Janet L. Gibson

Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris.

Rhodopseudomonas palustris is among the most metabolically versatile bacteria known. It uses light, inorganic compounds, or organic compounds, for energy. It acquires carbon from many types of green plant–derived compounds or by carbon dioxide fixation, and it fixes nitrogen. Here we describe the genome sequence of R. palustris, which consists of a 5,459,213-base-pair (bp) circular chromosome with 4,836 predicted genes and a plasmid of 8,427 bp. The sequence reveals genes that confer a remarkably large number of options within a given type of metabolism, including three nitrogenases, five benzene ring cleavage pathways and four light harvesting 2 systems. R. palustris encodes 63 signal transduction histidine kinases and 79 response regulator receiver domains. Almost 15% of the genome is devoted to transport. This genome sequence is a starting point to use R. palustris as a model to explore how organisms integrate metabolic modules in response to environmental perturbations.

The σE stress response is required for stress-induced mutation and amplification in Escherichia coli

Pathways of mutagenesis are induced in microbes under adverse conditions controlled by stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e. are stressed. Stress‐induced mutagenesis in the Escherichia coli Lac assay occurs either by ‘point’ mutation or gene amplification. Point mutagenesis is associated with DNA double‐strand‐break (DSB) repair and requires DinB error‐prone DNA polymerase and the SOS DNA‐damage‐ and RpoS general‐stress responses. We report that the RpoE envelope‐protein‐stress response is also required. In a screen for mutagenesis‐defective mutants, we isolated a transposon insertion in the rpoE P2 promoter. The insertion prevents rpoE induction during stress, but leaves constitutive expression intact, and allows cell viability. rpoE insertion and suppressed null mutants display reduced point mutagenesis and maintenance of amplified DNA. Furthermore, σE acts independently of stress responses previously implicated: SOS/DinB and RpoS, and of σ32, which was postulated to affect mutagenesis. I‐SceI‐induced DSBs alleviated much of the rpoE phenotype, implying that σE promoted DSB formation. Thus, a third stress response and stress input regulate DSB‐repair‐associated stress‐induced mutagenesis. This provides the first report of mutagenesis promoted by σE, and implies that extracytoplasmic stressors may affect genome integrity and, potentially, the ability to evolve.

Residues that influence in vivo and in vitro CbbR function in Rhodobacter sphaeroides and identification of a specific region critical for co‐inducer recognition

CbbR is a LysR‐type transcriptional regulator (LTTR) that is required to activate transcription of the cbb operons, responsible for CO2 fixation, in Rhodobacter sphaeroides. LTTR proteins often require a co‐inducer to regulate transcription. Previous studies suggested that ribulose 1,5‐bisphosphate (RuBP) is a positive effector for CbbR function in this organism. In the current study, RuBP was found to increase the electrophoretic mobility of the CbbR/cbbI promoter complex. To define and analyse the co‐inducer recognition region of CbbR, constitutively active mutant CbbR proteins were isolated. Under growth conditions that normally maintain transcriptionally inactive cbb operons, the mutant CbbR proteins activated transcription. Fourteen of the constitutively active mutants resulted from a single amino acid substitution. One mutant was derived from amino acid substitutions at two separate residues that appeared to act synergistically. Different mutant proteins showed both sensitivity and insensitivity to RuBP and residues that conferred constitutive transcriptional activity could be highlighted on a three‐dimensional model, with several residues unique to CbbR shown to be at locations critical to LTTR function. Many of the constitutive residues clustered in or near two specific loops in the LTTR tertiary structure, corresponding to a proposed site of co‐inducer binding.

Differential Expression of the CO2 Fixation Operons of Rhodobacter sphaeroides by the Prr/Reg Two-Component System during Chemoautotrophic Growth

In Rhodobacter sphaeroides, the two cbb operons encoding duplicated Calvin-Benson Bassham (CBB) CO2 fixation reductive pentose phosphate cycle structural genes are differentially controlled. In attempts to define the molecular basis for the differential regulation, the effects of mutations in genes encoding a subunit of Cbb3 cytochrome oxidase, ccoP, and a global response regulator, prrA (regA), were characterized with respect to CO2 fixation (cbb) gene expression by using translational lac fusions to the R. sphaeroides cbb(I) and cbb(II) promoters. Inactivation of the ccoP gene resulted in derepression of both promoters during chemoheterotophic growth, where cbb expression is normally repressed; expression was also enhanced over normal levels during phototrophic growth. The prrA mutation effected reduced expression of cbb(I) and cbb(II) promoters during chemoheterotrophic growth, whereas intermediate levels of expression were observed in a double ccoP prrA mutant. PrrA and ccoP1 prrA strains cannot grow phototrophically, so it is impossible to examine cbb expression in these backgrounds under this growth mode. In this study, however, we found that PrrA mutants of R. sphaeroides were capable of chemoautotrophic growth, allowing, for the first time, an opportunity to directly examine the requirement of PrrA for cbb gene expression in vivo under growth conditions where the CBB cycle and CO2 fixation are required. Expression from the cbb(II) promoter was severely reduced in the PrrA mutants during chemoautotrophic growth, whereas cbb(I) expression was either unaffected or enhanced. Mutations in ccoQ had no effect on expression from either promoter. These observations suggest that the Prr signal transduction pathway is not always directly linked to Cbb3 cytochrome oxidase activity, at least with respect to cbb gene expression. In addition, lac fusions containing various lengths of the cbb(I) promoter demonstrated distinct sequences involved in positive regulation during photoautotrophic versus chemoautotrophic growth, suggesting that different regulatory proteins may be involved. In Rhodobacter capsulatus, ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) expression was not affected by cco mutations during photoheterotrophic growth, suggesting that differences exist in signal transduction pathways regulating cbb genes in the related organisms.

Genetic Analysis of CO2 Fixation Genes

Purple photosynthetic bacteria assimilate CO2 primarily via the Calvin reductive pentose phosphate pathway. Depending on the growth conditions, the amount of CO2 fixation required for growth and the level of Calvin cycle enzymes can vary considerably. Although numerous biochemical studies have demonstrated induction or derepression of the enzymes, the mechanisms underlying this control have remained elusive. Cloning and expression of Calvin cycle structural and regulatory genes from different photosynthetic bacteria have led to significant advances in our understanding of CO2 fixationat the molecular level. cbb genes have been shown to be clustered in several bacteria, including Rhodobacter sphaeroides, in which genes within two cbb gene clusters appear to be organized within operons thus coordinating expression at the level of transcription. Mutagenesis of cbb genes in Rb. sphaeroides has revealed independent and interdependent regulatory relationships between the two different operons that appear to be mediated through a common transcriptional regulatory protein, CbbR. Transcriptional regulation and cbbR genes have also been demonstrated in Chromatium vinosum and Rhodospirillum rubrum. Characterization of the CbbR proteins should provide clues to the identity of the molecular signal that serves as sensor of the environment to control expression of enzymes involved in CO2 assimilation. Alternate routes of CO2 fixationhave unexpectedly been exposed in Rubis CO deletion mutants constructed in Rb. sphaeroides and Rs. rubrum opening new avenues of research in CO2 fixation in the photosynthetic bacteria. The RubisCO mutants have also been used as host strains for analyzing foreign RubisCO enzymes. In Cm. vinosum, two distinct RubisCO coding sequences were identified by hybridization and expression in E. coli allowed characterization of both enzymes. Finally, nucleotide sequence comparisons of cbb structural and regulatory genes have raised provocative evolutionary questions concerning relationships between photosynthetic bacteria and other organisms.

An ultra-dense library resource for rapid deconvolution of mutations that cause phenotypes in Escherichia coli

With the wide availability of whole-genome sequencing (WGS), genetic mapping has become the rate-limiting step, inhibiting unbiased forward genetics in even the most tractable model organisms. We introduce a rapid deconvolution resource and method for untagged causative mutations after mutagenesis, screens, and WGS in Escherichia coli. We created Deconvoluter—ordered libraries with selectable insertions every 50 kb in the E. coli genome. The Deconvoluter method uses these for replacement of untagged mutations in the genome using a phage-P1-based gene-replacement strategy. We validate the Deconvoluter resource by deconvolution of 17 of 17 phenotype-altering mutations from a screen of N-ethyl-N-nitrosourea-induced mutants. The Deconvoluter resource permits rapid unbiased screens and gene/function identification and will enable exploration of functions of essential genes and undiscovered genes/sites/alleles not represented in existing deletion collections. This resource for unbiased forward-genetic screens with mapping-by-sequencing (‘forward genomics’) demonstrates a strategy that could similarly enable rapid screens in many other microbes.

Atypical Role for PhoU in Mutagenic Break Repair under Stress in Escherichia coli

Mechanisms of mutagenesis activated by stress responses drive pathogen/host adaptation, antibiotic and anti-fungal-drug resistance, and perhaps much of evolution generally. In Escherichia coli, repair of double-strand breaks (DSBs) by homologous recombination is high fidelity in unstressed cells, but switches to a mutagenic mode using error-prone DNA polymerases when the both the SOS and general (σS) stress responses are activated. Additionally, the σE response promotes spontaneous DNA breakage that leads to mutagenic break repair (MBR). We identified the regulatory protein PhoU in a genetic screen for functions required for MBR. PhoU negatively regulates the phosphate-transport and utilization (Pho) regulon when phosphate is in excess, including the PstB and PstC subunits of the phosphate-specific ABC transporter PstSCAB. Here, we characterize the PhoU mutation-promoting role. First, some mutations that affect phosphate transport and Pho transcriptional regulation decrease mutagenesis. Second, the mutagenesis and regulon-expression phenotypes do not correspond, revealing an apparent new function(s) for PhoU. Third, the PhoU mutagenic role is not via activation of the σS, SOS or σE responses, because mutations (or DSBs) that restore mutagenesis to cells defective in these stress responses do not restore mutagenesis to phoU cells. Fourth, the mutagenesis defect in phoU-mutant cells is partially restored by deletion of arcA, a gene normally repressed by PhoU, implying that a gene(s) repressed by ArcA promotes mutagenic break repair. The data show a new role for PhoU in regulation, and a new regulatory branch of the stress-response signaling web that activates mutagenic break repair in E. coli.

Molecular Control and Biochemistry of CO2 Fixation in Photosynthetic Bacteria

Photosynthetic bacteria are capable of diverse modes of CO2 assimilation, employing either the Calvin reductive pentose phosphate, reductive tricarboxylic acid, or hydroxyproprionate cycles, or in some cases various ancillary reactions. In purple bacteria and cyanobacteria, the Calvin reductive pentose phosphate pathway predominates (Tabita 1988, 1994, 1995), while green sulfur bacteria use the reductive TCA cycle and green nonsulfur bacteria employ the hydroxypropionate path (Sirevag 1995). Biochemical studies in our laboratory have focused on the enzymology of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) and phosphoribulokinase (PRK), the key enzymes of the Calvin cycle, and ATP-citrate lyase and the ferrodoxin-linked reactions of the reductive TCA cycle. The molecular control of CO2 fixation in nonsulfur purple bacteria has been a continuous interest (Gibson 1995), while the organism Chlorobium tepidum (Wahlund et al. 1991) has recently provided us with a useful and genetically tractable model system to study regulation of the reductive TCA cycle.