Acetyl-Phosphate Is Not a Global Regulatory Bridge between Virulence and Central Metabolism in Borrelia burgdorferi

In B. burgdorferi, the Rrp2-RpoN-RpoS signaling cascade is a distinctive system that coordinates the expression of virulence factors required for successful transition between its arthropod vector and mammalian hosts. Rrp2 (BB0763), an RpoN specific response regulator, is essential to activate this regulatory pathway. Previous investigations have attempted to identify the phosphate donor of Rrp2, including the cognate histidine kinase, Hk2 (BB0764), non-cognate histidine kinases such as Hk1, CheA1, and CheA2, and small molecular weight P-donors such as carbamoyl-phosphate and acetyl-phosphate (AcP). In a report by Xu et al., exogenous sodium acetate led to increased expression of RpoS and OspC and it was hypothesized this effect was due to increased levels of AcP via the enzyme AckA (BB0622). Genome analyses identified only one pathway that could generate AcP in B. burgdorferi: the acetate/mevalonate pathway that synthesizes the lipid, undecaprenyl phosphate (C55-P, lipid I), which is essential for cell wall biogenesis. To assess the role of AcP in Rrp2-dependent regulation of RpoS and OspC, we used a unique selection strategy to generate mutants that lacked ackA (bb0622: acetate to AcP) or pta (bb0589: AcP to acetyl-CoA). These mutants have an absolute requirement for mevalonate and demonstrate that ackA and pta are required for cell viability. When the ΔackA or Δpta mutant was exposed to conditions (i.e., increased temperature or cell density) that up-regulate the expression of RpoS and OspC, normal induction of those proteins was observed. In addition, adding 20mM acetate or 20mM benzoate to the growth media of B. burgdorferi strain B31 ΔackA induced the expression of RpoS and OspC. These data suggest that AcP (generated by AckA) is not directly involved in modulating the Rrp2-RpoN-RpoS regulatory pathway and that exogenous acetate or benzoate are triggering an acid stress response in B. burgdorferi

Binary Quasars at High Redshift I: 24 New Quasar Pairs at z ~ 3-4

The clustering of quasars on small scales yields fundamental constraints on models of quasar evolution and the buildup of supermassive black holes. This paper describes the first systematic survey to discover high redshift binary quasars. Using color-selection and photometric redshift techniques, we searched 8142 deg^2 of SDSS imaging data for binary quasar candidates, and confirmed them with follow-up spectroscopy. Our sample of 27 high redshift binaries (24 of them new discoveries) at redshifts 2.9 < z < 4.3 with proper transverse separations 10 kpc < R_{\perp} < 650 kpc increases the number of such objects known by an order of magnitude. Eight members of this sample are very close pairs with R_{\perp} 3.5. The completeness and efficiency of our well-defined selection algorithm are quantified using simulated photometry and we find that our sample is ~ 50% complete. Our companion paper uses this knowledge to make the first measurement of the small scale clustering (R < 1 Mpc/h comoving) of high-redshift quasars. High redshift binaries constitute exponentially rare coincidences of two extreme (M >~ 10^9 Msun) supermassive black holes. At z ~ 4 there is about one close binary per 10 Gpc^3, thus these could be the highest sigma peaks, the analogs of superclusters, in the early Universe.Comment: Submitted to Ap

Tryptophan-Accelerated Electron Flow Through Proteins

Energy flow in biological structures often requires submillisecond charge transport over long molecular distances. Kinetics modeling suggests that charge-transfer rates can be greatly enhanced by multistep electron tunneling in which redox-active amino acid side chains act as intermediate donors or acceptors. We report transient optical and infrared spectroscopic experiments that quantify the extent to which an intervening tryptophan residue can facilitate electron transfer between distant metal redox centers in a mutant Pseudomonas aeruginosa azurin. CuI oxidation by a photoexcited ReI-diimine at position 124 on a histidine(124)-glycine(123)-tryptophan(122)-methionine(121) β strand occurs in a few nanoseconds, fully two orders of magnitude faster than documented for single-step electron tunneling at a 19 angstrom donor-acceptor distance

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The Drosophila melanogaster Genetic Reference Panel

A major challenge of biology is understanding the relationship between molecular genetic variation and variation in quantitative traits, including fitness. This relationship determines our ability to predict phenotypes from genotypes and to understand how evolutionary forces shape variation within and between species. Previous efforts to dissect the genotype&ndash;phenotype map were based on incomplete genotypic information. Here, we describe the Drosophila melanogaster Genetic Reference Panel (DGRP), a community resource for analysis of population genomics and quantitative traits. The DGRP consists of fully sequenced inbred lines derived from a natural population. Population genomic analyses reveal reduced polymorphism in centromeric autosomal regions and the X chromosome, evidence for positive and negative selection, and rapid evolution of the X chromosome. Many variants in novel genes, most at low frequency, are associated with quantitative traits and explain a large fraction of the phenotypic variance. The DGRP facilitates genotype&ndash;phenotype mapping using the power of Drosophila genetics

Weak Organic Acids Decrease Borrelia burgdorferi Cytoplasmic pH, Eliciting an Acid Stress Response and Impacting RpoN- and RpoS-Dependent Gene Expression

The spirochete Borrelia burgdorferi survives in its tick vector, Ixodes scapularis, or within various hosts. To transition between and survive in these distinct niches, B. burgdorferi changes its gene expression in response to environmental cues, both biochemical and physiological. Exposure of B. burgdorferi to weak monocarboxylic organic acids, including those detected in the blood meal of fed ticks, decreased the cytoplasmic pH of B. burgdorferi in vitro. A decrease in the cytoplasmic pH induced the expression of genes encoding enzymes that have been shown to restore pH homeostasis in other bacteria. These include putative coupled proton/cation exchangers, a putative Na+/H+ antiporter, a neutralizing buffer transporter, an amino acid deaminase and a proton exporting vacuolar-type VoV1 ATPase. Data presented in this report suggested that the acid stress response triggered the expression of RpoN- and RpoS-dependent genes including important virulence factors such as outer surface protein C (OspC), BBA66, and some BosR (Borreliaoxidative stress regulator)-dependent genes. Because the expression of virulence factors, like OspC, are so tightly connected by RpoS to general cellular stress responses and cell physiology, it is difficult to separate transmission-promoting conditions in what is clearly a multifactorial and complex regulatory web

Genomic and phenotypic characterization of <i>Borrelia afzelii</i> BO23 and <i>Borrelia garinii</i> CIP 103362

<div><p>In recent years, the number of Lyme disease or borreliosis cases in Eurasia has been dramatically increasing. This tick-borne disease is caused by <i>Borrelia burgdorferi</i> sensu lato, which includes <i>B</i>. <i>burgdorferi</i> sensu stricto, the main species found in North America, and <i>B</i>. <i>afzelii</i> and <i>B</i>. <i>garinii</i>, which are primarily responsible for the disease in Eurasia. Currently, research on Lyme disease has focused mainly on <i>B</i>. <i>burgdorferi</i> while <i>B</i>. <i>afzelii</i> and <i>B</i>. <i>garinii</i>, which cause disease with distinctly different symptoms, are less studied. The purpose of this study is to evaluate <i>B</i>. <i>afzelii</i> BO23 and <i>B</i>. <i>garinii</i> CIP 103362 as model organisms to study Eurasian Lyme disease. To begin our analyses, we sequenced, annotated the chromosomes of both species and compared them to <i>B</i>. <i>burgdorferi</i> strain B31. We also assayed shuttle vector, pBSV2, for transformation efficacy and demonstrated that these strains can be cultured on solid media. In addition, we characterized how physicochemical parameters (e.g., oxygen, osmolarity, oxidative stress) affect both growth and motility of the bacteria. Finally, we describe each strain’s antibiotic susceptibility and accessed their ability to infect mice. In conclusion, <i>B</i>. <i>afzelii</i> BO23 was more practical for <i>in vitro</i> and <i>in vivo</i> studies than <i>B</i>. <i>garinii</i> CIP 103362.</p></div

Immunoblot of <i>B</i>. <i>burgdorferi</i> B31-A3, <i>B</i>. <i>garinii</i> CIP 103362 and <i>B</i>. <i>afzelii</i> BO23 strains incubated with mouse sera.

<p><i>Borrelia</i> strains were grown in BSK-II to mid-log phase and cell lysates were analyzed by immunoblotting. Cell lysates were incubated with serum obtained from mice either before (Pre-bleed, PB) or 6 weeks after needle inoculation (Post-infection, PI) with <i>B</i>. <i>burgdorferi</i> (lanes 1 to 3), <i>B</i>. <i>garinii</i> (lanes 4 to 6) and <i>B</i>. <i>afzelii</i> (lanes 7 to 9).</p

Comparison of the genome of the three <i>Borrelia</i> strains.

<p>Comparison of the genome of the three <i>Borrelia</i> strains.</p