Proposal of three novel species of soil bacteria, <Emphasis Type="Italic">Variovorax ureilyticus,Variovorax rhizosphaerae</Emphasis>, and <Emphasis Type="Italic">Variovorax robiniae</Emphasis>, in the family <Emphasis Type="Italic">Comamonadaceae</Emphasis>

Three novel bacterial strains (UCM-2^T, UCM-G28^T, and UCM-G35^T) were obtained while isolating soil bacteria for the development of antibiotics. Cells of these strains were Gram-negative, non-spore forming, motile by means of a single flagellum, and rod shaped. In all strains, the predominant isoprenoid quinone was ubiquinone-8 (Q-8). Cells contained C_16:0, summed feature 3 (C_16:1ω7c and/or C_16:1ω6c), summed feature 8 (C_18:1ω7c and/or C_18:1ω6c), and C_17:0 cyclo as the major fatty acids, and C_10:0 3-OH as the major hydroxy fatty acid. The polar lipid profiles of the three novel strains were dominated by diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. The genomic DNA G + C contents of strains UCM-2^T, UCM-G28^T, and UCMG35^T were 67.5, 65.9, and 66.4 mol%, respectively. Phylogenetic analyses based on 16S rRNA sequences showed that strain UCM-2^T was most closely related to Variovorax soli NBRC 106424^T, whereas strains UCM-G28^T and UCM-G35^T were most similar to Variovorax ginsengisoli Gsoil 3165^T. Values indicating DNA-DNA hybridization between the novel isolates and closely related species in the genus Variovorax were lower than the 70% cut-off point. These phenotypic, chemotaxonomic, and phylogenetic data indicate that the three isolates should be classified as new members of the genus Variovorax, for which the names Variovorax ureilyticus sp. nov., Variovorax rhizosphaerae sp. nov., and Variovorax robiniae sp. nov. are proposed. The type strains are UCM-2^T (= KACC 18899^T = NBRC 112306^T), UCMG28^T (= KACC 18900^T = NBRC 112307^T), and UCM-G35^T (= KACC 18901^T = NBRC 112308^T), respectively.     

Multiple signaling systems target a core set of transition metal homeostasis genes using similar binding motifs

Bacterial response to metals can require complex regulation. We report an overlapping regulation for copper and zinc resistance genes in the denitrifying bacterium, Pseudomonas stutzeri RCH2, by three two‐component regulatory proteins CopR1, CopR2 and CzcR. We conducted genome‐wide evaluations to identify gene targets of two paralogous regulators, CopR1 and CopR2, annotated for copper signaling, and compared the results with the gene targets for CzcR, implicated in zinc signaling. We discovered that the CopRs and CzcR have largely common targets, and crossregulate a core set of P. stutzeri copper and zinc responsive genes. We established that this crossregulation is enabled by a conserved binding motif in the upstream regulatory regions of the target genes. The crossregulation is physiologically relevant as these regulators synergistically and antagonistically target multicopper oxidases, metal efflux and sequestration systems. CopR1 and CopR2 upregulate two cop operons encoding copper tolerance genes, while all three regulators downregulate a putative copper chaperone, Psest_1595. CzcR also upregulated the oprD gene and the CzcIABC Zn²⁺ efflux system, while CopR1 and CopR2 downregulated these genes. Our study suggests that crossregulation of copper and zinc homeostasis can be advantageous, and in P. stutzeri this is enabled by shared binding motifs for multiple response regulators.     

Investigating CFTR and KCa3.1 Protein/Protein Interactions

In epithelia, Cl^- channels play a prominent role in fluid and electrolyte transport. Of particular importance is the cAMP-dependent cystic fibrosis transmembrane conductance regulator Cl^- channel (CFTR) with mutations of the CFTR encoding gene causing cystic fibrosis. The bulk transepithelial transport of Cl^- ions and electrolytes needs however to be coupled to an increase in K⁺ conductance in order to recycle K⁺ and maintain an electrical driving force for anion exit across the apical membrane. In several epithelia, this K⁺ efflux is ensured by K⁺ channels, including KCa3.1, which is expressed at both the apical and basolateral membranes. We show here for the first time that CFTR and KCa3.1 can physically interact. We first performed a two-hybrid screen to identify which KCa3.1 cytosolic domains might mediate an interaction with CFTR. Our results showed that both the N-terminal fragment M1-M40 of KCa3.1 and part of the KCa3.1 calmodulin binding domain (residues L345-A400) interact with the NBD2 segment (G1237-Y1420) and C- region of CFTR (residues T1387-L1480), respectively. An association of CFTR and F508del-CFTR with KCa3.1 was further confirmed in co-immunoprecipitation experiments demonstrating the formation of immunoprecipitable CFTR/KCa3.1 complexes in CFBE cells. Co-expression of KCa3.1 and CFTR in HEK cells did not impact CFTR expression at the cell surface, and KCa3.1 trafficking appeared independent of CFTR stimulation. Finally, evidence is presented through cross-correlation spectroscopy measurements that KCa3.1 and CFTR colocalize at the plasma membrane and that KCa3.1 channels tend to aggregate consequent to an enhanced interaction with CFTR channels at the plasma membrane following an increase in intracellular Ca²⁺ concentration. Altogether, these results suggest 1) that the physical interaction KCa3.1/CFTR can occur early during the biogenesis of both proteins and 2) that KCa3.1 and CFTR form a dynamic complex, the formation of which depends on internal Ca²⁺.     

An Examination of the Plastid DNA of Hypohaploid Nicotiana plumbaginifolia Plants

DNA was extracted from different morphological types of hypohaploid Nicotiana plumbaginifolia plants. The cellular levels of chloroplast DNA (expressed as percent of total DNA) were found to be approximately two- to threefold higher in two albino hypohaploids than in a green hypohaploid. The level of chloroplast DNA in the green hypohaploid was not significantly different from either in vitro or in vivo grown haploid N. plumbaginifolia plants. Molecular hybridization with DNA probes for the large subunit of ribulose bisphosphate carboxylase from spinach and with Pvull fragments representing the entire Nicotiana tabacum chloroplast genome revealed no gross qualitative differences in the chloroplast DNAs of hypohaploid plants. Based on these observations we have concluded that the lack of chloroplast function observed in the albino forms of hypohaploid N. plumbaginifolia plants is not due to changes in the chloroplast genome.     

Altered PGE2 production by glomeruli and papilla of rats with hereditary diabetes insipidus

PGE₂ synthesis rate was studied in vitro in isolated glomeruli and in papillary homogenates prepared from kidneys of Brattleboro rats with hereditary diabetes insipidus (DI) (no ADH) and of Brattleboro heterozygous control rats. Incubations were carried out in isotonic buffer at 37°C in the presence or absence of arachidonic acid for 15, 30, 60 and 90 min. PGE₂ production was measured in the supernatant by specific radioimmunoassay. Results were compared by analysis of variance. PGE₂ production was significantly decreased in the papilla (p < 0.01) and increased in the glomeruli (p < 0.01) of DI rats compared to controls. Stimulation by arachidonic acid was similar in both groups. Chronic ADH deficiency thus modifies the ability of the kidney to produce PGE₂ in vitro. The opposite effects observed in glomeruli and papilla suggest a different hormonal control of PGE₂ synthesis in both tissues.     

Expression and characterization of an active and thermally more stable recombinant antifreeze polypeptide from ocean pout, Macrozoarces americanus, in Escherichia coli: improved expression by the modification of the secondary structure of the mRNA.

The cDNA clone coding for the ocean pout antifreeze polypeptide (AFP) was modified to improve translation of its mRNA in Escherichia coli. A recombinant AFP (rAFP), MetLys-AFP-Lys, was expressed successfully using the lambda PL promoter, and constituted 1-2% of total bacterial proteins. The rAFP was purified to homogeneity from the soluble fractions of bacterial extracts. Its identity was confirmed by amino acid analysis, automated Edman degradation, immuno-blot and activity measurements. Although the rAFP is indistinguishable from the authentic AFP in its secondary structure, thermal hysteretic activity and the alteration of ice crystal structure, it is, however, thermally more stable (approximately 4.5 degrees C increase in Tm) and is more effective in inhibiting ice growth along the a-axis. These investigations indicate that the extra amino acids in rAFP significantly improve the thermal stability and ice-binding activity of the polypeptide.     

Immunoreactive calmodulin in supernatant fraction of platelet homogenates from humans, guinea pigs, rats, and rabbits

Immunoreactive Calmodulin (CM) was measured in the supernatant fraction of homogenates of platelets obtained from humans, rats, guinea pigs and rabbits, using a commercial RIA kit. The levels (microgram/g wet weight of platelets) of immunoreactive CM were 6.8 +/- 0.5, 6.9 +/- 0.4, 8.6 +/- 1.8 and 9.7 +/- 3.1 (mean +/- SEM) for rat, human, rabbit and guinea pig, respectively. There was no statistically significant difference in values between these four species. The refractoriness of rat platelets to aggregate to certain agonists such as platelet activating factor (PAF) cannot be explained on differences in amount of immunoreactive CM.