cr ~ and r ~2 are two heat-and ethanol-inducible g-factors in Escherichia coli. The cr 32 regulon is also induced by unfolded and misfolded proteins in the cytoplasm, and the function of many of the proteins in the cr 32 regulon is to bind to cytoplasmic proteins and assist them in folding or unfolding. To further understand the function of the cr F regulon, we searched for mutants that affected cr E activity. Our results indicate that a signal generated by expression of outer membrane proteins modulates cr E activity. Specifically, r ~ activity is induced by increased expression of OMPs and is reduced by decreased expression of OMPs. In addition, mutations that cause misfolded OMPs induce cr E activity. This signal is generated after the fate of OMPs and periplasmic proteins diverge in the secretory pathway and is not the result of an accumulation of OMP precursors in the cytoplasm. Our results indicate that this effect of OMPs is specific to the r E regnlon, because none of the above mutations affect r 32 activity. We propose that the ~r ~ regnlon is involved in processes that occur in extracytoplasmic compartments and that these two heat-inducible regulons may have distinct but complementary roles of monitoring the state of proteins in the cytoplasm (or 32) and outer membrane ((]rE).[Key Words: (r-Factors; protein export; outer membrane proteins; heat shock; (rE] Received September 13, 1993; revised version accepted October 14, 1993.In bacterial cells the (r-subunit directs RNA polymerase to initiate transcription at promoter sites on the DNA (Burgess et al. 1969). The primary (r-factor in the cell is responsible for transcription of most genes during exponential growth. In addition, alternative (r-factors direct transcription of sets of genes whose products are needed for specific functions, such as sporulation, nitrogen fixation, or flagella synthesis {Gross et al. 1992). Alternative (r-factors are often activated by changes in environmental or cellular conditions that generate morphological and/or molecular cues, signaling the need for the gene products in the regulon under control of a particular (r-factor. Elucidation of these signal-transduction pathways provides insights about global control of gene activity in prokaryotic cells.The activity of two Escherichia coli alternative (r-factors, (r32 and (re ((r24), increases after temperature upshift or exposure to ethanol {Grossman et al. 1984;Erickson et al. 1987;Straus et al. 1987;Erickson and Gross 1989;Wang and Kaguni 1989}. RNA polymerase (E) containing o ~2 (E(r 32) transcribes the heat shock genes with products that consist primarily of chaperones and proteases.

A transcriptional response to singlet oxygen in Rhodobacter sphaeroides is controlled by the group IV sigma factor sigma(E) and its cognate anti-sigma ChrR. Crystal structures of the sigma(E)/ChrR complex reveal a modular, two-domain architecture for ChrR. The ChrR N-terminal anti-sigma domain (ASD) binds a Zn(2+) ion, contacts sigma(E), and is sufficient to inhibit sigma(E)-dependent transcription. The ChrR C-terminal domain adopts a cupin fold, can coordinate an additional Zn(2+), 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 anti-sigmas. 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 sigma factor.

Cultivation methods have contributed to our present knowledge about the presence and diversity of microbes in naturally occurring communities. However, it is well established that only a small fraction of prokaryotes have been cultivated by standard methods and, therefore, the prokaryotes that are cultivated may not ref lect the composition and diversity within those communities. Of the two prokaryotic phylogenetic domains, Bacteria and Archaea, members of the former have been shown to be ubiquitous in nature, with ample evidence of vast assemblages of uncultured organisms. There is also now increasingly compelling evidence that the Archaea, which were once thought to occupy a limited number of environments, are also globally widespread. Here we report the use of molecular phylogenetic techniques, which are independent of microbial cultivation, to conduct an assessment of Archaea in a soil microbial community. Small subunit ribosomal RNA genes of Archaea were amplified from soil and cloned. Phylogenetic and nucleotide signature analyses of these cloned small subunit ribosomal RNA gene sequences revealed a cluster of Archaea from a soil microbial community that diverge deeply from the crenarchaeotal line of descent and has the closest affiliation to the lineage of planktonic Archaea. The identification and phylogenetic classification of this archaeal lineage from soil contributes to our understanding of the ecological significance of Archaea as a component of microbial communities in non-extreme environments.

The ability of phototrophs to convert light into biological energy is critical for life on Earth. However, there can be deleterious consequences associated with this bioenergetic conversion, including the production of toxic byproducts. For example, singlet oxygen ( 1 O2) can be formed during photosynthesis by energy transfer from excited triplet-state chlorophyll pigments to O2. By monitoring gene expression and growth in the presence of 1 O2, we show that the phototrophic bacterium Rhodobacter sphaeroides mounts a transcriptional response to this reactive oxygen species (ROS) that requires the alternative factor, E . An increase in E activity is seen when cells are exposed to 1 O2 generated either by photochemistry within the photosynthetic apparatus or the photosensitizer, methylene blue. Wavelengths of light responsible for the generating triplet-state chlorophyll pigments in the photosynthetic apparatus are sufficient for a sustained increase in E activity. Continued exposure to 1 O2 is required to maintain this transcriptional response, and other ROS do not cause a similar increase in E -dependent gene expression. When a E mutant produces low levels of carotenoids, 1 O2 is bacteriocidal, suggesting that this response is essential for protecting cells from this ROS. In addition, global gene expression analysis identified Ϸ180 genes (Ϸ60 operons) whose RNA levels increase >3-fold in cells with increased E activity. Gene products encoded by four newly identified E -dependent operons are predicted to be involved in stress response, protecting cells from 1 O2 damage, or the conservation of energy.factor ͉ reactive oxygen species ͉ Rhodobacter sphaeroides ͉ photochemistry ͉ carotenoids

Herbivores can gain indirect access to recalcitrant carbon present in plant cell walls through symbiotic associations with lignocellulolytic microbes. A paradigmatic example is the leaf-cutter ant (Tribe: Attini), which uses fresh leaves to cultivate a fungus for food in specialized gardens. Using a combination of sugar composition analyses, metagenomics, and whole-genome sequencing, we reveal that the fungus garden microbiome of leaf-cutter ants is composed of a diverse community of bacteria with high plant biomass-degrading capacity. Comparison of this microbiome's predicted carbohydrate-degrading enzyme profile with other metagenomes shows closest similarity to the bovine rumen, indicating evolutionary convergence of plant biomass degrading potential between two important herbivorous animals. Genomic and physiological characterization of two dominant bacteria in the fungus garden microbiome provides evidence of their capacity to degrade cellulose. Given the recent interest in cellulosic biofuels, understanding how large-scale and rapid plant biomass degradation occurs in a highly evolved insect herbivore is of particular relevance for bioenergy.

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 Rhodobacter sphaeroides cytochrome c2 functions as a mobile electron carrier in both aerobic and photosynthetic electron transport chains. Synthetic deoxyoligonucleotide probes, based on the known amino acid sequence of this protein (Mr 14,000), were used to identify and clone the cytochrome c2 structural gene (cycA The facultative photoheterotrophic bacterium Rhodobacter sphaeroides (recently redefined from the genus Rhodopseudomonas [23]) is an excellent model system for studying membrane bioenergetics (18a), photosynthesis (24), membrane biogenesis (17a), and the physiological control of gene expression for components of the inducible photosynthetic membrane (10, 52). When growing chemoheterotrophically, R. sphaeroides contains a typical gram-negative outer membrane and a cytoplasmic membrane. Under these conditions energy is generated by an aerobic respiratory chain whose components are structurally and functionally similar to those found in mitochondria (51). The removal of oxygen from a chemoheterotrophic culture induces a differentiation of the cytoplasmic membrane resulting in the synthesis of the intracytoplasmic membrane (ICM). The ICM exists as structurally contiguous but functionally distinct invaginations of the cytoplasmic membrane which constitute the photosynthetic apparatus of the cell (17a, 24). Although photopigments and the bacteriochlorophyll (Bchl)-binding proteins are found exclusively in the ICM of photosynthetic cells, the redox components of the respiratory pathways and the proton-translocating ATPase are located in both the cytoplasmic membrane and ICM of photosynthetic cells (2, 24).Cytochrome c2 (cyt c2) is a constitutive redox protein located in the periplasmic space of R. sphaeroides (39). This soluble cytochrome (Mr 14,000) mediates electron transfer from the membrane-bound ubiquinol:cyt c2 oxidoreductase (19) to the photochemical reaction center (48) in the cyclic photosynthetic electron transport chain (12). In aerobically grown cells cyt c2 transfers electrons from the membranebound oxidoreductase complex to a cyt aa3-type oxidase similar to that found in mitochondria (1,20 (17,36). This conservation in amino acid sequence and polypeptide structure has been the basis for the proposal that aerobic respiration in both procaryotes and eucaryotes arose from an ancestor containing a dual-function photosynthetic and respiratory electron transport chain similar to that found in organisms such as R. sphaeroides (17).Despite considerable information on the structure (17, 36), function (13, 18a, 48), and localization (39)

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 ( 1 O 2 ) is controlled by the group IV σ factor σ E and the anti-σ factor ChrR. In this study, we integrated various large datasets to identify genes within the 1 O 2 stress response that contain σ Edependent promoters both within R. sphaeroides and across the bacterial phylogeny. Transcript pattern clustering and a σ E -binding sequence model were used to predict candidate promoters that respond to 1 O 2 stress in R. sphaeroides. These candidate promoters were experimentally validated to nine R. sphaeroides σ E -dependent promoters that control the transcription of 15 1 O 2 -activated genes. Knowledge of the R. sphaeroides response to 1 O 2 and its regulator σ E -ChrR was combined with large-scale phylogenetic and sequence analyses to predict the existence of a core set of approximately eight conserved σ E -dependent genes in α-proteobacteria and γ-proteobacteria. The bacteria predicted to contain this conserved response to 1 O 2 include photosynthetic species, as well as free-living and symbiotic/pathogenic nonphotosynthetic species. Our analysis also predicts that the response to 1 O 2 evolved within the time frame of the accumulation of atmospheric molecular oxygen on this planet.