Use of the Meganuclease I-SceI of Saccharomyces cerevisiae to select for gene deletions in actinomycetes

The search for new natural products is leading to the isolation of novel actinomycete species, many of which will ultimately require genetic analysis. Some of these isolates will likely exhibit low intrinsic frequencies of homologous recombination and fail to sporulate under laboratory conditions, exacerbating the construction of targeted gene deletions and replacements in genetically uncharacterised strains. To facilitate the genetic manipulation of such species, we have developed an efficient method to generate gene or gene cluster deletions in actinomycetes by homologous recombination that does not introduce any other changes to the targeted organism's genome. We have synthesised a codon optimised I-SceI gene for expression in actinomycetes that results in the production of the yeast I-SceI homing endonuclease which produces double strand breaks at a unique introduced 18 base pair recognition sequence. Only those genomes that undergo homologous recombination survive, providing a powerful selection for recombinants, approximately half of which possess the desired mutant genotype. To demonstrate the efficacy and efficiency of the system, we deleted part of the gene cluster for the red-pigmented undecylprodiginine complex of compounds in Streptomyces coelicolor M1141. We believe that the system we have developed will be broadly applicable across a wide range of actinomycetes

Two-component signal transduction in Corynebacterium glutamicum and other corynebacteria: on the way towards stimuli and targets

In bacteria, adaptation to changing environmental conditions is often mediated by two-component signal transduction systems. In the prototypical case, a specific stimulus is sensed by a membrane-bound histidine kinase and triggers autophosphorylation of a histidine residue. Subsequently, the phosphoryl group is transferred to an aspartate residue of the cognate response regulator, which then becomes active and mediates a specific response, usually by activating and/or repressing a set of target genes. In this review, we summarize the current knowledge on two-component signal transduction in Corynebacterium glutamicum. This Gram-positive soil bacterium is used for the large-scale biotechnological production of amino acids and can also be applied for the synthesis of a wide variety of other products, such as organic acids, biofuels, or proteins. Therefore, C. glutamicum has become an important model organism in industrial biotechnology and in systems biology. The type strain ATCC 13032 possesses 13 two-component systems and the role of five has been elucidated in recent years. They are involved in citrate utilization (CitAB), osmoregulation and cell wall homeostasis (MtrAB), adaptation to phosphate starvation (PhoSR), adaptation to copper stress (CopSR), and heme homeostasis (HrrSA). As C. glutamicum does not only face changing conditions in its natural environment, but also during cultivation in industrial bioreactors of up to 500 m3 volume, adaptability can also be crucial for good performance in biotechnological production processes. Detailed knowledge on two-component signal transduction and regulatory networks therefore will contribute to both the application and the systemic understanding of C. glutamicum and related species

A Kinetic Model of Dopamine- and Calcium-Dependent Striatal Synaptic Plasticity

Corticostriatal synapse plasticity of medium spiny neurons is regulated by glutamate input from the cortex and dopamine input from the substantia nigra. While cortical stimulation alone results in long-term depression (LTD), the combination with dopamine switches LTD to long-term potentiation (LTP), which is known as dopamine-dependent plasticity. LTP is also induced by cortical stimulation in magnesium-free solution, which leads to massive calcium influx through NMDA-type receptors and is regarded as calcium-dependent plasticity. Signaling cascades in the corticostriatal spines are currently under investigation. However, because of the existence of multiple excitatory and inhibitory pathways with loops, the mechanisms regulating the two types of plasticity remain poorly understood. A signaling pathway model of spines that express D1-type dopamine receptors was constructed to analyze the dynamic mechanisms of dopamine- and calcium-dependent plasticity. The model incorporated all major signaling molecules, including dopamine- and cyclic AMP-regulated phosphoprotein with a molecular weight of 32 kDa (DARPP32), as well as AMPA receptor trafficking in the post-synaptic membrane. Simulations with dopamine and calcium inputs reproduced dopamine- and calcium-dependent plasticity. Further in silico experiments revealed that the positive feedback loop consisted of protein kinase A (PKA), protein phosphatase 2A (PP2A), and the phosphorylation site at threonine 75 of DARPP-32 (Thr75) served as the major switch for inducing LTD and LTP. Calcium input modulated this loop through the PP2B (phosphatase 2B)-CK1 (casein kinase 1)-Cdk5 (cyclin-dependent kinase 5)-Thr75 pathway and PP2A, whereas calcium and dopamine input activated the loop via PKA activation by cyclic AMP (cAMP). The positive feedback loop displayed robust bi-stable responses following changes in the reaction parameters. Increased basal dopamine levels disrupted this dopamine-dependent plasticity. The present model elucidated the mechanisms involved in bidirectional regulation of corticostriatal synapses and will allow for further exploration into causes and therapies for dysfunctions such as drug addiction

Two Coregulated Efflux Transporters Modulate Intracellular Heme and Protoporphyrin IX Availability in Streptococcus agalactiae

Streptococcus agalactiae is a major neonatal pathogen whose infectious route involves septicemia. This pathogen does not synthesize heme, but scavenges it from blood to activate a respiration metabolism, which increases bacterial cell density and is required for full virulence. Factors that regulate heme pools in S. agalactiae are unknown. Here we report that one main strategy of heme and protoporphyrin IX (PPIX) homeostasis in S. agalactiae is based on a regulated system of efflux using two newly characterized operons, gbs1753 gbs1752 (called pefA pefB), and gbs1402 gbs1401 gbs1400 (called pefR pefC pefD), where pef stands for ‘porphyrin-regulated efflux’. In vitro and in vivo data show that PefR, a MarR-superfamily protein, is a repressor of both operons. Heme or PPIX both alleviate PefR-mediated repression. We show that bacteria inactivated for both Pef efflux systems display accrued sensitivity to these porphyrins, and give evidence that they accumulate intracellularly. The ΔpefR mutant, in which both pef operons are up-regulated, is defective for heme-dependent respiration, and attenuated for virulence. We conclude that this new efflux regulon controls intracellular heme and PPIX availability in S. agalactiae, and is needed for its capacity to undergo respiration metabolism, and to infect the host

The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence

Trost E, Ott L, Schneider J, et al. The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence. BMC Genomics. 2010;11(1): 728

Somatostatin Receptor 1 and 5 Double Knockout Mice Mimic Neurochemical Changes of Huntington's Disease Transgenic Mice

Selective degeneration of medium spiny neurons and preservation of medium sized aspiny interneurons in striatum has been implicated in excitotoxicity and pathophysiology of Huntington's disease (HD). However, the molecular mechanism for the selective sparing of medium sized aspiny neurons and vulnerability of projection neurons is still elusive. The pathological characteristic of HD is an extensive reduction of the striatal mass, affecting caudate putamen. Somatostatin (SST) positive neurons are selectively spared in HD and Quinolinic acid/N-methyl-D-aspartic acid induced excitotoxicity, mimic the model of HD. SST plays neuroprotective role in excitotoxicity and the biological effects of SST are mediated by five somatostatin receptor subtypes (SSTR1-5). and R6/2 mice. Conversely, the expression of somatostatin receptor subtypes, enkephalin and phosphatidylinositol 3-kinases were strain specific. SSTR1/5 appears to be important in regulating NMDARs, DARPP-32 and signaling molecules in similar fashion as seen in HD transgenic mice.This is the first comprehensive description of disease related changes upon ablation of G- protein coupled receptor gene. Our results indicate that SST and SSTRs might play an important role in regulation of neurodegeneration and targeting this pathway can provide a novel insight in understanding the pathophysiology of Huntington's disease

A simulation model approach to analysis of the business case for eliminating health care disparities

<p>Abstract</p> <p>Background</p> <p>Purchasers can play an important role in eliminating racial and ethnic disparities in health care. A need exists to develop a compelling "business case" from the employer perspective to put, and keep, the issue of racial/ethnic disparities in health care on the quality improvement agenda for health plans and providers.</p> <p>Methods</p> <p>To illustrate a method for calculating an employer business case for disparity reduction and to compare the business case in two clinical areas, we conducted analyses of the direct (medical care costs paid by employers) and indirect (absenteeism, productivity) effects of eliminating known racial/ethnic disparities in mammography screening and appropriate medication use for patients with asthma. We used Markov simulation models to estimate the consequences, for defined populations of African-American employees or health plan members, of a 10% increase in HEDIS mammography rates or a 10% increase in appropriate medication use among either adults or children/adolescents with asthma.</p> <p>Results</p> <p>The savings per employed African-American woman aged 50-65 associated with a 10% increase in HEDIS mammography rate, from direct medical expenses and indirect costs (absenteeism, productivity) combined, was $50. The findings for asthma were more favorable from an employer point of view at approximately$1,660 per person if raising medication adherence rates in African-American employees or dependents by 10%.</p> <p>Conclusions</p> <p>For the employer business case, both clinical scenarios modeled showed positive results. There is a greater potential financial gain related to eliminating a disparity in asthma medications than there is for eliminating a disparity in mammography rates.</p

Crystallization and preliminary X-ray analysis of AbsC, a novel regulator of antibiotic production in Streptomyces coelicolor.

A novel regulator of antibiotic production in S. coelicolor, AbsC, has been crystallized in space group P212121. X-ray data to 2.25 Å resolution were collected on station PX 14.1 at Daresbury