A majority of Rhodobacter sphaeroides promoters lack a crucial RNA polymerase recognition feature, enabling coordinated transcription activation.

Using an in vitro transcription system with purified RNA polymerase (RNAP) to investigate rRNA synthesis in the photoheterotrophic α-proteobacterium Rhodobacter sphaeroides, we identified a surprising feature of promoters recognized by the major holoenzyme. Transcription from R. sphaeroides rRNA promoters was unexpectedly weak, correlating with absence of -7T, the very highly conserved thymine found at the last position in -10 elements of promoters in most bacterial species. Thymine substitutions for adenine at position -7 in the three rRNA promoters strongly increased intrinsic promoter activity, indicating that R. sphaeroides RNAP can utilize -7T when present. rRNA promoters were activated by purified R. sphaeroides CarD, a transcription factor found in many bacterial species but not in ß- and γ-proteobacteria. Overall, CarD increased the activity of 15 of 16 native R. sphaeroides promoters tested in vitro that lacked -7T, whereas it had no effect on three of the four native promoters that contained -7T. Genome-wide bioinformatic analysis of promoters from R. sphaeroides and two other α-proteobacterial species indicated that 30 to 43% contained -7T, whereas 90 to 99% of promoters from non-α-proteobacteria contained -7T. Thus, promoters lacking -7T appear to be widespread in α-proteobacteria and may have evolved away from consensus to enable their coordinated regulation by transcription factors like CarD. We observed a strong reduction in R. sphaeroides CarD levels when cells enter stationary phase, suggesting that reduced activation by CarD may contribute to inhibition of rRNA transcription when cells enter stationary phase, the stage of growth when bacterial ribosome synthesis declines.

Genome-Wide Identification of Transcription Start Sites in Two Alphaproteobacteria, Rhodobacter sphaeroides 2.4.1 and Novosphingobium aromaticivorans DSM 12444.

Here, we report the genome-wide identification of transcription start sites (TSSs) from two Alphaproteobacteria grown under conditions that result in significant changes in gene expression. TSSs that were identified as present in one condition or both will be an important resource for future studies of these, and possibly other, Alphaproteobacteria.

The NtrYX Two-Component System Regulates the Bacterial Cell Envelope.

Activity of the NtrYX two-component system has been associated with important processes in diverse bacteria, ranging from symbiosis to nitrogen and energy metabolism. In the facultative alphaproteobacterium Rhodobacter sphaeroides, loss of the two-component system NtrYX results in increased lipid production and sensitivity to some known cell envelope-active compounds. In this study, we show that NtrYX directly controls multiple properties of the cell envelope. We find that the response regulator NtrX binds upstream of cell envelope genes, including those involved in peptidoglycan biosynthesis and modification and in cell division. We show that loss of NtrYX impacts the cellular levels of peptidoglycan precursors and lipopolysaccharide and alters cell envelope structure, increasing cell length and the thickness of the periplasm. Cell envelope function is also disrupted in the absence of NtrYX, resulting in increased outer membrane permeability. Based on the properties of R. sphaeroides cells lacking NtrYX and the target genes under direct control of this two-component system, we propose that NtrYX plays a previously undescribed, and potentially conserved, role in the assembly, structure, and function of the cell envelope in a variety of bacteria.IMPORTANCE The bacterial cell envelope provides many important functions. It protects cells from harsh environments, serves as a selective permeability barrier, houses bioenergetic functions, defines sensitivity to antibacterial agents, and plays a crucial role in biofilm formation, symbiosis, and virulence. Despite the important roles of this cellular compartment, we lack a detailed understanding of the biosynthesis and remodeling of the cell envelope. Here, we report that the R. sphaeroides two-component signaling system NtrYX is a previously undescribed regulator of cell envelope processes, providing evidence that it is directly involved in controlling transcription of genes involved in cell envelope assembly, structure, and function in this and possibly other bacteria. Thus, our data report on a newly discovered process used by bacteria to assemble and remodel the cell envelope.

Mutations That Alter the Bacterial Cell Envelope Increase Lipid Production.

Lipids from microbes offer a promising source of renewable alternatives to petroleum-derived compounds. In particular, oleaginous microbes are of interest because they accumulate a large fraction of their biomass as lipids. In this study, we analyzed genetic changes that alter lipid accumulation in Rhodobacter sphaeroides By screening an R. sphaeroides Tn5 mutant library for insertions that increased fatty acid content, we identified 10 high-lipid (HL) mutants for further characterization. These HL mutants exhibited increased sensitivity to drugs that target the bacterial cell envelope and changes in shape, and some had the ability to secrete lipids, with two HL mutants accumulating ~60% of their total lipids extracellularly. When one of the highest-lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to that of oleaginous microbes, with the majority of the lipids secreted into the medium. Based on the properties of these HL mutants, we conclude that alterations of the cell envelope are a previously unreported approach to increase microbial lipid production. We also propose that this approach may be combined with knowledge about biosynthetic pathways, in this or other microbes, to increase production of lipids and other chemicals.IMPORTANCE This paper reports on experiments to understand how to increase microbial lipid production. Microbial lipids are often cited as one renewable replacement for petroleum-based fuels and chemicals, but strategies to increase the yield of these compounds are needed to achieve this goal. While lipid biosynthesis is often well understood, increasing yields of these compounds to industrially relevant levels is a challenge, especially since genetic, synthetic biology, or engineering approaches are not feasible in many microbes. We show that altering the bacterial cell envelope can be used to increase microbial lipid production. We also find that the utility of some of these alterations can be enhanced by growing cells in bioreactor configurations that can be used industrially. We propose that our findings can inform current and future efforts to increase production of microbial lipids, other fuels, or chemicals that are currently derived from petroleum.

Oxygen-dependent regulation of bacterial lipid production.

UNLABELLED: Understanding the mechanisms of lipid accumulation in microorganisms is important for several reasons. In addition to providing insight into assembly of biological membranes, lipid accumulation has important applications in the production of renewable fuels and chemicals. The photosynthetic bacterium Rhodobacter sphaeroides is an attractive organism to study lipid accumulation, as it has the ability to increase membrane production at low O2 tensions. Under these conditions, R. sphaeroides develops invaginations of the cytoplasmic membrane to increase its membrane surface area for housing of the membrane-bound components of its photosynthetic apparatus. Here we use fatty acid levels as a reporter of membrane lipid content. We show that, under low-O2 and anaerobic conditions, the total fatty acid content per cell increases 3-fold. We also find that the increases in the amount of fatty acid and photosynthetic pigment per cell are correlated as O2 tensions or light intensity are changed. To ask if lipid and pigment accumulation were genetically separable, we analyzed strains with mutations in known photosynthetic regulatory pathways. While a strain lacking AppA failed to induce photosynthetic pigment-protein complex accumulation, it increased fatty acid content under low-O2 conditions. We also found that an intact PrrBA pathway is required for low-O2-induced fatty acid accumulation. Our findings suggest a previously unknown role of R. sphaeroides transcriptional regulators in increasing fatty acid and phospholipid accumulation in response to decreased O2 tension. IMPORTANCE: Lipids serve important functions in living systems, either as structural components of membranes or as a form of carbon storage. Understanding the mechanisms of lipid accumulation in microorganisms is important for providing insight into the assembly of biological membranes and additionally has important applications in the production of renewable fuels and chemicals. In this study, we investigate the ability of Rhodobacter sphaeroides to increase membrane production at low O2 tensions in order to house its photosynthetic apparatus. We demonstrate that this bacterium has a mechanism to increase lipid content in response to decreased O2 tension and identify a transcription factor necessary for this response. This is significant because it identifies a transcriptional regulatory pathway that can increase microbial lipid content.

Curation accuracy of model organism databases.

Manual extraction of information from the biomedical literature-or biocuration-is the central methodology used to construct many biological databases. For example, the UniProt protein database, the EcoCyc Escherichia coli database and the Candida Genome Database (CGD) are all based on biocuration. Biological databases are used extensively by life science researchers, as online encyclopedias, as aids in the interpretation of new experimental data and as golden standards for the development of new bioinformatics algorithms. Although manual curation has been assumed to be highly accurate, we are aware of only one previous study of biocuration accuracy. We assessed the accuracy of EcoCyc and CGD by manually selecting curated assertions within randomly chosen EcoCyc and CGD gene pages and by then validating that the data found in the referenced publications supported those assertions. A database assertion is considered to be in error if that assertion could not be found in the publication cited for that assertion. We identified 10 errors in the 633 facts that we validated across the two databases, for an overall error rate of 1.58%, and individual error rates of 1.82% for CGD and 1.40% for EcoCyc. These data suggest that manual curation of the experimental literature by Ph.D-level scientists is highly accurate. Database URL: http://ecocyc.org/, http://www.candidagenome.org//

A Rhodobacter sphaeroides protein mechanistically similar to Escherichia coli DksA regulates photosynthetic growth.

ABSTRACT DksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the "stringent response" to nutrient starvation in the gammaproteobacterium Escherichia coli and for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly related Rhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length to E. coli DksA but lacks the Zn finger motif of the E. coli DksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of an E. coli strain lacking the dksA gene and modulates transcription in vitro with E. coli RNA polymerase (RNAP) similarly to E. coli DksA. RSP2654 reduces RNAP-promoter complex stability in vitro with RNAPs from E. coli or R. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity to E. coli DksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsp has distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp. IMPORTANCE The role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis in Rhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

SIX1 induces lymphangiogenesis and metastasis via upregulation of VEGF-C in mouse models of breast cancer.

An association between lymph node metastasis and poor prognosis in breast cancer was observed decades ago. However, the mechanisms by which tumor cells infiltrate the lymphatic system are not completely understood. Recently, it has been proposed that the lymphatic system has an active role in metastatic dissemination and that tumor-secreted growth factors stimulate lymphangiogenesis. We therefore investigated whether SIX1, a homeodomain-containing transcription factor previously associated in breast cancer with lymph node positivity, was involved in lymphangiogenesis and lymphatic metastasis. In a model in which human breast cancer cells were injected into immune-compromised mice, we found that SIX1 expression promoted peritumoral and intratumoral lymphangiogenesis, lymphatic invasion, and distant metastasis of breast cancer cells. SIX1 induced transcription of the prolymphangiogenic factor VEGF-C, and this was required for lymphangiogenesis and lymphatic metastasis. Using a mouse mammary carcinoma model, we found that VEGF-C was not sufficient to mediate all the metastatic effects of SIX1, indicating that SIX1 acts through additional, VEGF-C-independent pathways. Finally, we verified the clinical significance of this prometastatic SIX1/VEGF-C axis by demonstrating coexpression of SIX1 and VEGF-C in human breast cancer. These data define a critical role for SIX1 in lymphatic dissemination of breast cancer cells, providing a direct mechanistic explanation for how VEGF-C expression is upregulated in breast cancer, resulting in lymphangiogenesis and metastasis.