Identification of Rhizobium-specific intergenic mosaic elements within an essential two-component regulatory system of Rhizobium species.

Analysis of the DNA regions upstream of the phosphoenolpyruvate carboxykinase gene (pckA) in Rhizobium meliloti and Rhizobium sp. strain NGR234 identified an open reading frame which was highly homologous to the Agrobacterium tumefaciens chromosomal virulence gene product ChvI. A second gene product, 500 bp downstream of the chvI-like gene in R. meliloti, was homologous to the A. tumefaciens ChvG protein. The homology between the R. meliloti and A. tumefaciens genes was confirmed, because the R. meliloti chvI and chvG genes complemented A. tumefaciens chvI and chvG mutants for growth on complex media. We were unable to construct chvI or chvG insertion mutants of R. meliloti, whereas mutants carrying insertions outside of these genes were readily obtained. A 108-bp repeat element characterized by two large palindromes was identified in the chvI and chvG intergenic regions of both Rhizobium species. This element was duplicated in Rhizobium sp. strain NGR234. Another structurally similar element with a size of 109 bp was present in R. meliloti but not in Rhizobium sp. strain NGR234. These elements were named rhizobium-specific intergenic mosaic elements (RIMEs), because their distribution seems to be limited to members of the family Rhizobiaceae. A homology search in GenBank detected six more copies of the first element (RIME1), all in Rhizobium species, and three extra copies of the second element (RIME2), only in R. meliloti. Southern blot analysis with a probe specific to RIME1 showed the presence of several copies of the element in the genome of R. meliloti, Rhizobium sp. strain NGR234, Rhizobium leguminosarum, and Agrobacterium rhizogenes, but none was present in A. tumefaciens and Bradyrhizobium japonicum.     

Site-directed mutagenesis and DNA sequence of pckA of Rhizobium NGR234, encoding phosphoenolpyruvate carboxykinase: gluconeogenesis and host-dependent symbiotic phenotype.

Summary We have cloned and sequenced the pckA gene of Rhizobium sp. NGR234, a broad host-range strain. The gene encodes phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of gluconeogenesis. The locus was isolated and subcloned from a genomic library of NGR234 employing hybridization with an R. meliloti pck gene probe and complementation of a Tn5 mutant in this species. The DNA sequence of pckA (NGR234) was determined and encoded a PEPCK protein of 535 amino acids with a molecular weight of 58.4 kDa. The deduced polypeptide sequence was compared to those of three known ATP-dependent PEPCKs. Slightly higher homology was observed with yeast and trypanosome polypeptides than with that of Escherichia coli. We have identified several regions that are conserved in all four PEPCK proteins. A mutant constructed in the pck gene by site-directed mutagenesis with interposon failed to grow on succinate, malate and arabinose but grew on glucose and glycerol as sole carbon sources. These data show that NGR234 requires PEPCK-driven gluconeogenesis to grow on TCA cycle intermediates. A host-dependent effect of the pckA mutation was observed on nodule development and nitrogen fixation. Nodules formed by the site-directed mutant on Leucaena leucocephala and Macroptilium atropurpureum were Fix^Red, but on Vigna unguiculata were Fix^–. The expression of the gene was positively regulated in free-living cells of NGR234 by either succinate or host-plant exudates, and was subject to catabolite repression by glucose.     

Growth factor-dependent differentiation along the myeloid and lymphoid lineages in an immature acute T lymphocytic leukemia

Bone marrow cells from a child with an immature (CD2+, CD5+, CD7+) acute T lymphocytic leukemia (T-ALL) were cultured in the presence and absence of human rIL-2, IL-3, or granulocyte-macrophage (GM)-CSF. Cells cultured without growth factors failed to divide and those initiated in the presence of IL-2 or GM-CSF underwent maturation and terminal T lymphoid or myelomonocytic differentiation, respectively. In contrast, a permanent growth factor-dependent cell line, designated TALL-103/3, was established upon culture in IL-3. The TALL-103/3 cells gradually lost the T cell-specific markers and acquired a myeloid phenotype (CD15+, CD33+). Switching of the IL-3-dependent cells at an early passage to medium containing only human rIL-2 resulted in the establishment of a subline, named TALL-103/2, with a T lymphoid phenotype (CD3+, CD8+, TCR-gamma delta +, CD7+). The TALL-103/2 cells strictly require IL-2 for growth, are irreversibly committed to the lymphoid lineage, and cannot survive in the presence of any other hemopoietic growth factor tested so far. In contrast, the IL-3-dependent TALL-103/3 cells could be adapted to grow in synthetic (serum-free) medium also in the presence of either GM-CSF or IL-5, in which they retain a myeloid phenotype. Interestingly, after 18 mo in culture in IL-3, the TALL-103/3 cells can still be phenotypically converted to the lymphoid lineage upon addition of IL-2, thus maintaining its bipotentiality. Despite the marked phenotypic differences, the TALL-103/2 and TALL-103/3 cell lines show the same karyotypes with multiple abnormalities present in the primary malignant clone and have identical rearrangements of the TCR-gamma and -delta loci, thus confirming their derivation from a common precursor cell. Together, these findings indicate that the phenotype of immature T-ALL cells can be drastically modified by the presence of specific hemopoietic growth factors in the environment, leading to either lymphoid or myeloid lineage commitment while leaving their karyotype and genotype intact.     

A phosphate transport system is required for symbiotic nitrogen fixation by Rhizobium meliloti.

The bacterium Rhizobium meliloti forms N2-fixing root nodules on alfalfa plants. The ndvF locus, located on the 1,700-kb pEXO megaplasmid of R. meliloti, is required for nodule invasion and N2 fixation. Here we report that ndvF contains four genes, phoCDET, which encode an ABC-type transport system for the uptake of Pi into the bacteria. The PhoC and PhoD proteins are homologous to the Escherichia coli phosphonate transport proteins PhnC and PhnD. The PhoT and PhoE proteins are homologous to each other and to the E. coli phosphonate transport protein PhnE. We show that the R. meliloti phoD and phoE genes are induced in response to phosphate starvation and that the phoC promoter contains two elements which are similar in sequence to the PHO boxes present in E. coli phosphate-regulated promoters. The R. meliloti ndvF mutants grow poorly at a phosphate concentration of 2 mM, and we hypothesize that their symbiotic phenotype results from their failure to grow during the nodule infection process. Presumably, the PhoCDET transport system is employed by the bacteria in the soil environment, where the concentration of available phosphate is normally 0.1 to 1 microM.     

PI(3)Kgamma has an important context-dependent role in neutrophil chemokinesis

The directional movement of cells in a gradient of external stimulus is termed chemotaxis and is important in many aspects of development and differentiated cell function. Phophoinositide 3-kinases (PI(3)Ks) are thought to have critical roles within the gradient-sensing machinery of a variety of highly motile cells, such as mammalian phagocytes, allowing these cells to respond quickly and efficiently to shallow gradients of soluble stimuli. Our analysis of mammalian neutrophil migration towards ligands such as fMLP shows that, although PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) accumulate in a PI(3)Kgamma-dependent fashion at the up-gradient leading-edge, this signal is not required for efficient gradient-sensing and gradient-biased movement. PI(3)Kgamma activity is however, a critical determinant of the proportion of cells that can move, that is, respond chemokinetically, in reaction to fMLP. Furthermore, this dependence of chemokinesis on PI(3)Kgamma activity is context dependent, both with respect to the state of priming of the neutrophils and the type of surface on which they are migrating. We propose this effect of PI(3)Kgamma is through roles in the regulation of some aspects of neutrophil polarization that are relevant to movement, such as integrin-based adhesion and the accumulation of polymerized (F)-actin at the leading-edge.     

A putative 3‐hydroxyisobutyryl‐CoA hydrolase is required for efficient symbiotic nitrogen fixation in Sinorhizobium meliloti and Sinorhizobium fredii NGR234

We report that the smb20752 gene of the alfalfa symbiont Sinorhizobium meliloti is a novel symbiotic gene required for full N₂‐fixation. Deletion of smb20752 resulted in lower nitrogenase activity and smaller nodules without impacting overall nodule morphology. Orthologs of smb20752 were present in all alpha and beta rhizobia, including the ngr_b20860 gene of Sinorhizobium fredii NGR234. A ngr_b20860 mutant formed Fix^? determinate nodules that developed normally to a late stage of the symbiosis on the host plants Macroptilium atropurpureum and Vigna unguiculata. However an early symbiotic defect was evident during symbiosis with Leucaena leucocephala, producing Fix^? indeterminate nodules. The smb20752 and ngr_b20860 genes encode putative 3‐hydroxyisobutyryl‐CoA (HIB‐CoA) hydrolases. HIB‐CoA hydrolases are required for l ‐valine catabolism and appear to prevent the accumulation of toxic metabolic intermediates, particularly methacrylyl‐CoA. Evidence presented here and elsewhere (Curson et al., 2014 , PLoS ONE 9:e97660) demonstrated that Smb20752 and NGR_b20860 can also prevent metabolic toxicity, are required for l ‐valine metabolism, and play an undefined role in 3‐hydroxybutyrate catabolism. We present evidence that the symbiotic defect of the HIB‐CoA hydrolase mutants is independent of the inability to catabolize l ‐valine and suggest it relates to the toxicity resulting from metabolism of other compounds possibly related to 3‐hydroxybutyric acid.     

Characterization of two members of a novel malic enzyme class.

The Gram-negative bacterium Rhizobium meliloti contains two distinct malic enzymes. We report the purification of the two isozymes to homogeneity, and their in vitro characterization. Both enzymes exhibit unusually high subunit molecular weights of about 82 kDa. The NAD(P)(+) specific malic enzyme [EC 1.1.1.39] exhibits positive co-operativity with respect to malate, but Michaelis-Menten type behavior with respect to the co-factors NAD(+) or NADP(+). The enzyme is subject to substrate inhibition, and shows allosteric regulation by acetyl-CoA, an effect that has so far only been described for some NADP(+) dependent malic enzymes. Its activity is positively regulated by succinate and fumarate. In contrast to the NAD(P)(+) specific malic enzyme, the NADP(+) dependent malic enzyme [EC 1.1.1.40] shows Michaelis-Menten type behavior with respect to malate and NADP(+). Apart from product inhibition, the enzyme is not subjected to any regulatory mechanism. Neither reductive carboxylation of pyruvate, nor decarboxylation of oxaloacetate, could be detected for either malic enzyme. Our characterization of the two R. meliloti malic enzymes therefore suggests a number of features uncharacteristic for malic enzymes described so far.     

Time perspective,psychological outcomes,and risky behavior among runaway adolescents

In this study, we compared the time perspectives of runaway and nonrunaway adolescents by examining relationships among time perspective, psychological outcomes, and risky behavior within runaways. Participants included 163 runaway and 581 nonrunaway adolescents who completed a self-report survey. Several dimensions of time perspective were measured including positive and negatives attitudes, orientations, and perceived relationships regarding the past, the present, and the future. Psychological outcomes included optimism, self-esteem, and hope; risky behavior was assessed with a composite scale. Results indicated that runaway adolescents reported less positive and more negative attitudes toward time and perceived time periods as less related than nonrunaways, even after controlling for differences in maternal education and academic achievement. Findings also showed that among runaways, psychological outcomes and risky behavior were associated with time perspective dimensions in expected directions. Results are discussed in light of implications for theory on time perspective and interventions with runaways.     

A Retrospective Observational Study of the Relationship between Single Nucleotide Polymorphisms Associated with the Risk of Developing Colorectal Cancer and Survival

BackgroundThere is variability in clinical outcome for patients with apparently the same stage colorectal cancer (CRC). Single nucleotide polymorphisms (SNPs) mapping to chromosomes 1q41, 3q26.2, 6p21, 8q23.3, 8q24.21, 10p14, 11q13, 11q23.1, 12q13.13, 14q22, 14q22.2, 15q13.3, 16q22.1, 18q21.1, 19q13.11, 20p12, 20p12.3, 20q13.33 and Xp22 have robustly been shown to be associated with the risk of developing CRC. Since germline variation can also influence patient outcome the relationship between these SNPs and patient survivorship from CRC was examined.MethodsAll enrolled into the National Study of Colorectal Cancer Genetics (NSCCG) were genotyped for 1q41, 3q26.2, 6p21, 8q23.3, 8q24.21, 10p14, 11q13, 11q23.1, 12q13.13, 14q22, 14q22.2, 15q13.3, 16q22.1, 18q21.1, 19q13.11, 20p12, 20p12.3, 20q13.33 and xp22 SNPs. Linking this information to the National Cancer Data Repository allowed patient genotype to be related to survival.ResultsThe linked dataset consisted of 4,327 individuals. 14q22.22 genotype defined by the SNP rs4444235 showed a significant association with overall survival. Specifically, the C allele was associated with poorer observed survival (per allele hazard ratio 1.13, 95% confidence interval 1.05–1.22, P = 0.0015).ConclusionThe CRC susceptibility SNP rs4444235 also appears to exert an influence in modulating patient survival and warrants further evaluation as a potential prognostic marker.