Combining metagenomics with metaproteomics and stable isotope probing reveals metabolic pathways used by a naturally occurring marine methylotroph

A variety of culture-independent techniques have been developed that can be used in conjunction with culture-dependent physiological and metabolic studies of key microbial organisms, in order to better understand how the activity of natural populations influences and regulates all major biogeochemical cycles. In this study, we combined DNA-stable isotope probing with metagenomics and metaproteomics to characterize an as yet uncultivated marine methylotroph that actively incorporated carbon from 13C-labeled methanol into biomass. By metagenomic sequencing of the heavy DNA, we retrieved virtually the whole genome of this bacterium and determined its metabolic potential. Through protein-stable isotope probing, the RuMP cycle was established as the main carbon assimilation pathway, and the classical methanol dehydrogenase-encoding gene mxaF, as well as three out of four identified xoxF homologues were found to be expressed. This proof-of-concept study is the first in which theculture-independent techniques of DNA- and protein-stable isotope probing have been used to characterize the metabolism of a naturally-ocurring Methylophaga-like bacterium in the marine environment (i.e. M. thiooxydans L4) and thus provides a powerful approach to access the genome and proteome of uncultivated microbes involved in key processes in the environment

Selective migration from deprived areas in Northern Ireland and the spatial distribution of inequalities: implications for monitoring health and inequalities in health

Much of the evidence suggesting that inequalities in health have been increasing over the last two decades has come from studies that compared the changes in relative health status of areas over time. Such studies ignore the movement of people between areas. This paper examines the population movement between small areas in Northern Ireland in the year prior to the 1991 census as well as the geographical distribution of migrants to Northern Ireland over the same period. It shows that deprived areas tended to become depopulated and that those who left these areas were the more affluent residents. While immigrants differed a little from the indigenous population, the overall effect of their distribution would be to maintain the geographical socio-economic status quo. The selective movement of people between areas would result in the distribution of health and ill-health becoming more polarized, i.e. produce a picture of widening inequalities between areas even though the distribution between individuals is unchanged. These processes suggest potential significant problems with the area-based approaches to monitoring health and inequalities in health.

The duty of loyalty in the cooperative law. A comparative study

Among the duties of the government bodies’ members of the different entities, which are connected to the good government, the duty of loyalty must be underlined. This duty, derived from the good-faith duty, obliges to act in the interest of the entity in case of conflict of interest, and to subordinate one’s own interests, except when there is an authorisation. Loyalty duty is applicable both to managers, who must manage the entity’s interest, and to partners, according to the common purpose derived from the company contract. This duty, at the same time, includes some particular rules, referred to transparency, remuneration, prohibition of competition, self-contracting... This essay compares the regulation of the duty of loyalty and its realizations in the different Cooperative Laws in Spain, both referring to the managers and to the partners, comparing this, at the same time, with the regulation of these aspects in companies’ general legislation, an in the Spanish Corporate Enterprises Act in particular, in order to obtain a general view of the issue, a necessary basis to go more deeply into it, and suggesting some preliminary conclusions or assessments.

New ways to break an old bond: the bacterial carbon-phosphorus hydrolases and their role in biogeochemical phosphorus cycling

Phosphonates are organophosphorus molecules that contain the highly stable C-P bond, rather than the more common, and more labile, C-O-P phosphate ester bond. They have ancient origins but their biosynthesis is widespread among more primitive organisms and their importance in the contemporary biosphere is increasingly recognized; for example phosphonate-P is believed to play a particularly significant role in the productivity of the oceans. The microbial degradation of phosphonates was originally thought to occur only under conditions of phosphate limitation, mediated exclusively by the poorly characterized C-P lyase multienzyme system, under Pho regulon control. However, more recent studies have demonstrated the Pho-independent mineralization by environmental bacteria of three of the most widely distributed biogenic phosphonates: 2-aminoethylphosphonic acid (ciliatine), phosphonoacetic acid, and 2-amino-3-phosphonopropionic acid (phosphonoalanine). The three phosphonohydrolases responsible have unique specificities and are members of separate enzyme superfamilies; their expression is regulated by distinct members of the LysR family of bacterial transcriptional regulators, for each of which the phosphonate substrate of the respective degradative operon serves as coinducer. Previously no organophosphorus compound was known to induce the enzymes required for its own degradation. Whole-genome and metagenome sequence analysis indicates that the genes encoding these newly described C-P hydrolases are distributed widely among prokaryotes. As they are able to function under conditions in which C-P lyases are inactive, the three enzymes may play a hitherto-unrecognized role in phosphonate breakdown in the environment and hence make a significant contribution to global biogeochemical P-cycling.

Ruthenium complexes of the 1,4-bis(diphenylphosphino)-1,3-butadiene-bridged diphosphine 1,2,3,4-Me-4-NUPHOS: Solvent-dependent interconversion of four- and six-electron donor coordination and transfer hydrogenation activity

In chloroform, [RuCl2(nbd)(py)(2)] (1) (nbd = norbornadiene; py = pyridine) reacts with 1,4-bis(diphenylphosphino)-1,2,3,4-tetramethyl-1,3-butadiene (1,2,3,4-Me-4-NUPHOS) to give the dimer [Ru2Cl3(eta(4)-1,2,3,4-Me-4-NUPHOS)(2)]Cl (2a), whereas, in THF [RuCl2(1,2,3,4-Me-4-NUPHOS)(PY)(2)] (3) is isolated as the sole product of reaction. Compound 2 exists as a 4:1 mixture of two noninterconverting isomers, the major with C, symmetry and the minor with either C, or C-2 symmetry. A single-crystal X-ray analysis of [Ru2Cl3 (eta(4)-1,2,3,4-Me-4-NUPHOS)(2)] [SbF6] (2b), the hexafluoroantimonate salt of 2a, revealed that the diphosphine coordinates in an unusual manner, as a eta(4)-six-electron donor, bonded through both P atoms and one of the double bonds of the butadiene tether. Compounds 2a and 3 react with 1,2-ethylenediamine (en) in THF to afford [RuCl2(1,2,3,4-Me-4-NUPHOS)(en)] (4), which rapidly dissociates a chloride ligand in chloroform to give [RuCl(eta(4)-1,2,3,4-Me-4-NUPHOS)(en)] [Cl] (5a). Complexes 4 and 5a cleanly and quantitatively interconvert in a solvent-dependent equilibrium, and in THF 5a readily adds chloride to displace the eta(2)-interaction and re-form 4. A single-crystal X-ray structure determination of [RuCl(eta(4)-1,2,3,4-Me-4-NUPHOS)(en)][ClO4] (5b) confirmed that the diphosphine coordinates in an eta(4)-manner as a facial six-electron donor with the eta(2)-coordinated double bond occupying the site trans to chloride. The eta(4)-bonding mode can be readily identified by the unusually high-field chemical shift associated with the phosphorus atom adjacent to the eta(2)-coordinated double bond. Complexes 2a, 2b, 4, and 5a form catalysts that are active for transfer hydrogenation of a range of ketones. In all cases, catalysts formed from precursors 2a and 2b are markedly more active than those formed from 4 and 5a.

Efficient modeling of novel uniplanar left-handed metamaterials

This paper presents an efficient. modeling technique for the derivation of the dispersion characteristics of novel uniplanar metallodielectric periodic structures. The analysis is based on the method of moments and an interpolation scheme, which significantly accelerates the computations. Triangular basis functions are used that allow for modeling of arbitrary shaped metallic elements. Based on this method, novel uniplanar left-handed (LH) metamaterials are proposed. Variations of the split rectangular-loop element printed on grounded dielectric substrate are demonstrated to possess LH propagation properties. Full-wave dispersion curves are presented. Based on the dual transmission-line concept, we study the distribution of the modal fields And the variation of series capacitance and shunt inductance for all the proposed elements. A verification of the left-handedness is presented by means of full-wave simulation of finite uniplanar arrays using commercial software (HFSS). The cell dimensions are a small fraction of the wavelength (approximately lambda/24) so that the structures can he considered as a homogeneous effective medium. The structures are simple, readily scalable to higher frequencies, and compatible with low-cost fabrication techniques.

Potential for phosphonoacetate utilization by marine bacteria in temperate coastal waters

Phosphonates are organic compounds that contain a C-P bond and are a poorly characterized component of the marine phosphorus cycle. They may represent a potential source of bioavailable phosphorus, particularly in oligotrophic conditions. This study has investigated the distribution of the phnA gene which encodes phosphonoacetate hydrolase, the enzyme that mineralizes phosphonoacetate. Using newly designed degenerate primers targeting the phnA gene we analysed the potential for phosphonoacetate utilization in DNA and cDNA libraries constructed from a phytoplankton bloom in the Western English Channel during July 2006. Total RNA was isolated and reverse transcribed and phosphonoacetate hydrolase (phnA) transcripts were PCR amplified from the cDNA with the degenerate primers, cloned and sequenced. Phylogenetic analysis demonstrated considerable diversity with 14 sequence types yielding five unique phnA protein groups. We also identified 28 phnA homologues in a 454-pyrosequencing metagenomic and metatranscriptomic study from a coastal marine mesocosm, indicating that > 3% of marine bacteria in this study contained phnA. phnA homologues were also present in a metagenomic fosmid library from this experiment. Finally, cultures of four isolates of potential coral pathogens belonging to the Vibrionaceae contained the phnA gene. In the laboratory, these isolates were able to grow with phosphonoacetate as sole P and C source. The fact that the capacity to utilize phosphonoacetate was evident in each of the three coastal environments suggests the potential for widespread utilization of this bioavailable P source.

Limits of life in hostile environments: no barriers to biosphere function?

Environments that are hostile to life are characterized by reduced microbial activity which results in poor soil- and plant-health, low biomass and biodiversity, and feeble ecosystem development. Whereas the functional biosphere may primarily be constrained by water activity (a w) the mechanism(s) by which this occurs have not been fully elucidated. Remarkably we found that, for diverse species of xerophilic fungi at a w values of = 0.72, water activity per se did not limit cellular function. We provide evidence that chaotropic activity determined their biotic window, and obtained mycelial growth at water activities as low as 0.647 (below that recorded for any microbial species) by addition of compounds that reduced the net chaotropicity. Unexpectedly we found that some fungi grew optimally under chaotropic conditions, providing evidence for a previously uncharacterized class of extremophilic microbes. Further studies to elucidate the way in which solute activities interact to determine the limits of life may lead to enhanced biotechnological processes, and increased productivity of agricultural and natural ecosystems in arid and semiarid regions.

Secreted cysteine proteases of the carcinogenic liver fluke, Opisthorchis viverrini: Regulation of cathepsin F activation by autocatalysis and trans-processing by cathepsin B

Opisthorchis viverrini is an important helminth pathogen of humans that is endemic in Thailand and Laos. Adult flukes reside within host bile ducts and feed on epithelial tissue and blood cells. Chronic opisthorchiasis is associated with severe hepatobiliary diseases such as cholangiocarcinoma. Here we report that adult O. viverrini secrete two major cysteine proteases: cathepsin F (Ov-CF-1) and cathepsin B1 (Ov-CB-1). Ov-CF-1 is secreted as an inactive zymogen that autocatalytically processes and activates to a mature enzyme at pH 4.5 via an intermolecular cleavage at the prosegment-mature domain junction. Ov-CB-1 is also secreted as a zymogen but, in contrast to Ov-CF-1, is fully active against peptide and macromolecular substrates despite retaining the N-terminal prosegment. The active Ov-CB-1 zymogen was capable of trans-activating Ov-CF-1 by proteolytic removal of its prosegment at pH 5.5, a pH at which the Ov-CF-1 zymogen cannot autocatalytically activate. Both cathepsins hydrolyse human haemoglobin but their combined action more efficiently degrades haemoglobin to smaller peptides than each enzyme alone. Ov-CF-1 degraded extracellular matrix proteins more effectively than Ov-CB-1 at physiological pH. We propose that Ov-CB-1 regulates Ov-CF-1 activity and that both enzymes work together to degrade host tissue contributing to the development of liver fluke-associated cholangiocarcinoma.

Burkholderia cenocepacia disrupts host cell actin cytoskeleton by inactivating Rac and Cdc42

Burkholderia cenocepacia, a member of the Burkholderia cepacia complex, is an opportunistic pathogen that causes devastating infections in patients with cystic fibrosis. The ability of B. cenocepacia to survive within host cells could contribute significantly to its virulence in immunocompromised patients. In this study, we explored the mechanisms that enable B. cenocepacia to survive inside macrophages. We found that B. cenocepacia disrupts the actin cytoskeleton of infected macrophages, drastically altering their morphology. Submembranous actin undergoes depolymerization, leading to cell retraction. The bacteria perturb actin architecture by inactivating Rho family GTPases, particularly Rac1 and Cdc42. GTPase inactivation follows internalization of viable B. cenocepacia and compromises phagocyte function: macropinocytosis and phagocytosis are markedly inhibited, likely impairing the microbicidal and antigen-presenting capability of infected macrophages. The type VI secretion system is essential for the bacteria to elicit these changes. This is the first report demonstrating inactivation of Rho family GTPases by a member of the B. cepacia complex.