Rhodobacter sphaeroides is a free-living, photoheterotrophic bacterium known for its genomic and metabolic complexity. We have discovered that this purple photosynthetic organism possesses a quorum-sensing system. Quorum sensing occurs in a number of eukaryotic host-associated gram-negative bacteria. In these bacteria there are two genes required for quorum sensing, the luxR and luxI homologs, and there is an acylhomoserine lactone signal molecule synthesized by the product of the luxI homolog. In R. sphaeroides, synthesis of a novel homoserine lactone signal, 7,8-cis-N-(tetradecenoyl)homoserine lactone, is directed by a luxI homolog termed cerI. Two open reading frames immediately upstream of cerI are proposed to be components of the quorumsensing system. The first of these is a luxR homolog termed cerR, and the second is a small open reading frame of 159 bp. Inactivation of cerI in R. sphaeroides results in mucoid colony formation on agar and formation of large aggregates of cells in liquid cultures. Clumping of CerI mutants in liquid culture is reversible upon addition of the acylhomoserine lactone signal and represents a phenotype unlike those controlled by quorum sensing in other bacteria.Rhodobacter sphaeroides 2.4.1 is a free-living, photoheterotrophic member of the ␣-3 subdivision of the Proteobacteria and has been widely studied due to its remarkable genomic and physiologic complexity. It has a unique genome architecture, possessing both a large (chromosome I, 3.0 Mbp) and a smaller (chromosome II, 0.9 Mbp) circular chromosome (42, 43) in addition to five other endogenous replicons (15). It can grow aerobically as a chemoheterotroph, and it can grow anaerobically by using photosynthetic electron transport (8, 24) or anaerobic respiration (13).We report that R. sphaeroides produces an acylhomoserine lactone. N-Acylhomoserine lactones (commonly called autoinducers) are produced by many gram-negative bacteria and serve as intercellular signals that facilitate a phenomenon termed quorum sensing (17,18,34,40). Quorum sensing allows a bacterial species to monitor its population density and activate specific sets of genes at sufficiently high cell numbers. Regulation of luminescence in Vibrio fischeri was the first reported example of N-acylhomoserine lactone-mediated quorum sensing (9, 25) and continues to serve as a model system (17, 18). In V. fischeri, the luxI gene encodes an autoinducer synthase that catalyzes the production of N-(3-oxohexanoyl) homoserine lactone (9). The luxR gene encodes a transcriptional regulator that activates expression of the luminescence genes in the presence of a sufficient concentration of N-(3-oxohexanoyl)homoserine lactone (11).This type of regulation is involved in the expression of genes encoding extracellular virulence factors in Pseudomonas aeruginosa (27,28), conjugal transfer in Agrobacterium tumefaciens (16,30,46), and antibiotic synthesis and extracellular enzyme and exopolysaccharide production in Erwinia species (2, 31) to name a few. In these quorum-sensing system...

This review describes some of the recent highlights taken from the studies of Rhodobacter sphaeroides 2.4.1. The review is not intended to be comprehensive, but to reflect the bias of the authors as to how the availability of a sequenced and annotated genome, a gene-chip, and proteomic profile as well as comparative genomic analyses can direct the progress of future research in this system.

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.

A spontaneous mutant (R/89) of photosynthetic purple bacterium Rhodobacter sphaeroides R-26 was selected for resistance to 200 μM atrazin. It showed increased resistance to interquinone electron transfer inhibitors of o-phenanthroline (resistance factor, RF=20) in UQo reconstituted isolated reaction centers and terbutryne in reaction centers (RF=55) and in chromatophores (RF=85). The amino acid sequence of the QB binding protein of the photosynthetic reaction center (the L subunit) was determined by sequencing the corresponding pufL gene and a single mutation was found (Ile(L229) → Met). The changed amino acid of the mutant strain is in van der Waals contact with the secondary quinone QB. The binding and redox properties of QB in the mutant were characterized by kinetic (charge recombination) and multiple turnover (cytochrome oxidation and semiquinone oscillation) assays of the reaction center. The free energy for stabilization of QAQB (-) with respect to QA (-)QB was ΔGAB=-60 meV and 0 meV in reaction centers and ΔGAB=-85 meV and -46 meV in chromatophores of R-26 and R/89 strains at pH 8, respectively. The dissociation constants of the quinone UQo and semiquinone UQo (-) in reaction centers from R-26 and R/89 showed significant and different pH dependence. The observed changes in binding and redox properties of quinones are interpreted in terms of differential effects (electrostatics and mesomerism) of mutation on the oxidized and reduced states of QB.

Low molecular weight (LMW) isoforms of cyclin E are posttranslationally generated in breast cancer cells and are associated with aggressive disease and poor prognosis. In this study, the specificity of LMW cyclin E to cancer cells was determined by measuring cyclin E expression in tumor and non-tumor tissue from 340 breast cancer patients. Our results reveal the LMW isoforms were detected significantly more frequently in breast tumor tissue than in adjacent non-tumor breast tissues (p < 0.0001). The biologic consequences of the LMW isoforms were studied using a non-tumorigenic mammary epithelial cell line transfected with the cyclin E isoforms and resulted in increased clonogenicity, the inability to enter quiescence in response to growth factor deprivation and genomic instability compared to the full-length cyclin E. Biochemical differences between the full-length and the LMW isoforms were also evident. Biacore analyses show that the LMW isoforms have more efficient binding to CDK2 compared to full-length cyclin E, which could account for the unique biologic consequences observed with the expression of LMW cyclin E. The LMW isoforms of cyclin E are tumor specific, and are biochemically and biologically distinct from the full-length cyclin E which could provide a novel role in breast cancer progression.