress response to a ROS of broad biological, agricultural, and biomedical importance.

Acoustic droplet ejection achieves precise, tipless, non-invasive transfer of diverse aqueous solutions, enabling nanolitre-scale crystallization trials. The rapid and scalable technique demonstrated successful crystal growth with diverse targets in drop volumes as small as 20 nl.

Focused acoustic energy allows accurate and precise liquid transfer on scales from picolitre to microlitre volumes. This technology was applied in protein crystallization, successfully transferring a diverse set of proteins as well as hundreds of precipitant solutions from custom and commercial crystallization screens and achieving crystallization in drop volumes as small as 20 nl. Only higher concentrations (>50%) of 2-­methyl-2,4-pentanediol (MPD) appeared to be systematically problematic in delivery. The acoustic technology was implemented in a workflow, successfully reproducing active crystallization systems and leading to the discovery of crystallization conditions for previously uncharacterized proteins. The technology offers compelling advantages in low-nanolitre crystallization trials by providing significant reagent savings and presenting seamless scalability for those crystals that require larger volume optimization experiments using the same vapor-diffusion format.

Bacteria need signal transducing systems to respond to environmental changes. Next to one- and two-component systems, alternative σ factors of the extra-cytoplasmic function (ECF) protein family represent the third fundamental mechanism of bacterial signal transduction. A comprehensive classification of these proteins identified more than 40 phylogenetically distinct groups, most of which are not experimentally investigated. Here, we present the characterization of such a group with unique features, termed ECF41. Among analyzed bacterial genomes, ECF41 σ factors are widely distributed with about 400 proteins from 10 different phyla. They lack obvious anti-σ factors that typically control activity of other ECF σ factors, but their structural genes are often predicted to be cotranscribed with carboxymuconolactone decarboxylases, oxidoreductases, or epimerases based on genomic context conservation. We demonstrate for Bacillus licheniformis and Rhodobacter sphaeroides that the corresponding genes are preceded by a highly conserved promoter motif and are the only detectable targets of ECF41-dependent gene regulation. In contrast to other ECF σ factors, proteins of group ECF41 contain a large C-terminal extension, which is crucial for σ factor activity. Our data demonstrate that ECF41 σ factors are regulated by a novel mechanism based on the presence of a fused regulatory domain.

This study explores the regulatory networks controlling anaerobic energy production by the facultative phototroph Rhodobacter sphaeroides. The specific aim was to determine why activity of the P2 promoter for the gene (cycA) encoding the essential photosynthetic electron carrier, cytochrome c2, is decreased when the alternative electron acceptor DMSO is added to photosynthetically grown cells. The presence of DMSO is believed to activate the DorR response regulator, which controls expression of proteins required to reduce DMSO. A DorR− strain showed no change in cycA P2 promoter activity when DMSO was added to photosynthetic cells, indicating that DorR was required for the decreased expression in wild-type cells. To test if DorR acted directly at this promoter to change gene expression, recombinant DorR was purified and studied in vitro. Preparations of DorR that were active at other target promoters showed no detectable interaction with cycA P2, suggesting that this protein is not a direct regulator of this promoter. We also found that cycA P2 activity in a DorA− strain was not decreased by the addition of DMSO to photosynthetic cells. A model is presented to explain why the presence of a functional DMSO reductase (DorA) is required for DMSO to decrease cycA P2 expression under photosynthetic conditions.

The Rhodobacter sphaeroides response regulator PrrA directly activates transcription of genes necessary for energy conservation at low O2 tensions and under anaerobic conditions. It is proposed that PrrA homologues contain a C-terminal DNA-binding domain (PrrA-CTD) that lacks significant amino acid sequence similarity to those found in other response regulators. To test this hypothesis, single amino acid substitutions were created at 12 residues in the PrrA-CTD. These mutant PrrA proteins were purified and tested for the ability to be phosphorylated by the low-molecular-mass phosphate donor acetyl phosphate, to activate transcription and to bind promoter DNA. Each mutant PrrA protein accepted phosphate from 32P-labelled acetyl phosphate. At micromolar concentrations of acetyl phosphate-treated wild-type PrrA, a single 20 bp region in the PrrA-dependent cycA P2 promoter was protected from DNase I digestion. Of the mutant PrrA proteins tested, only acetyl phosphate-treated PrrA-N168A and PrrA-I177A protected cycA P2 from DNase I digestion at similar protein concentrations compared to wild-type PrrA. The use of in vitro transcription assays with the PrrA-dependent cycA P2 and puc promoters showed that acetyl phosphate-treated PrrA-N168A produced transcript levels similar to that of wild-type PrrA at comparable protein concentrations. Using concentrations of acetyl phosphate-treated PrrA that are saturating for the wild-type protein, PrrA-H170A and PrrA-I177A produced<45%as much transcript as wild-type PrrA. Under identical conditions, the remaining mutant PrrA proteins produced little or no detectable transcripts from either promoter in vitro. Explanations are presented for why these amino acid side chains in the PrrA-CTD are important for its ability to activate transcription.

Anoxygenic photosynthetic bacteria have provided us with crucial insights into the process of solar energy capture, pathways of metabolic and societal importance, specialized differentiation of membrane domains, function or assembly of bioenergetic enzymes, and into the genetic control of these and other activities. Recent insights into the organization of this bioenergetic membrane system, the genetic control of this specialized domain of the inner membrane and the process by which potentially photosynthetic and non-photosynthetic cells protect themselves from an important class of reactive oxygen species will provide an unparalleled understanding of solar energy capture and facilitate the design of solar-powered microbial biorefineries.

In the photosynthetic bacterium Rhodobacter sphaeroides, a transcriptional response to the reactive oxygen species singlet oxygen (1O2) is mediated by ChrR, a zinc metalloprotein that binds to and inhibits activity of the alternative sigma factor, σE. We provide evidence that 1O2 promotes dissociation of σE from ChrR to activate transcription in vivo. To identify what is required for 1O2 to promote dissociation of σE/ChrR complexes, we analyzed the in vivo properties of variant ChrR proteins with amino acid changes in conserved residues of the C-terminal cupin-like domain (ChrR-CLD). We found that 1O2 was unable to promote detectable dissociation of σE/ChrR complexes when the ChrR-CLD zinc ligands (His141, His143, Glu147, and His177) were substituted with alanine, even though individual substitutions caused a 2- to 10-fold decrease in zinc affinity for this domain relative to that of wild-type ChrR (Kd ∼4.6 × 10−10 M). We conclude that the side chains of these invariant residues play a crucial role in the response to 1O2. Additionally, we found that cells containing variant ChrR proteins with single amino acid substitutions at Cys187 or Cys189 exhibited σE activity similar to those containing wild-type ChrR when exposed to 1O2, suggesting that these thiol side chains are not required for 1O2 to induce σE activity in vivo. Finally, we found that the same aspects of R. sphaeroides ChrR needed for a response to 1O2 are required for dissociation of σE/ChrR in the presence of the organic hydroperoxide, tert-butyl hydroperoxide (t-BOOH).

Timothy Donohue and Richard Cogdell discuss how microbiology can contribute towards the provision of clean solutions to the world′s energy needs.

Formaldehyde is an intermediate formed during the metabolism of methanol or other methylated compounds. Many Gram-negative bacteria generate formaldehyde from methanol via a periplasmic pyrroloquinoline quinone (PQQ)-dependent dehydrogenase in which the α subunit of an α2β2 tetramer has catalytic activity. The genome of the facultative formaldehyde-oxidizing bacterium Rhodobacter sphaeroides encodes XoxF, a homologue of the catalytic subunit of a proposed PQQ-containing dehydrogenase of Paracoccus denitrificans. R. sphaeroides xoxF is part of a gene cluster that encodes periplasmic c-type cytochromes, including CycI, isocytochrome c2 and CycB (a cyt c553i homologue), as well as adhI, a glutathione-dependent formaldehyde dehydrogenase (GSH-FDH), and gfa, a homologue of a glutathione–formaldehyde activating enzyme (Gfa). To test the roles of XoxF, CycB and Gfa in formaldehyde metabolism by R. sphaeroides, we monitored photosynthetic growth with methanol as a source of formaldehyde and whole-cell methanol-dependent oxygen uptake. Our data show that R. sphaeroides cells lacking XoxF or CycB do not exhibit methanol-dependent oxygen uptake and lack the capacity to utilize methanol as a sole photosynthetic carbon source. These results suggest that both proteins are required for formaldehyde metabolism. R. sphaeroides Gfa is not essential to activate formaldehyde, as cells lacking gfa are capable of both methanol-dependent oxygen uptake and growth with methanol as a photosynthetic carbon source.

We used global transcript analyses and mutant studies to investigate the pathways that impact H2 production in the photosynthetic bacterium Rhodobacter sphaeroides. We found that H2 production capacity is related to the levels of expression of the nitrogenase and hydrogenase enzymes and the enzymes of the Calvin-Benson-Bassham pathway.

Background

Rhodobacter sphaeroides is one of the best studied purple non-sulfur photosynthetic bacteria and serves as an excellent model for the study of photosynthesis and the metabolic capabilities of this and related facultative organisms. The ability of R. sphaeroides to produce hydrogen (H2), polyhydroxybutyrate (PHB) or other hydrocarbons, as well as its ability to utilize atmospheric carbon dioxide (CO2) as a carbon source under defined conditions, make it an excellent candidate for use in a wide variety of biotechnological applications. A genome-level understanding of its metabolic capabilities should help realize this biotechnological potential.

Results

Here we present a genome-scale metabolic network model for R. sphaeroides strain 2.4.1, designated iRsp1095, consisting of 1,095 genes, 796 metabolites and 1158 reactions, including R. sphaeroides-specific biomass reactions developed in this study. Constraint-based analysis showed that iRsp1095 agreed well with experimental observations when modeling growth under respiratory and phototrophic conditions. Genes essential for phototrophic growth were predicted by single gene deletion analysis. During pathway-level analyses of R. sphaeroides metabolism, an alternative route for CO2 assimilation was identified. Evaluation of photoheterotrophic H2 production using iRsp1095 indicated that maximal yield would be obtained from growing cells, with this predicted maximum ~50% higher than that observed experimentally from wild type cells. Competing pathways that might prevent the achievement of this theoretical maximum were identified to guide future genetic studies.

Conclusions

iRsp1095 provides a robust framework for future metabolic engineering efforts to optimize the solar- and nutrient-powered production of biofuels and other valuable products by R. sphaeroides and closely related organisms.

chipD is a web server that facilitates design of DNA oligonucleotide probes for high-density tiling arrays, which can be used in a number of genomic applications such as ChIP-chip or gene-expression profiling. The server implements a probe selection algorithm that takes as an input, in addition to the target sequences, a set of parameters that allow probe design to be tailored to specific applications, protocols or the array manufacturer’s requirements. The algorithm optimizes probes to meet three objectives: (i) probes should be specific; (ii) probes should have similar thermodynamic properties; and (iii) the target sequence coverage should be homogeneous and avoid significant gaps. The output provides in a text format, the list of probe sequences with their genomic locations, targeted strands and hybridization characteristics. chipD has been used successfully to design tiling arrays for bacteria and yeast. chipD is available at http://chipd.uwbacter.org/.

Rhodobacter sphaeroides σE is a member of the extra cytoplasmic function sigma factor (ECF) family, whose members have been shown to regulate gene expression in response to a variety of signals. The functions of ECF family members are commonly regulated by a specific, reversible interaction with a cognate anti-sigma factor. In R. sphaeroides, σE activity is inhibited by ChrR, a member of a newly discovered family of zinc containing anti-sigma factors. We used gel filtration chromatography to gain insight into the mechanism by which ChrR inhibits σE activity. We found that formation of the σE:ChrR complex inhibits the ability of σE to form a stable complex with core RNA polymerase. Since the σE:ChrR complex inhibits the ability of the sigma factor to bind RNA polymerase, we sought to identify amino acid substitutions in σE that altered the sensitivity of this sigma factor to inhibition by ChrR. This analysis identified single amino acid changes in conserved region 2.1 of σE that either increased or decreased the sensitivity of σE for inhibition by ChrR. Many of the amino acid residues that alter the sensitivity of σE to ChrR are located within regions known to be important for interacting with core RNA polymerase in other members of the s70 superfamily. Our results suggest a model where solvent-exposed residues with region 2.1 of σE interact with ChrR to sterically occlude this sigma factor from binding core RNA polymerase and to inhibit target gene expression.

The analysis of proteomes from aerobic and photosynthetic Rhodobacter sphaeroides 2.4.1 cell cultures by liquid chromatography-mass spectrometry yielded approximately 6,500 high confidence peptides representing 1,675 gene products (39% of the predicted proteins). The identified proteins corresponded primarily to open reading frames (ORFs) contained within the two chromosomal elements of this bacterium, but a significant number were also observed from ORFs associated with 5 naturally occurring plasmids. Using the accurate mass and time (AMT) tag approach, comparative studies showed that a number of proteins were uniquely detected within the photosynthetic cell culture. The estimated abundances of proteins observed in both aerobic respiratory and photosynthetic grown cultures were compared to provide insights into bioenergetic models for both modes of growth. Additional emphasis was placed on gene products annotated as hypothetical to gain information as to their potential roles within these two growth conditions. Where possible, transcriptome and proteome data for R. sphaeroides obtained under the same culture conditions were also compared.

The high-throughput accurate mass and time (AMT) tag proteomic approach was utilized to characterize the proteomes for cytoplasm, cytoplasmic membrane, periplasm, and outer membrane fractions from aerobic and photosynthetic cultures of the gram-nagtive bacterium Rhodobacter sphaeroides 2.4.1. In addition, we analyzed the proteins within purified chromatophore fractions that house the photosynthetic apparatus from photosynthetically grown cells. In total, 8300 peptides were identified with high confidence from at least one subcellular fraction from either cell culture. These peptides were derived from 1514 genes or 35% percent of proteins predicted to be encoded by the genome. A significant number of these proteins were detected within a single subcellular fraction and their localization was compared to in silico predictions. However, the majority of proteins were observed in multiple subcellular fractions, and the most likely subcellular localization for these proteins was investigated using a Z-score analysis of estimated protein abundance along with clustering techniques. Good (81%) agreement was observed between the experimental results and in silico predictions. The AMT tag approach provides localization evidence for those proteins that have no predicted localization information, those annotated as putative proteins, and/or for those proteins annotated as hypothetical and conserved hypothetical.

Singlet oxygen is one of several reactive oxygen species that can destroy biomolecules, microorganisms and other cells. Traditionally, the response to singlet oxygen has been termed photo-oxidative stress, as light-dependent processes in photosynthetic cells are major biological sources of singlet oxygen. Recent work identifying a core set of singlet oxygen stress response genes across various bacterial species highlights the importance of this response for survival by both photosynthetic and non-photosynthetic cells. Here, we review how bacterial cells mount a transcriptional response to photo-oxidative stress in the context of what is known about bacterial stress responses to other reactive oxygen species.

The appearance of atmospheric oxygen from photosynthetic activity led to the evolution of aerobic respiration and responses to the resulting reactive oxygen species. In Rhodobacter sphaeroides, a photosynthetic α-proteobacterium, a transcriptional response to the reactive oxygen species singlet oxygen (1O2) is controlled by the group IV σ factor σE and the anti-σ factor ChrR. In this study, we integrated various large datasets to identify genes within the 1O2 stress response that contain σE-dependent promoters both within R. sphaeroides and across the bacterial phylogeny. Transcript pattern clustering and a σE-binding sequence model were used to predict candidate promoters that respond to 1O2 stress in R. sphaeroides. These candidate promoters were experimentally validated to nine R. sphaeroides σE-dependent promoters that control the transcription of 15 1O2-activated genes. Knowledge of the R. sphaeroides response to 1O2 and its regulator σE–ChrR was combined with large-scale phylogenetic and sequence analyses to predict the existence of a core set of approximately eight conserved σE-dependent genes in α-proteobacteria and γ-proteobacteria. The bacteria predicted to contain this conserved response to 1O2 include photosynthetic species, as well as free-living and symbiotic/pathogenic nonphotosynthetic species. Our analysis also predicts that the response to 1O2 evolved within the time frame of the accumulation of atmospheric molecular oxygen on this planet.

Summary

Since their classification as members of the σ70 super-family, Group IV alternative σ factors have been found to control gene expression in response to diverse environmental or stress signals. Activity of the Streptomyces coelicolor Group IV family member, σR (SigR), is increased by changes in the oxidation-reduction state of cytoplasmic disulphide bonds. Once released by its cognate anti-σ factor RsrA, σR activates expression of gene products that help cells reduce cytoplasmic disulphide bonds. In this issue of Molecular Microbiology, Kim and co-workers provide new insights into positive and negative control of σR activity. The authors show that a transcript derived from the inducible σR-dependent sigRrsrA p2 promoter operon encodes a σR protein of a higher molecular weight (termed σR′) than is found in uninduced cells. One major difference between σR′ and the smaller σR protein found in uninduced cells is the rapid proteolysis of σR′ by the ClpP1/P2 protease system. The genes for the ClpP1/ClpP2 protease subunits are themselves members of the σR regulon. The newly identified positive (σR′ synthesis) and negative control (selective σR′ turnover) aspects of this circuit are either found or predicted to exist in other related Group IV σ factor family members.

In the facultatively phototrophic proteobacterium Rhodobacter sphaeroides, formation of the photosynthetic apparatus is oxygen dependent. When oxygen tension decreases, the response regulator PrrA of the global two-component PrrBA system is believed to directly activate transcription of the puf, puh, and puc operons, encoding structural proteins of the photosynthetic complexes, and to indirectly upregulate the photopigment biosynthesis genes bch and crt. Decreased oxygen also results in inactivation of the photosynthesis-specific repressor PpsR, bringing about derepression of the puc, bch, and crt operons. We uncovered a hierarchical relationship between these two regulatory systems, earlier thought to function independently. We also more accurately assessed the spectrum of gene targets of the PrrBA system. First, expression of the appA gene, encoding the PpsR antirepressor, is PrrA dependent, which establishes one level of hierarchical dominance of the PrrBA system over AppA-PpsR. Second, restoration of the appA transcript to the wild-type level is insufficient for rescuing phototrophic growth impairment of the prrA mutant, whereas inactivation of ppsR is sufficient. This suggests that in addition to controlling appA transcription, PrrA affects the activity of the AppA-PpsR system via an as yet unidentified mechanism(s). Third, PrrA directly activates several bch and crt genes, traditionally considered to be the PpsR targets. Therefore, in R. sphaeroides, the global PrrBA system regulates photosynthesis gene expression (i) by rigorous control over the photosynthesis-specific AppA-PpsR regulatory system and (ii) by extensive direct transcription activation of genes encoding structural proteins of photosynthetic complexes as well as genes encoding photopigment biosynthesis enzymes.

Biological systems are in a continual state of flux, which necessitates an understanding of the dynamic nature of protein abundances. The study of protein abundance dynamics has become feasible with recent improvements in mass spectrometry-based quantitative proteomics. However, a number of challenges still remain related to how best to extract biological information from dynamic proteomics data, for example, challenges related to extraneous variability, missing abundance values, and the identification of significant temporal patterns. This paper describes a strategy that addresses these issues and demonstrates its values for analyzing temporal bottom-up proteomics data using data from a Rhodobacter sphaeroides 2.4.1 time-course study.

PrrA is a global trancription regulator activated upon phosphorylation by its cognate kinase PrrB in response to low oxygen conditions in Rhodobacter sphaeroides. Here we show by gel filtration, analytical ultracentrifugation and NMR diffusion measurements that treatment of PrrA by a phosphate analogue, BeF3-, results in dimerization of the protein, producing a protein that binds DNA. No dimeric species was observed in the absence of BeF3-. On addition of BeF3-, the inhibitory activity of the N-terminal domain on the C-terminal DNA-binding domain is relieved, after which PrrA becomes capable of binding DNA as a dimer. The interaction surface of the DNA-binding domain with the regulatory domain of PrrA is identified by NMR as being a well conserved region centered on helix α6, which is on the opposite face to the DNA recognition helix. This suggests that there is no direct blockage of DNA binding in the inactive state, but rather that PrrA dimerization promotes a correct arrangement of two adjacent DNA binding domains to recognise specific DNA binding sequences.

SUMMARY

A transcriptional response to singlet oxygen in Rhodobacter sphaeroides is controlled by the group IV σ factor σE and its cognate anti-σ ChrR. Crystal structures of the σE/ChrR complex reveal a modular, two-domain architecture for ChrR. The ChrR N-terminal anti-σ domain (ASD) binds a Zn2+ ion, contacts σE, and is sufficient to inhibit σE-dependent transcription. The ChrR C-terminal domain adopts a cupin fold, can coordinate an additional Zn2+, and is required for the transcriptional response to singlet oxygen. Structure-based sequence analyses predict that the ASD defines a common structural fold among predicted group IV antiσs. These ASDs are fused to diverse C-terminal domains that are likely involved in responding to specific environmental signals that control the activity of their cognate σ factor.

The Rhodobacter sphaeroides intracytoplasmic membrane (ICM) is an inducible membrane that is dedicated to the major events of bacterial photosynthesis, including harvesting light energy, separating primary charges, and transporting electrons. In this study, multichromatographic methods coupled with Fourier transform ion cyclotron resonance mass spectrometry, combined with subcellular fractionation, was used to test the hypothesis that the photosynthetic membrane of R. sphaeroides 2.4.1 contains a significant number of heretofore unidentified proteins in addition to the integral membrane pigment-protein complexes, including light-harvesting complexes 1 and 2, the photochemical reaction center, and the cytochrome bc1 complex described previously. Purified ICM vesicles are shown to be enriched in several abundant, newly identified membrane proteins, including a protein of unknown function (AffyChip designation RSP1760) and a possible alkane hydroxylase (RSP1467). When the genes encoding these proteins are mutated, specific photosynthetic phenotypes are noted, illustrating the potential new insights into solar energy utilization to be gained by this proteomic blueprint of the ICM. In addition, proteins necessary for other cellular functions, such as ATP synthesis, respiration, solute transport, protein translocation, and other physiological processes, were also identified to be in association with the ICM. This study is the first to provide a more global view of the protein composition of a photosynthetic membrane from any source. This protein blueprint also provides insights into potential mechanisms for the assembly of the pigment-protein complexes of the photosynthetic apparatus, the formation of the lipid bilayer that houses these integral membrane proteins, and the possible functional interactions of ICM proteins with activities that reside in domains outside this specialized bioenergetic membrane.

We have identified a second RpoH homolog, RpoHII, in the α-proteobacterium Rhodobacter sphaeroides. Primary amino acid sequence comparisons demonstrate that R. sphaeroides RpoHII belongs to a phylogenetically distinct group with RpoH orthologs from α-proteobacteria that contain two rpoH genes. Like its previously identified paralog, RpoHI, RpoHII is able to complement the temperature-sensitive phenotype of an Escherichia coli σ32 (rpoH) mutant. In addition, we show that recombinant RpoHI and RpoHII each transcribe two E. coli σ32-dependent promoters (rpoD PHS 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, groESL1, rpoD PHS, dnaK P1, hslO, and ecfE), all of which resemble the E. coli σ32 promoter consensus. While RpoHI reconstituted with R. sphaeroides core RNA polymerase transcribed all six promoters, RpoHII produced detectable transcripts from only four promoters (cycA P1, groESL1, hslO, and ecfE). These results, in combination with previous work demonstrating that an RpoHI mutant mounts a typical heat shock response, suggest that while RpoHI and RpoHII have redundant roles in response to heat, they may also have roles in response to other environmental stresses.