GABA (γ-Aminobutyric Acid) Uptake Via the GABA Permease GabP Represses Virulence Gene Expression in Pseudomonas syringae pv. tomato DC3000.

The nonprotein amino acid γ-aminobutyric acid (GABA) is the most abundant amino acid in the tomato (Solanum lycopersicum) leaf apoplast and is synthesized by Arabidopsis thaliana in response to infection by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (hereafter called DC3000). High levels of exogenous GABA have previously been shown to repress the expression of the type III secretion system (T3SS) in DC3000, resulting in reduced elicitation of the hypersensitive response (HR) in the nonhost plant tobacco (Nicotiana tabacum). This study demonstrates that the GABA permease GabP provides the primary mechanism for GABA uptake by DC3000 and that the gabP deletion mutant ΔgabP is insensitive to GABA-mediated repression of T3SS expression. ΔgabP displayed an enhanced ability to elicit the HR in young tobacco leaves and in tobacco plants engineered to produce increased levels of GABA, which supports the hypothesis that GABA uptake via GabP acts to regulate T3SS expression in planta. The observation that P. syringae can be rendered insensitive to GABA through loss of gabP but that gabP is retained by this bacterium suggests that GabP is important for DC3000 in a natural setting, either for nutrition or as a mechanism for regulating gene expression. [Formula: see text] Copyright © 2016 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Local adaptation is associated with zinc tolerance in Pseudomonas endophytes of the metal-hyperaccumulator plant Noccaea caerulescens.

Metal-hyperaccumulating plants, which are hypothesized to use metals for defence against pests and pathogens, provide a unique context in which to study plant-pathogen coevolution. Previously, we demonstrated that the high concentrations of zinc found in leaves of the hyperaccumulator Noccaea caerulescens provide protection against bacterial pathogens, with a potential trade-off between metal-based and pathogen-induced defences. We speculated that an evolutionary arms race between zinc-based defences in N. caerulescens and zinc tolerance in pathogens might have driven the development of the hyperaccumulation phenotype. Here, we investigate the possibility of local adaptation by bacteria to the zinc-rich environment of N. caerulescens leaves and show that leaves sampled from the contaminated surroundings of a former mine site harboured endophytes with greater zinc tolerance than those within plants of an artificially created hyperaccumulating population. Experimental manipulation of zinc concentrations in plants of this artificial population influenced the zinc tolerance of recovered endophytes. In laboratory experiments, only endophytic bacteria isolated from plants of the natural population were able to grow to high population densities in any N. caerulescens plants. These findings suggest that long-term coexistence with zinc-hyperaccumulating plants leads to local adaptation by endophytic bacteria to the environment within their leaves.

Mutations in aquaporin-1 in phenotypically normal humans without functional CHIP water channels.

The gene aquaporin-1 encodes channel-forming integral protein (CHIP), a member of a large family of water transporters found throughout nature. Three rare individuals were identified who do not express CHIP-associated Colton blood group antigens and whose red cells exhibit low osmotic water permeabilities. Genomic DNA analyses demonstrated that two individuals were homozygous for different nonsense mutations (exon deletion or frameshift), and the third had a missense mutation encoding a nonfunctioning CHIP molecule. Surprisingly, none of the three suffers any apparent clinical consequence, which raises questions about the physiological importance of CHIP and implies that other mechanisms may compensate for its absence.

Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein.

Water rapidly crosses the plasma membrane of red blood cells (RBCs) and renal tubules through specialized channels. Although selective for water, the molecular structure of these channels is unknown. The CHIP28 protein is an abundant integral membrane protein in mammalian RBCs and renal proximal tubules and belongs to a family of membrane proteins with unknown functions. Oocytes from Xenopus laevis microinjected with in vitro-transcribed CHIP28 RNA exhibited increased osmotic water permeability; this was reversibly inhibited by mercuric chloride, a known inhibitor of water channels. Therefore it is likely that CHIP28 is a functional unit of membrane water channels.

Human red cell aquaporin CHIP. I. Molecular characterization of ABH and Colton blood group antigens.

Blood group antigens are structural variants in surface carbohydrate or amino acid polymorphisms on extracellular domains of membrane proteins. The red cell water channel-forming integral protein (Aquaporin CHIP) is a homotetramer with only one N-glycosylated subunit, however no CHIP-associated blood group antigens have yet been identified. Immunoblotting, monosaccharide composition analysis, and selective glycosidase digestions revealed that the CHIP-associated oligosaccharide contains ABH determinants and resembles a band 3-type glycan that cannot be cleaved from intact membranes by Peptide:N-glycosidase F. The molecular structure of the Colton antigens was previously unknown, but CHIP was selectively immunoprecipitated with anti-Coa or anti-Co(b). The DNA sequence from Colton-typed individuals predicted that residue 45 is alanine in the Co(a+b-) phenotype and valine in the Co(a-b+) phenotype. The nucleotide polymorphism corresponds to a PflMI endonuclease digestion site in the DNA from Co(a-b+) individuals. These studies have defined antigens within two blood group systems on CHIP: (a) an ABH-bearing polylactosaminoglycan attached to a poorly accessible site in the native membrane; and (b) the Colton antigen polymorphism which may permit the identification of rare individuals with defective water channel expression.

Human red cell Aquaporin CHIP. II. Expression during normal fetal development and in a novel form of congenital dyserythropoietic anemia.

Channel-forming integral protein (CHIP) is the archetypal member of the Aquaporin family of water channels. Delayed CHIP expression was shown recently in perinatal rat (Smith, B. L., R. Baumgarten, S. Nielsen, D. Raben, M. L. Zeidel, and P. Agre. 1993. J. Clin. Invest. 92:2035-2041); here we delineate the human patterns. Compared with adult, second and third trimester human fetal red cells had lower CHIP/spectrin ratios (0.72 +/- 0.12, 0.94 +/- 0.22 vs 1.18 +/- 0.11) and reduced osmotic water permeability (0.029, 0.026 vs 0.037 cm/s); CHIP was already present in human renal tubules by the second trimester. A patient with a novel form of congenital dyserythropoietic anemia (CDA) with persistent embryonic and fetal globins and absent red cell CD44 protein was studied because of reduced CHIP-associated Colton antigens. Novel CDA red cells contained < 10% of the normal level of CHIP and had remarkably low osmotic water permeability (< 0.01 cm/s), but no mutation was identified in Aquaporin-1, the gene encoding CHIP. These studies demonstrate: (a) unlike rat, human CHIP expression occurs early in fetal development; (b) red cell water channels are greatly reduced in a rare phenotype; and (c) disrupted expression of red cell CHIP and CD44 suggests an approach to the molecular defect in a novel form of CDA.

Transcriptional and posttranscriptional regulation of the rat prolactin gene by calcium.

The rat prolactin gene is expressed at a high basal level in the pituitary tumor GH3 cell line. Culturing GH3 cells in a low-Ca2+, serum-free medium (SFM) depresses prolactin mRNA levels, and subsequent addition of Ca2+ to the SFM results in a specific, gradual, and sustained increase in prolactin mRNA levels. We have now examined whether the observed increase in prolactin mRNA levels can be attributed solely to an increase in the transcriptional rate of the prolactin gene. Treatment of GH3 cells in SFM with 0.4 mM CaCl2 for 24 to 48 h increased cytoplasmic prolactin mRNA levels by 5- to 10-fold, whereas the transcriptional rate of the prolactin gene was increased by less than twofold over values for SFM controls. Prolactin mRNA levels increased progressively during the 24-h period after Ca2+ addition, whereas prolactin gene transcription never exceeded a twofold increase over values for SFM controls. The activities of nuclear extracts from control and Ca2(+)-induced cells were examined in an in vitro transcription assay. The two extracts directed transcription from the prolactin promoter and the adenovirus major late promoter equally well. Cycloheximide had no effect on the ability of Ca2+ to increase or maintain prolactin mRNA levels. In dactinomycin mRNA clearance experiments, prolactin mRNA was cleared at the same rate in the absence and presence of Ca2+. These results demonstrate that although Ca2+ has a small effect on the transcriptional rate of the prolactin gene, Ca2+ produces a significant increase in prolactin mRNA levels by acting at a posttranscriptional site(s). Furthermore, Ca(2+) appears to increase prolactin mRNA levels by posttranslational modification of a stable protein, probably at a nuclear site.

Detection of two chromatin proteins which bind specifically to the 5'-flanking region of the rat prolactin gene.

We employed a protein gel blotting procedure to search for nuclear proteins from rat pituitary cells that bind preferentially to the 5'-flanking region of the rat prolactin gene. By gel blots of chromatin proteins from GH3 rat pituitary tumor cells with a 32P-labeled prolactin genomic clone, we detected two major binding proteins with molecular weights of approximately 44,000 and 48,000, designated NP44 and NP48, respectively. Both NP44 and NP48 are minor chromatin proteins which are extracted at low salt concentrations (0.4 M NaCl) and exhibit a range of slightly acidic isoelectric variants. NP44 and NP48 were detected at similar levels in chromatin extracts of GH3 cells, the prolactin-negative GC cell variant of the GH3 cells, and normal rat pituitary tissue. Considerably lower levels of these two proteins were found in chromatin extracts from rat liver and rat C6 glial cells. NP44 and NP48 exhibit DNA sequence specificity, as evidenced by their strong binding to the upstream flanking region of the prolactin gene, but only very weak binding to plasmid DNA, rat prolactin or growth hormone cDNAs, or upstream flanking regions of two other rat genes. By analyzing subclones of a rat prolactin genomic clone, we established that NP44 and NP48 bind to at least two sites, which are located between 0.4 and 2.0 kilobases (region I) and between 2.0 and 4.8 kilobases (region II) upstream of the transcription initiation site. These findings are discussed in the context of a possible functional association between the strong binding of NP44 and NP48 to the prolactin 5'-flanking region and pituitary-specific expression of the prolactin gene.

Bacterial genomics and adaptation to life on plants: implications for the evolution of pathogenicity and symbiosis.

Many bacteria form intimate associations with plants. Despite the agricultural and biotechnological significance of these bacteria, no whole genome sequences have yet been described. Plant-associated bacteria form a phylogenetically diverse group, with representative species from many major taxons. Sequence information from genomes of closely related bacteria, in combination with technological developments in the field of functional genomics, provides new opportunities for determining the origin and evolution of traits that contribute to bacterial fitness and interactions with plant hosts.

Expression of multiple water channel activities in Xenopus oocytes injected with mRNA from rat kidney.

To test the hypothesis that renal tissue contains multiple distinct water channels, mRNA prepared from either cortex, medulla, or papilla of rat kidney was injected into Xenopus oocytes. The osmotic water permeability (Pf) of oocytes injected with either 50 nl of water or 50 nl of renal mRNA (1 microgram/microliter) was measured 4 d after the injection. Pf was calculated from the rate of volume increase on exposure to hyposmotic medium. Injection of each renal mRNA preparation increased the oocyte Pf. This expressed water permeability was inhibited by p-chloromercuriphenylsulfonate and had a low energy of activation, consistent with the expression of water channels. The coinjection of an antisense oligonucleotide for CHIP28 protein, at an assumed > 100-fold molar excess, with either cortex, medulla, or papilla mRNA reduced the expression of the water permeability by approximately 70, 100, and 30%, respectively. Exposure of the oocyte to cAMP for 1 h resulted in a further increase in Pf only in oocytes injected with medulla mRNA. This cAMP activation was not altered by the CHIP28 antisense oligonucleotide. These results suggest that multiple distinct water channels were expressed in oocytes injected with mRNA obtained from sections of rat kidney: (a) CHIP28 water channels in cortex and medulla, (b) cAMP-activated water channels in medulla, and (c) cAMP-insensitive water channels in papilla.

In vivo expression technology strategies: valuable tools for biotechnology.

Whole genome sequences have shown that bacteria possess a significant number of genes that have no known function. It is probable that many of these are required for survival in environments other than the agar plate. In vivo selection strategies provide a means of obtaining genes active in complex natural environments. Direct access to these genes is essential for understanding ecological performance and provides novel opportunities for biotechnology.

Evidence for a role of topoisomerase II in the Ca2+-dependent basal level expression of the rat prolactin gene.

The PRL gene is expressed at a high basal level in rat pituitary tumor GH3 cells, and this basal level enhancement of PRL gene expression is maintained through a Ca2+-calmodulin-dependent mechanism. We have now examined whether the enzyme, DNA topoisomerase II, which has been shown to be phosphorylated by a Ca2+-calmodulin-dependent protein kinase, plays a role in the Ca2+-calmodulin-dependent basal level enhancement of PRL gene expression. The topoisomerase II inhibitor, novobiocin, at concentrations in the range of 35-140 microM, effectively blocked the ability of Ca2+ to increase PRL mRNA levels. Examination of the effects of novobiocin on the levels of protein synthesis, glucose-regulated protein (GRP) 78 mRNA, histone 3 mRNA, and 18S ribosomal RNA indicated that the drug selectivity inhibited PRL gene expression. Two other topoisomerase II inhibitors, m-AMSA and VM26, also diminished the Ca2+-induced levels of PRL mRNA at concentrations (100-400 nM) that did not lower total mRNA levels. We then examined whether topoisomerase II interacted nonrandomly with DNA from the 5' transcribed and 5'-flanking region of the rat PRL gene by in vitro mapping of topoisomerase II DNA cleavage sites. In initial assays with a 10.5 kilobase (kb) PRL genomic DNA fragment containing 3.5 kb of 5'-transcribed DNA and 7 kb of 5'-flanking DNA, we detected 4 major cleavage sites in the following regions: site 1, +1500 to +1600; site 2, +1 to -100; site 3, -1200 to -1300; and site 4, -2900 to -3000.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of degenerate oligonucleotide primers and the polymerase chain reaction to clone gene family members.

As more and more genes are cloned and sequenced, it is apparent that nearly all genes are related to other genes. Similar genes are grouped into families. Examples of gene families include the collagen, globin, and myosin gene families. There are also gene superfamilies. Gene superfamilies are composed of genes that have areas of high homology and areas of high divergence. Examples of gene superfamilies include the oncogenes, homeotic genes, and a newly recognized gene superfamily of transmembrane proteins related to the lens fiber cells major intrinsic protein, or the MIP gene superfamily (1). In most cases, the different members of a gene family carry out related functions.

Effects of the DNA topoisomerase II inhibitor, VM26, on transcriptional initiation in vitro.

Inhibitors of DNA topoisomerase II have been shown to influence the expression of specific genes in vivo, leading investigators to suggest that the topological state of certain genes may affect their expression. In the present study, we have examined the effects of the topoisomerase II inhibitor, VM26, on transcriptional initiation in vitro. VM26 inhibited transcriptional initiation from the rat prolactin and adenovirus major late promoters in an in vitro transcription assay using rat pituitary tumor GH3 cell nuclear extracts. Transcriptional initiation from both supercoiled and linear DNA templates was equally inhibited by VM26 in a dose dependent manner. In order to identify sequences around the prolactin promoter recognized by topoisomerase II, we have mapped topoisomerase II cleavage sites using GH3 cell nuclear extracts as a source for the endogenous enzyme. Using a 910 bp genomic DNA fragment containing 423 bp of 5'-flanking sequences from the prolactin gene, we observed five VM26-enhanced topoisomerase II cleavage sites. Some of these sites map to positions near or within previously described regulatory DNA elements, and thus, may provide the structural basis for the sensitivity of prolactin gene expression to topoisomerase II inhibitors in vivo. These data suggest that inhibition of transcriptional initiation was a consequence of VM26 induced cleavage in the promoter region.

The intracellular calcium antagonist, TMB-8, inhibits prolactin gene expression in GH3 cells.

We examined the effects of the drug, TMB-8, which promotes sequestration of intracellular Ca2+, on the ability of extracellular Ca2+ to stimulate prolactin gene expression in GH3 cells. TMB-8 inhibited prolactin mRNA levels in a dose-dependent manner in the concentration range of 2.5-10 microM. Prolactin mRNA levels were increased about 18-fold by the addition of 0.1 mM CaCl2, and about 25-fold by the addition of 0.4 mM CaCl2. Addition of 10 microM TMB-8 reduced these levels to about 4-fold and 7-fold, respectively. At 10 microM TMB-8 did not effect total protein synthesis or the Ca2+-induced aggregation of the cells, indicating a selective inhibition by the drug of prolactin gene expression. Both TMB-8 and the calmodulin inhibitor, calmidazolium, reversed the effects of Ca2+ on prolactin mRNA levels in cells that had been pretreated for 2 days with 0.4 mM CaCl2.

ABH and Colton blood group antigens on aquaporin-1, the human red cell water channel protein.

The recent identification of the red cell water transporter (AQP1) has led to the identification of the "aquaporins", a new class of membrane proteins which function as water-selective transport proteins and are involved in many physiological processes. Identification of the chromosomal localization of the corresponding gene led to the recognition that AQP1 is the structural basis of the Colton blood group antigens. Analysis of individuals with the Colton null phenotype led to the recognition that homozygosity for knockout mutations in the corresponding gene is exceedingly rare but is without a significant clinical phenotype, predicting a redundancy in expression of other aquaporin homologs. These studies demonstrate the importance which molecular studies in red cell blood group antigens may play in diverse areas of biomedical research. Moreover, they provide another example that blood group antigens may be polymorphisms in functionally important proteins on the red cell surface.