Abundance and phylogenetic distribution of eight key enzymes of the phosphorus biogeochemical cycle in grassland soils.

Grasslands are one of the most diverse and widely distributed biomes on the Earth's surface. Nutrient cycling is one of the main ecosystem services provided by grasslands. The organic fraction of phosphorus (P) represents over half of the total P in soil and is a valuable reservoir. Soil microorganisms, involved in the P cycle, mediate organic P release through three enzyme families: alkaline phosphatases, nonspecific acid phosphatases, and phytases (P-enzymes). This study aimed, through a metagenomic approach, to assess the abundance and phylogenetic distribution of prokaryotic P-enzymes in a wide distribution of grass biomes across the globe and how they are related with environmental variables. To generate a functional perspective of phosphorus cycling, 43 soil metagenomes from 17 sites/projects representing different environmental conditions were examined for eight key P-enzymes. Multivariate analyses showed that Tmax, pH and evapotranspiration were highly associated with P-enzymes abundance and diversity. In addition, they tend to respond in a correlated manner to these variables suggesting an intricate relationship of abundance and diversity between them. On the other hand, their association with the general functional profiles was more idiosyncratic

The Flagellar Regulator fliT Represses Salmonella Pathogenicity Island 1 through flhDC and fliZ

Salmonella pathogenicity island 1 (SPI1), comprising a type III section system that translocates effector proteins into host cells, is essential for the enteric pathogen Salmonella to penetrate the intestinal epithelium and subsequently to cause disease. Using random transposon mutagenesis, we found that a Tn10 disruption in the flagellar fliDST operon induced SPI1 expression when the strain was grown under conditions designed to repress SPI1, by mimicking the environment of the large intestine through the use of the intestinal fatty acid butyrate. Our genetic studies showed that only fliT within this operon was required for this effect, and that exogenous over-expression of fliT alone significantly reduced the expression of SPI1 genes, including the invasion regulator hilA and the sipBCDA operon, encoding type III section system effector proteins, and Salmonella invasion of cultured epithelial cells. fliT has been known to inhibit the flagellar machinery through repression of the flagellar master regulator flhDC. We found that the repressive effect of fliT on invasion genes was completely abolished in the absence of flhDC or fliZ, the latter previously shown to induce SPI1, indicating that this regulatory pathway is required for invasion control by fliT. Although this flhDC-fliZ pathway was necessary for fliT to negatively control invasion genes, fliZ was not essential for the repressive effect of fliT on motility, placing fliT high in the regulatory cascade for both invasion and motility

Salmonella prevalence serotypes and patterns of antimicrobial resistance in cohorts of nursery and finishing pigs

Salmonella are now being monitored by the Food Safety Inspection Service of the USDA for purposes of process control in slaughter plants, and as a sentinel organism for emerging antimicrobial resistance (I). Public concerns about the emergence of foodborne bacteria with multi-resistant phenotypes, and about the use of antimicrobials in food animals, are increasing. Despite considerable research, the epidemiology of asymptomatic Salmonella in pigs is poorly understood (2). The purpose of this paper is to examine the association between nursery prevalence of Salmonella and finisher prevalence, and to describe patterns of antimicrobial use and antimicrobial resistant Salmonella in modern swine rearing facilities

Determinants of the voltage dependence of G protein modulation within calcium channel β subunits

CaVβ subunits of voltage-gated calcium channels contain two conserved domains, a src-homology-3 (SH3) domain and a guanylate kinase-like (GK) domain with an intervening HOOK domain. We have shown in a previous study that, although Gβγ-mediated inhibitory modulation of CaV2.2 channels did not require the interaction of a CaVβ subunit with the CaVα1 subunit, when such interaction was prevented by a mutation in the α1 subunit, G protein modulation could not be removed by a large depolarization and showed voltage-independent properties (Leroy et al., J Neurosci 25:6984–6996, 2005). In this study, we have investigated the ability of mutant and truncated CaVβ subunits to support voltage-dependent G protein modulation in order to determine the minimal domain of the CaVβ subunit that is required for this process. We have coexpressed the CaVβ subunit constructs with CaV2.2 and α2δ-2, studied modulation by the activation of the dopamine D2 receptor, and also examined basal tonic modulation. Our main finding is that the CaVβ subunit GK domains, from either β1b or β2, are sufficient to restore voltage dependence to G protein modulation. We also found that the removal of the variable HOOK region from β2a promotes tonic voltage-dependent G protein modulation. We propose that the absence of the HOOK region enhances Gβγ binding affinity, leading to greater tonic modulation by basal levels of Gβγ. This tonic modulation requires the presence of an SH3 domain, as tonic modulation is not supported by any of the CaVβ subunit GK domains alone

Engineering the Salmonella type III secretion system to export spider silk monomers

The type III secretion system (T3SS) exports proteins from the cytoplasm, through both the inner and outer membranes, to the external environment. Here, a system is constructed to harness the T3SS encoded within Salmonella Pathogeneity Island 1 to export proteins of biotechnological interest. The system is composed of an operon containing the target protein fused to an N-terminal secretion tag and its cognate chaperone. Transcription is controlled by a genetic circuit that only turns on when the cell is actively secreting protein. The system is refined using a small human protein (DH domain) and demonstrated by exporting three silk monomers (ADF-1, -2, and -3), representative of different types of spider silk. Synthetic genes encoding silk monomers were designed to enhance genetic stability and codon usage, constructed by automated DNA synthesis, and cloned into the secretion control system. Secretion rates up to 1.8 mg l−1 h−1 are demonstrated with up to 14% of expressed protein secreted. This work introduces new parts to control protein secretion in Gram-negative bacteria, which will be broadly applicable to problems in biotechnology

BarA-UvrY Two-Component System Regulates Virulence of Uropathogenic E. coli CFT073

Uropathogenic Escherichia coli (UPEC), a member of extraintestinal pathogenic E. coli, cause ∼80% of community-acquired urinary tract infections (UTI) in humans. UPEC initiates its colonization in epithelial cells lining the urinary tract with a complicated life cycle, replicating and persisting in intracellular and extracellular niches. Consequently, UPEC causes cystitis and more severe form of pyelonephritis. To further understand the virulence characteristics of UPEC, we investigated the roles of BarA-UvrY two-component system (TCS) in regulating UPEC virulence. Our results showed that mutation of BarA-UvrY TCS significantly decreased the virulence of UPEC CFT073, as assessed by mouse urinary tract infection, chicken embryo killing assay, and cytotoxicity assay on human kidney and uroepithelial cell lines. Furthermore, mutation of either barA or uvrY gene reduced the production of hemolysin, lipopolysaccharide (LPS), proinflammatory cytokines (TNF-α and IL-6) and chemokine (IL-8). The virulence phenotype was restored similar to that of wild-type by complementation of either barA or uvrY gene in trans. In addition, we discussed a possible link between the BarA-UvrY TCS and CsrA in positively and negatively controlling virulence in UPEC. Overall, this study provides the evidences for BarA-UvrY TCS regulates the virulence of UPEC CFT073 and may point to mechanisms by which virulence regulations are observed in different ways may control the long-term survival of UPEC in the urinary tract

Characterization of the role of ribonucleases in Salmonella small RNA decay

In pathogenic bacteria, a large number of sRNAs coordinate adaptation to stress and expression of virulence genes. To better understand the turnover of regulatory sRNAs in the model pathogen, Salmonella typhimurium, we have constructed mutants for several ribonucleases (RNase E, RNase G, RNase III, PNPase) and Poly(A) Polymerase I. The expression profiles of four sRNAs conserved among many enterobacteria, CsrB, CsrC, MicA and SraL, were analysed and the processing and stability of these sRNAs was studied in the constructed strains. The degradosome was a common feature involved in the turnover of these four sRNAs. PAPI-mediated polyadenylation was the major factor governing SraL degradation. RNase III was revealed to strongly affect MicA decay. PNPase was shown to be important in the decay of these four sRNAs. The stability of CsrB and CsrC seemed to be independent of the RNA chaperone, Hfq, whereas the decay of SraL and MicA was Hfq-dependent. Taken together, the results of this study provide initial insight into the mechanisms of sRNA decay in Salmonella, and indicate specific contributions of the RNA decay machinery components to the turnover of individual sRNAs

Crosstalk between Virulence Loci: Regulation of Salmonella enterica Pathogenicity Island 1 (SPI-1) by Products of the std Fimbrial Operon

Invasion of intestinal epithelial cells is a critical step in Salmonella infection and requires the expression of genes located in Salmonella pathogenicity island 1 (SPI-1). A key factor for SPI-1 expression is DNA adenine (Dam) methylation, which activates synthesis of the SPI-1 transcriptional activator HilD. Dam-dependent regulation of hilD is postranscriptional (and therefore indirect), indicating the involvement of unknown cell functions under Dam methylation control. A genetic screen has identified the std fimbrial operon as the missing link between Dam methylation and SPI-1. We show that all genes in the std operon are part of a single transcriptional unit, and describe three previously uncharacterized ORFs (renamed stdD, stdE, and stdF). We present evidence that two such loci (stdE and stdF) are involved in Dam-dependent control of Salmonella SPI-1: in a Dam− background, deletion of stdE or stdF suppresses SPI-1 repression; in a Dam+ background, constitutive expression of StdE and/or StdF represses SPI-1. Repression of SPI-1 by products of std operon explains the invasion defect of Salmonella Dam− mutants, which constitutively express the std operon. Dam-dependent repression of std in the ileum may be required to permit invasion, as indicated by two observations: constitutive expression of StdE and StdF reduces invasion of epithelial cells in vitro (1,000 fold) and attenuates Salmonella virulence in the mouse model (>60 fold). In turn, crosstalk between std and SPI-1 may play a role in intestinal infections by preventing expression of SPI-1 in the caecum, an intestinal compartment in which the std operon is known to be expressed

Proteomic analysis of Salmonella enterica serovar Enteritidis following propionate adaptation

<p>Abstract</p> <p>Background</p> <p><it>Salmonella </it>Enteritidis is a highly prevalent and persistent foodborne pathogen and is therefore a leading cause of nontyphoidal gastrointestinal disease worldwide. A variety of stresses are endured throughout its infection cycle, including high concentrations of propionate (PA) within food processing systems and within the gut of infected hosts. Prolonged PA exposure experienced in such milieus may have a drastic effect on the proteome of <it>Salmonella </it>Enteritidis subjected to this stress.</p> <p>Results</p> <p>In this study, we used 2 D gel electrophoresis to examine the proteomes of PA adapted and unadapted <it>S</it>. Enteritidis and have identified five proteins that are upregulated in PA adapted cultures using standard peptide mass fingerprinting by MALDI-TOF-MS and sequencing by MALDI LIFT-TOF/TOF tandem mass spectrometry. Of these five, two significant stress-related proteins (Dps and CpxR) were shown (via qRT-PCR analysis) to be upregulated at the transcriptional level as well. Unlike the wild type when adapted to PA (which demonstrates significant acid resistance), PA adapted <it>S</it>. Enteritidis ∆<it>dps </it>and <it>S</it>. Enteritidis ∆<it>cpxR </it>were at a clear disadvantage when challenged to a highly acidic environment. However, we found the acid resistance to be fully restorable after genetic complementation.</p> <p>Conclusions</p> <p>This work reveals a significant difference in the proteomes of PA adapted and unadapted <it>S</it>. Enteritidis and affirms the contribution of Dps and CpxR in PA induced acid resistance.</p

Gene Transcription and Splicing of T-Type Channels Are Evolutionarily-Conserved Strategies for Regulating Channel Expression and Gating

T-type calcium channels operate within tightly regulated biophysical constraints for supporting rhythmic firing in the brain, heart and secretory organs of invertebrates and vertebrates. The snail T-type gene, LCav3 from Lymnaea stagnalis, possesses alternative, tandem donor splice sites enabling a choice of a large exon 8b (201 aa) or a short exon 25c (9 aa) in cytoplasmic linkers, similar to mammalian homologs. Inclusion of optional 25c exons in the III–IV linker of T-type channels speeds up kinetics and causes hyperpolarizing shifts in both activation and steady-state inactivation of macroscopic currents. The abundant variant lacking exon 25c is the workhorse of embryonic Cav3 channels, whose high density and right-shifted activation and availability curves are expected to increase pace-making and allow the channels to contribute more significantly to cellular excitation in prenatal tissue. Presence of brain-enriched, optional exon 8b conserved with mammalian Cav3.1 and encompassing the proximal half of the I–II linker, imparts a ∼50% reduction in total and surface-expressed LCav3 channel protein, which accounts for reduced whole-cell calcium currents of +8b variants in HEK cells. Evolutionarily conserved optional exons in cytoplasmic linkers of Cav3 channels regulate expression (exon 8b) and a battery of biophysical properties (exon 25c) for tuning specialized firing patterns in different tissues and throughout development