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.

Osmoregulated periplasmic glucans (OPGs) of Rhodobacter sphaeroides are anionic cyclic molecules that accumulate in large amounts in the periplasmic space in response to low osmolarity of the medium. Their anionic character is provided by the substitution of the glucosidic backbone by succinyl residues. A wild-type strain was subject to transposon mutagenesis, and putative mutant clones were screened for changes in OPGs by thin layer chromatography. One mutant deficient in succinyl substitution of the OPGs was obtained and the gene inactivated in this mutant was characterized and named opgC. opgC is located downstream of three ORFs, opgGIH, two of which are similar to the Escherichia coli operon, mdoGH, governing OPG backbone synthesis. Inactivation of opgG, opgI or opgH abolished OPG production and complementation analysis indicated that the three genes are necessary for backbone synthesis. In contrast, inactivation of a gene similar to ndvB, encoding the OPG-glucosyl transferase in Sinorhizobium meliloti, had no consequence on OPG synthesis in Rhodobacter sphaeroides. Cassette insertions in opgH had a polar effect on glucan substitution, indicating that opgC is in the same transcription unit. Expression of opgIHC in E. coli mdoB/mdoC and mdoH mutants allowed the production of slightly anionic and abnormally long linear glucans.

Background Early stage HER2 amplified breast cancer has a generally favorable prognosis with over 95% of patients showing 2-year disease free survival (DFS) when treated with adjuvant trastuzumab. However, a subset of these tumors are refractory to treatment and present a challenge for the oncologist, particularly when clinical and histologic parameters, including the patient’s nodal and hormonal status, are indicative of lower-risk HER2 positive disease. In this study we describe the genomic landscape of three clinically lower-risk patients with HER2 amplified tumors who relapsed on adjuvant docetaxel and cyclophosphamide plus 1 year of trastuzumab in a phase 2 study (Jones et al, Lancet Oncology 14: 1121, 2013). All patients’ tumors showed high-level HER2 amplification ratios by FISH (8.51-14.46) and were analyzed in parallel with a fourth clinically matched 2-year DFS patient’s tumor from the same trial with high HER2 gene amplification (FISH ratio 11.38). Methods Primary tumor genomic DNA analysis was performed from archival tissue by next generation sequencing (NGS) on the Illumina HiSeq 2500 platform in a CLIA certified laboratory. Tumors were screened for point mutations and copy number alterations (CNAs) by NGS using a targeted-whole exome 613 gene panel. CNAs detected by NGS were confirmed on a DNA microarray featuring high-density probe coverage of the same 613 genes on the targeted panel. CGH chromosome ratio plots were overlaid with algorithmically derived NGS copy number data to generate a map of the chromosomal genomic landscape for each patient’s tumor. Results High-level HER2 gene amplification status was confirmed, and co-amplified chromosome 17 genomic regions were detected in all three relapsed patients’ tumors. High-level HER2 amplification was also confirmed in the non-relapsed patient’s tumor but co-amplified regions were not detected on chromosome 17 or elsewhere in this patient’s tumor genome. Two of the relapsed ER negative tumors shared focal high-level CCNE1 gene amplifications on chromosome 19. High-level MAP3K3 gene amplification on chromosome 17 was detected in the one ER positive tumor from a relapsed patient. Focal PIK3CA gene amplifications were not identified in any of these tumors, but two tumors (one from the relapse group and one non-relapse) were positive for activating H1047R mutations. Conclusions Combined NGS and CGH analysis of HER2 positive early stage breast cancer can be performed in the clinical laboratory to reveal the tumor’s chromosomal genomic landscape. Combined with other test results, this tumor map can help identify patients at high-risk for relapse and reveal alternative predictive biomarkers of therapeutic response. Citation Format: Shelly Gunn, Chris McCaskill, Linda Daley, Agnes Puskas, Lina Asmar, Yunfei Wang, Stephen Jones. The chromosomal genomic landscape and targetable co-amplified genes in HER2 positive breast cancer patients who relapsed on an adjuvant trastuzumab chemotherapy trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-10-18.

Fluorescence induction characteristics have been studied in wild-type and atrazine-resistant mutant of Rliodobacter capsulatus. Fluorescence induction was found to be a useful technique to monitor the altered electron transfer in the atrazine-resistant mutants as well as in the different membrane fractions of wild-type R. capsulatus. In both cases, the proportion of the fast rise of variable fluorescence was increased indicating the enhancement of QA. I`n the mutant strain, the I50 value of triazine herbicide terbutryn was increased by 100-fold whereas the natural resistance of R. capsulatus against diuron was abolished by the mutation.