Effects of DNase production, plasmid size, and restriction barriers on transformation of Vibrio cholerae by electroporation and osmotic shock

Attempts to transform wild type strains of V. cholerae with plasmid DNA by traditional osmotic shock methods were not successful. A mutant of V. cholerae that was deficient in extracellular DNase was transformed with plasmid DNA by osmotic shock, demonstrating directly that extracellular DNase is a major barrier to transformation of V. cholerae. Transformation of wild type and DNase-negative strains of V. cholerae was accomplished by electroporation. Efficiency of transformation by electroporation increased with field strength, decreased with plasmid size, and was relatively insensitive to changes in the electrolyte composition of the buffer as long as isotonic sucrose was present. Host-controlled modification/restriction systems also affected transformation efficiency in V. cholerae.     

Molecular dissection of the interaction between the small G proteins Rac1 and RhoA and protein kinase C-related kinase 1 (PRK1)

PRK1 is a serine/threonine kinase that belongs to the protein kinase C superfamily. It can be activated either by members of the Rho family of small G proteins, by proteolysis, or by interaction with lipids. Here we investigate the binding of PRK1 to RhoA and Rac1, two members of the Rho family. We demonstrate that PRK1 binds with a similar affinity to RhoA and Rac1. We present the solution structure of the second HR1 domain from the regulatory N-terminal region of PRK1, and we show that it forms an anti-parallel coiled-coil. In addition, we have used NMR to map the binding contacts of the HR1b domain with Rac1. These are compared with the contacts known to form between HR1a and RhoA. We have used mutagenesis to define the residues in Rac that are important for binding to HR1b. Surprisingly, as well as residues adjacent to Switch I, in Switch II, and in helix alpha5, it appears that the C-terminal stretch of basic amino acids in Rac is required for a high affinity interaction with HR1b.     

Developing pulmonary vasculopathy in systemic sclerosis, detected with non-invasive cardiopulmonary exercise testing

Background

Patients with systemic sclerosis (SSc) may develop exercise intolerance due to musculoskeletal involvement, restrictive lung disease, left ventricular dysfunction, or pulmonary vasculopathy (PV). The latter is particularly important since it may lead to lethal pulmonary arterial hypertension (PAH). We hypothesized that abnormalities during cardiopulmonary exercise testing (CPET) in patients with SSc can identify PV leading to overt PAH.

Methods

Thirty SSc patients from the Harbor-UCLA Rheumatology clinic, not clinically suspected of having significant pulmonary vascular disease, were referred for this prospective study. Resting pulmonary function and exercise gas exchange were assessed, including peakVO₂, anaerobic threshold (AT), heart rate- VO₂ relationship (O₂-pulse), exercise breathing reserve and parameters of ventilation-perfusion mismatching, as evidenced by elevated ventilatory equivalent for CO₂ (VE/VCO₂) and reduced end-tidal pCO₂ (P_ETCO₂) at the AT.

Results

Gas exchange patterns were abnormal in 16 pts with specific cardiopulmonary disease physiology: Eleven patients had findings consistent with PV, while five had findings consistent with left-ventricular dysfunction (LVD). Although both groups had low peak VO₂ and AT, a higher VE/VCO₂ at AT and decreasing P_ETCO₂ during early exercise distinguished PV from LVD.

Conclusions

Previously undiagnosed exercise impairments due to LVD or PV were common in our SSc patients. Cardiopulmonary exercise testing may help to differentiate and detect these disorders early in patients with SSc.     

Enhancement of Survival and Electricity Production in an Engineered Bacterium by Light-Driven Proton Pumping

Microorganisms can use complex photosystems or light-dependent proton pumps to generate membrane potential and/or reduce electron carriers to support growth. The discovery that proteorhodopsin is a light-dependent proton pump that can be expressed readily in recombinant bacteria enables development of new strategies to probe microbial physiology and to engineer microbes with new light-driven properties. Here, we describe functional expression of proteorhodopsin and light-induced changes in membrane potential in the bacterium Shewanella oneidensis strain MR-1. We report that there were significant increases in electrical current generation during illumination of electrochemical chambers containing S. oneidensis expressing proteorhodopsin. We present evidence that an engineered strain is able to consume lactate at an increased rate when it is illuminated, which is consistent with the hypothesis that proteorhodopsin activity enhances lactate uptake by increasing the proton motive force. Our results demonstrate that there is coupling of a light-driven process to electricity generation in a nonphotosynthetic engineered bacterium. Expression of proteorhodopsin also preserved the viability of the bacterium under nutrient-limited conditions, providing evidence that fulfillment of basic energy needs of organisms may explain the widespread distribution of proteorhodopsin in marine environments.Classic experiments in microbial bioenergetics used light-driven reactions from halobacterial bacteriorhodopsin or the photosynthetic reaction center to provide a temporary driving force for understanding transport and chemiosmotic coupling (6, 7, 19, 35). However, light-driven reactions have not been used in metabolic engineering to alter microbial physiology and production of chemicals. The recent discovery of proteorhodopsin (PR) in ocean microorganisms and the ease with which this membrane protein can be functionally expressed by recombinant bacteria have made possible many engineering strategies previously not available (1, 16). In this paper, we describe progress toward the goal of integrating light-driven reactions with biocatalysis.In contrast to the situation for established industrial microorganisms, such as Escherichia coli, our current understanding of less-studied algal and phototrophic bacteria may limit metabolic engineering strategies which require genetic manipulation. Metabolic engineering strategies using photosynthetic bacteria have focused largely on methods to increase hydrogen production, and improvements rely mainly on engineering of nitrogenase and hydrogenase to produce H₂. Algae appear to be suited to large-scale cultivation for lipid production, but so far little has been done to engineer these organisms (36). In principle, platform microbial hosts capable of producing a diverse range of products could be boosted by addition of light-driven processes from phototrophic metabolism.To demonstrate the feasibility of transferring a light-driven process into a nonphotosynthetic bacterium, we chose to study proteorhodopsin (PR) first because it is one of the simplest mechanisms for harnessing the energy from light. The proteorhodopsins are a group of transmembrane proteins that use the light-induced isomerization of retinal, the oxidative cleavage product of the carotenoid β-carotene, either to initiate signaling pathways or to catalyze the transfer of ions across cell membranes (8). PR was discovered by metagenomic analysis of marine samples (1) and is related to the well-studied bacteriorhodopsin of archaea (33) and rhodopsin (34), a eukaryotic light-sensing protein. The membrane potential generated by light-driven proton pumping by PR has been confirmed to drive ATP synthesis in a heterologous system (25). However, bacteria expressing heterologous PR were shown not to benefit from this pumping activity, as no significant increases in growth rates were observed (9). This led to the suggestion that PR may benefit the organism only under starvation conditions. In agreement with this hypothesis, Gomez-Consarnau et al. (10) have reported that the light-dependent growth rates of a marine flavobacterium that has a native PR are increased only when the organism is cultured under energy-limited conditions.Studies of both native and recombinant systems in which rhodopsins are expressed have generated light-dependent membrane potentials. In membrane vesicles isolated from a native host, the light-dependent membrane potential generated by bacteriorhodopsin provides the driving force for ATP synthesis (35) and uptake of leucine and glutamate (20, 22). More recently, studies of recombinant systems have coupled the membrane potential to other transport processes. In one example, the membrane potential-dependent export of specific toxic molecules increased when E. coli cells expressing both an archaeal rhodopsin and a specific efflux pump were exposed to light (17). In another experiment, starved E. coli cells expressing PR increased the swimming motion of their flagella when they were illuminated (44). Based upon measurements of flagellar motion as a function of light intensity and azide concentration, the proton motive force generated by PR was estimated to be −0.2 V, a value similar to the value for aerobic respiration in E. coli (42).As a nonphotosynthetic host for recombinant PR expression, we chose the dissimilatory metal-reducing bacterium Shewanella oneidensis strain MR-1, which is genetically tractable for engineering and is able to use a variety of terminal electron acceptors, including insoluble metal oxides (11, 30). Key to the ability of this bacterium to reduce metal oxides is a multicomponent extracellular respiratory pathway that transports electrons from menaquinol to cytochromes in the outer membrane. This pathway is composed of a cytoplasmic membrane tetraheme protein (CymA), a periplasmic decaheme protein (MtrA), an integral outer membrane protein (MtrB), and a decaheme lipoprotein (MtrC) that is associated with MtrB (14, 37, 40). The ability of S. oneidensis to reduce extracellular metal oxides has made it possible to harvest electrons from this organism by coupling it to an electrode which serves as the electron acceptor (21). The electron flow to the outer surface allows respiration rates to be measured directly by electrochemistry.In the current work, we introduced PR into an electricity-generating bacterium, S. oneidensis strain MR-1, and demonstrated that there was integration of a light-driven process into the metabolism of a previously nonphotosynthetic organism that resulted in a useful output. We show here that PR allows cells to survive for extended periods in stationary phase and that the presence of light results in an increase in electricity generation. A possible physiological model to explain these effects is discussed.     

BRCA1 Pathway Function in Basal-Like Breast Cancer Cells

Sporadic basal-like cancers (BLCs) are a common subtype of breast cancer that share multiple biological properties with BRCA1-mutated breast tumors. Despite being BRCA1^+/+, sporadic BLCs are widely viewed as phenocopies of BRCA1-mutated breast cancers, because they are hypothesized to manifest a BRCA1 functional defect or breakdown of a pathway(s) in which BRCA1 plays a major role. The role of BRCA1 in the repair of double-strand DNA breaks by homologous recombination (HR) is its best understood function and the function most often implicated in BRCA1 breast cancer suppression. Therefore, it is suspected that sporadic BLCs exhibit a defect in HR. To test this hypothesis, multiple DNA damage repair assays focused on several types of repair were performed on a group of cell lines classified as sporadic BLCs and on controls. The sporadic BLC cell lines failed to exhibit an overt HR defect. Rather, they exhibited defects in the repair of stalled replication forks, another BRCA1 function. These results provide insight into why clinical trials of poly(ADP-ribose) polymerase (PARP) inhibitors, which require an HR defect for efficacy, have been unsuccessful in sporadic BLCs, unlike cisplatin, which elicits DNA damage that requires stalled fork repair and has shown efficacy in sporadic BLCs.     

Comparative Phylogenetic Studies on Schistosoma japonicum and Its Snail Intermediate Host Oncomelania hupensis: Origins,Dispersal and Coevolution

Background

Schistosoma japonicum causes major public health problems in China and the Philippines; this parasite, which is transmitted by freshwater snails of the species Oncomelania hupensis, causes the disease intestinal schistosomiasis in humans and cattle. Researchers working on Schistosoma in Africa have described the relationship between the parasites and their snail intermediate hosts as coevolved or even as an evolutionary arms race. In the present study this hypothesis of coevolution is evaluated for S. japonicum and O. hupensis. The origins and radiation of the snails and the parasite across China, and the taxonomic validity of the sub-species of O. hupensis, are also assessed.

Methodology/Principal Findings

The findings provide no evidence for coevolution between S. japonicum and O. hupensis, and the phylogeographical analysis suggests a heterochronous radiation of the parasites and snails in response to different palaeogeographical and climatic triggers. The results are consistent with a hypothesis of East to West colonisation of China by Oncomelania with a re-invasion of Japan by O. hupensis from China. The Taiwan population of S. japonicum appears to be recently established in comparison with mainland Chinese populations.

Conclusions/Significance

The snail and parasite populations of the western mountain region of China (Yunnan and Sichuan) appear to have been isolated from Southeast Asian populations since the Pleistocene; this has implications for road and rail links being constructed in the region, which will breach biogeographical barriers between China and Southeast Asia. The results also have implications for the spread of S. japonicum. In the absence of coevolution, the parasite may more readily colonise new snail populations to which it is not locally adapted, or even new intermediate host species; this can facilitate its dispersal into new areas. Additional work is required to assess further the risk of spread of S. japonicum.     

Biochemical characterization of recombinant guaA-encoded guanosine monophosphate synthetase (EC 6.3.5.2) from Mycobacterium tuberculosis H37Rv strain

Administration of the current tuberculosis (TB) vaccine to newborns is not a reliable route for preventing TB in adults. The conversion of XMP to GMP is catalyzed by guaA-encoded GMP synthetase (GMPS), and deletions in the Shiguella flexneri guaBA operon led to an attenuated auxotrophic strain. Here we present the cloning, expression, and purification of recombinant guaA-encoded GMPS from Mycobacterium tuberculosis (MtGMPS). Mass spectrometry data, oligomeric state determination, steady-state kinetics, isothermal titration calorimetry (ITC), and multiple sequence alignment are also presented. The homodimeric MtGMPS catalyzes the conversion of XMP, MgATP, and glutamine into GMP, ADP, PP(i), and glutamate. XMP, NH(4)(+), and Mg(2+) displayed positive homotropic cooperativity, whereas ATP and glutamine displayed hyperbolic saturation curves. The activity of ATP pyrophosphatase domain is independent of glutamine amidotransferase domain, whereas the latter cannot catalyze hydrolysis of glutamine to NH(3) and glutamate in the absence of substrates. ITC data suggest random order of binding of substrates, and PP(i) is the last product released. Sequence comparison analysis showed conservation of both Cys-His-Glu catalytic triad of N-terminal Class I amidotransferase and of amino acid residues of the P-loop of the N-type ATP pyrophosphatase family.     

Radioprotective Effect of Aminothiol PrC-210 on Irradiated Inner Ear of Guinea Pig

Radiotherapy of individuals suffering with head & neck or brain tumors subserve the risk of sensorineural hearing loss. Here, we evaluated the protective effect of Aminothiol PrC-210 (3-(methyl-amino)-2-((methylamino)methyl)propane-1-thiol) on the irradiated inner ear of guinea pigs. An intra-peritoneal or intra-tympanic dose of PrC-210 was administered prior to receiving a dose of gamma radiation (3000 cGy) to each ear. Auditory Brainstem Responses (ABRs) were recorded one week and two weeks after the radiation and compared with the sham animal group. ABR thresholds of guinea pigs that received an intra-peritoneal dose of PrC-210 were significantly better compared to the non-treated, control animals at one week post-radiation. Morphologic analysis of the inner ear revealed significant inflammation and degeneration of the spiral ganglion in the irradiated animals not treated with PrC-210. In contrast, when treated with PrC-210 the radiation effect and injury to the spiral ganglion was significantly alleviated. PrC-210 had no apparent cytotoxic effect in vivo and did not affect the morphology or count of cochlear hair cells. These findings suggest that aminothiol PrC-210 attenuated radiation-induced cochlea damage for at least one week and protected hearing.     

The character and use of the Soros Hill Obsidian source,Antiparos (Greece)

This article details the geological and elemental character of the obsidian from the Soros Hill source on the Cycladic island of Antiparos, Greece. EDXRF was used to analyse 40 geological geo-referenced samples. The products are clearly chemically discriminated from those of the other Aegean sources, and those from the Carpathians and central Anatolia. While the obsidian is of excellent tool-making quality, the small size of its nodules seems to have made it a less attractive raw material, attested at only a handful of prehistoric sites in the central Cyclades.