Role of Ca2+ in the activity of rhicadhesin from Rhizobium leguminosarum biovar viciae,which mediates the first step in attachment of Rhizobiaceae cells to plant root hair tips

The first step in attachment of Rhizobiaceae cells to plant root hair tips is mediated by a Ca²⁺-dependent, Ca²⁺-binding protein, rhicadhesin. The possible role of Ca²⁺ in synthesis, anchoring and activity of rhicadhesin was investigated. Growth of Rhizobium leguminosarum biovar viciae cells under Ca²⁺-limitation was found to result in loss of attachment ability. Under these conditions, rhicadhesin could not be usolated from the bacterial cell surface, but was found to be excreted in the growth medium. Divalent ions appeared to be essential for the ability of purified rhicadhesin to inhibit attachment of R. leguminosarum biovar viciae cells to pea root hair tips. Calcium ions were found not to be involved in binding of rhicadhesin to the plant surface, but appeared to be involved in anchoring of the adhesin to the bacterial cell surface. A model for the role of Ca²⁺ in activity of rhicadhesin is presented.     

Roles of flagella, lipopolysaccharide, and a Ca2+-dependent cell surface protein in attachment of Rhizobium leguminosarum biovar viciae to pea root hair tips.

The relationship between Ca2+-dependent cell surface components of Rhizobium leguminosarum biovar viciae, motility, and ability to attach to pea root hair tips was investigated. In contrast to flagella and lipopolysaccharide, a small protein located on the cell surface was identified as the Ca2+-dependent adhesin.     

Involvement of both cellulose fibrils and a Ca2+-dependent adhesin in the attachment of Rhizobium leguminosarum to pea root hair tips.

We have previously described an assay for the attachment of Rhizobium bacteria to pea root hair tips (cap formation) which was used as a model to study the attachment step in the nodulation process. Under all conditions tested, a positive correlation was observed between the percentage of fibrillated cells and the ability of these bacteria to form caps and to adhere to glass, suggesting that fibrils play a role in the attachment of Rhizobium leguminosarum to pea root hair tips and to glass (G. Smit, J. W. Kijne, and B. J. J. Lugtenberg, J. Bacteriol. 168:821-827, 1986). In the present paper the chemical and functional characterization of the fibrils of R. leguminosarum is described. Characterization of purified fibrils by infrared spectroscopy and cellulase treatment followed by thin-layer chromatography showed that the fibrils are composed of cellulose. Purified cellulose fibrils, as well as commercial cellulose, inhibited cap formation when present during the attachment assay. Incubation of the bacteria with purified cellulase just before the attachment assay strongly inhibited cap formation, indicating that the fibrils are directly involved in the attachment process. Tn5-induced fibril-overproducing mutants showed a greatly increased ability to form caps, whereas Tn5-induced fibril-negative mutants lost this ability. None of these Tn5 insertions appeared to be located on the Sym plasmid. Both types of mutants showed normal nodulation properties, indicating that cellulose fibrils are not a prerequisite for successful nodulation under the conditions used. The ability of the fibril-negative mutants to attach to glass was not affected by the mutations, indicating that attachment to pea root hair tips and attachment to glass are (partly) based on different mechanisms. However, growth of the rhizobia under low Ca2+ conditions strongly reduced attachment to glass and also prevented cap formation, although it had no negative effect on fibril synthesis. This phenomenon was found for several Rhizobium spp. It was concluded that both cellulose fibrils and a Ca2+ -dependent adhesin(s) are involved in the attachment of R. leguminosarum to pea root hair tips. A model cap formation as a two-step process is discussed.     

Correlation between extracellular fibrils and attachment of Rhizobium leguminosarum to pea root hair tips.

As part of a project meant to characterize molecules involved in nodulation, a semiquantitative microscopic assay was developed for measuring attachment of Rhizobium leguminosarum cells to pea root hair tips, i.e., the site at which R. leguminosarum initiates nodulation. This form of attachment, designated as cap formation, was dependent on the incubation pH and growth phase, with optimal attachment at pH 7.5 and with bacteria in the early stationary phase of growth. Addition of glucose to the growth medium delayed the initiation of the stationary phase and cap formation, suggesting a correlation between cap formation and carbon limitation. Attachment of R. leguminosarum was not inhibited by pea lectin haptens which makes it unlikely that lectins are involved under the tested conditions. Moreover, heterologous fast-growing rhizobia adhered equally well to pea root hair tips. Since the attachment characteristics of a Sym plasmid-cured derivative were indistinguishable from those of the wild-type strain, the Sym plasmidborne nodulation genes are not necessary for attachment. Sodium chloride and various other salts abolished attachment when present during the attachment assay in final concentrations of 100 mM. R. leguminosarum produced extracellular fibrils. A positive correlation between the percentage of fibrillated cells and the ability of the bacteria to form caps and to adhere to glass and erythrocytes was observed under various conditions, suggesting that these fibrils play a role in attachment of the bacteria to pea root hair tips, to glass, and to erythrocytes.     

Lectin-enhanced accumulation of manganese-limited Rhizobium leguminosarum cells on pea root hair tips.

The ability of Rhizobium leguminosarum 248 to attach to developing Pisum sativum root hairs was investigated during various phases of bacterial growth in yeast extract-mannitol medium. Direct cell counting revealed that growth of the rhizobia transiently stopped three successive times during batch culture in yeast extract-mannitol medium. These interruptions of growth, as well as the simultaneous autoagglutination of the bacteria, appeared to be caused by manganese limitation. Rhizobia harvested during the transient phases of growth inhibition appeared to have a better attachment ability than did exponentially growing rhizobia. The attachment characteristics of these manganese-limited rhizobia were compared with those of carbon-limited rhizobia (G. Smit, J. W. Kijne, and B. J. J. Lugtenberg, J. Bacteriol. 168:821-827, 1986, and J. Bacteriol. 169:4294-4301, 1987). In contrast to the attachment of carbon-limited cells, accumulation of manganese-limited rhizobia (cap formation) was already in full progress after 10 min of incubation; significantly delayed by 3-O-methyl-D-glucose, a pea lectin haptenic monosaccharide; partially resistant to sodium chloride; and partially resistant to pretreatment of the bacteria with cellulase. Binding of single bacteria to the root hair tips was not inhibited by 3-O-methyl-D-glucose. Whereas attachment of single R. leguminosarum cells to the surface of pea root hair tips seemed to be similar for both carbon- and manganese-limited cells, the subsequent accumulation of manganese-limited rhizobia at the root hair tips is apparently accelerated by pea lectin molecules. Moreover, spot inoculation tests with rhizobia grown under various culture conditions indicated that differences in attachment between manganese- and carbon-limited R. leguminosarum cells are correlated with a significant difference in infectivity in that manganese-limited rhizobia, in contrast to carbon-limited rhizobia, are infective. This growth-medium-dependent behavior offers and explanation for the seemingly conflicting data on the involvement of host plant lectins in attachment of rhizobia to root hairs of leguminous plants. Sym plasmid-borne genes do not play a role in manganese-limitation-induced attachment of R. leguminosarum.     

Construction and characterization of a Rhizobium leguminosarum biovar viciae strain designed to assess horizontal gene transfer in the environment

Abstract An integration vector was developed which inserts cloned DNA in a non-essential site of the Rhizobium leguminosarum biovar viciae chromosome. The expression of integrated genes is under the control of the constitutive neomycin phosphotransferase II ( npt II) promotor of transposon Tn5. The design of the vector ensures that loss of vector sequences can be detected, enabling selection of progeny containing only the requisite DNA. The newly constructed vector was employed to insert the Escherichia coli gusA gene conferring GUS activity into R. leguminosarum bv. viciae strain LRS39401 which is cured of its symbiotic plasmid (pSym). One GUS-positive transconjugant, strain CT0370, was shown to have lost all vector sequences. Conjugal transfer of pSym2004 (a Tn5-tagged derivative of symbiotic plasmid pRL1JI, which specifies pea nodulation and symbiotic nitrogen fixation) to CT0370, restored the GUS-positive strain's symbiotic proficiency. Strain CT0370 is presently being used in a field release experiment in order to assess the extent of pSym transfer in a natural R. leguminosarum bv. viciae population under environmental conditions.     

Protein composition of the peribacteriod space in root nodules of Pisum sativum and Vicia faba induced by Rhizobium leguminosarum biovar viciae

Proteins in the peribacteroid space (PBS) between the bacteroid outer membrane and the peribacteroid membrane in root nodules of Pisum sativum and Vicia faba induced by Rhizobium leguminosarum PRE were analysed by two-dimensional (2-D) gel electrophoresis. Most of the detectable proteins were found to migrate to identical positions; however the level of accumulation of some of these appear to be determined by the host plant. When a different R. leguminosarum strain (RB1) was used to inoculate P. sativum , the majority of the isolated PBS proteins were found to migrate in the 2-D gel to identical positions as those of the other two combinations ( R. leguminosarum PRE x P. sativum and R. leguminosarum PRE x V. faba ).     

Purification and characterization of the Ca2+ plus Mg2+-dependent endodeoxyribonuclease from calf thymus chromatin

Ca2+ plus Mg2+-dependent endodeoxyribonuclease was extracted from calf thymus chromatin and purified to a state free from contamination by other DNases. This DNase required both Ca2+ and Mg2+, or Mn2+ alone for its activity and the optimum pH for activity was at 6.5-7.5. No specificity for the 5'-base was observed. The molecular weight of the DNase was estimated to be about 25,000-30,000 by glycerol gradient centrifugation. Actin and antibody for pancreatic DNase (DNase I) did not inhibit the enzyme, whereas both strongly inhibited DNase I, suggesting that these two DNases are different enzymes.     

Purification and characterization of a cytosolic, 42-kDa and Ca2+-dependent phospholipase A2 from bovine red blood cells: its involvement in Ca2+-dependent release of arachidonic acid from mammalian red blood cells

It has become evident that a Ca(2+)-dependent release of arachidonic acid (AA) and subsequent formation of bioactive lipid mediators such as prostaglandins and leukotrienes in red blood cells (RBCs) can modify physiological functions of neighboring RBCs and platelets. Here we identified a novel type of cytosolic PLA(2) in bovine and human RBCs and purified it to apparent homogeneity with a 14,000-fold purification. The purified enzyme, termed rPLA(2), has a molecular mass of 42 kDa and reveals biochemical properties similar to group IV cPLA(2), but shows different profiles from cPLA(2) in several column chromatographies. Moreover, rPLA(2) did not react with any of anti-cPLA(2) and anti-sPLA(2) antibodies and was identified as an unknown protein in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Divalent metal ions tested exhibited similar effects between rPLA(2) and cPLA(2), whereas mercurials inhibited cPLA(2) but had no effect on rPLA(2). Antibody against the 42-kDa protein not only precipitated the rPLA(2) activity, but also reacted with the 42-kDa protein from bovine and human RBCs in immunoblot analysis. The 42-kDa protein band was selectively detected in murine fetal liver cells known as a type of progenitor cells of RBCs. It was found that EA4, a derivative of quinone newly developed as an inhibitor for rPLA(2), inhibited a Ca(2+) ionophore-induced AA release from human and bovine RBCs, indicating that this enzyme is responsible for the Ca(2+)-dependent AA release from mammalian RBCs. Finally, erythroid progenitor cell assay utilizing diaminobenzidine staining of hemoglobinized fetal liver cells showed that rPLA(2) detectable in erythroid cells was down-regulated when differentiated to non-erythroid cells. Together, our results suggest that the 42-kDa rPLA(2) identified as a novel form of Ca(2+)-dependent PLA(2) may play an important role in hemostasis, thrombosis, and/or erythropoiesis through the Ca(2+)-dependent release of AA.     

Role of plant root exudate and Sym plasmid-localized nodulation genes in the synthesis by Rhizobium leguminosarum of Tsr factor, which causes thick and short roots on common vetch.

In a previous paper it was shown that cocultivation of Rhizobium leguminosarum with the plant Vicia sativa subsp. nigra on solid medium causes a changed mode of growth of the plant roots, resulting in thick and short roots (Tsr). The Sym plasmid present in the bacterium appeared to be essential for causing Tsr (A. A. N. van Brussel, T. Tak, A. Wetselaar, E. Pees, and C. A. Wijffelman, Plant Sci. Lett. 27:317-325, 1982). In the present paper, we show that a role in causing Tsr is general for Sym plasmids of R. leguminosarum and Rhizobium trifolii. Moreover, mutants with transposon insertions in the Sym plasmid-localized nodulation genes nodA, B, C, and D are unable to cause Tsr, in contrast to nodulation mutants localized in other parts of the Sym plasmid. The observation that Tsr could also be brought about in liquid medium enabled us to show that Tsr is caused by a soluble factor. Experiments in which plants and bacteria were grown separately in the sterile supernatant fluids of each other resulted in establishing the following sequence of events. (i) The plant produces a factor, designated as factor A. (ii) Factor A causes the Sym plasmid-harboring bacteria to produce Tsr factor. (iii) Growth of young plants in the presence of Tsr factor results in the Tsr phenotype. Models explaining this example of molecular signalling between bacteria and plants are discussed.     

Purification and characterization of a Ca2+-dependent membrane peptidase involved in the signaling of mating pheromone in Rhodosporidium toruloides.

A mating-type-specific, membrane thiol peptidase (referred to as trigger peptidase) that seems to play a key role in the transmembrane signaling of the lipopeptidyl mating pheromone rhodotorucine A at the cell surface of mating type a cells of Rhodosporidium toruloides (T. Miyakawa, M. Kaji, T. Yasutake, Y.K. Jeong, E. Tsuchiya, and S. Fukui, J. Bacteriol. 162:294-299, 1985) was purified to homogeneity and characterized. The following lines of evidence support the contention that the enzyme we purified was the trigger peptidase: the identical specificity of hydrolysis at the Arg-Asn sequence of rhodotorucine A and the sensitivity of the reaction to sulfhydryl-blocking reagents; the identical specificity for the substrate, with a strict requirement for the presence of the lipid moiety; and the absence of the corresponding activity in the pheromone-producing strain (mating type A) and in a sterile mutant strain, M-39 (type a), that lacks trigger peptidase activity in vivo. The apparent molecular weight of trigger peptidase was estimated to be 68,000 by Sepharose 6B gel filtration in the presence of octylglucoside and 63,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Trigger peptidase alone was inactive but exhibited enzymatic activity with the simultaneous addition of Ca2+, membrane phospholipids, and a nonionic detergent such as octylglucoside. The concentration of Ca2+ required for maximum activation was approximately 1 mM. Only Mn2+ could replace Ca2+ at comparable concentrations. Among the phospholipids tested, only phosphatidylserine and phosphatidylethanolamine supported trigger peptidase activation. Solubilized trigger peptidase was strongly inhibited by antipain and phosphoramidon.     

Purification and characterization of the Ca2+-ATPase of plasma membranes from Ehrlich ascites mammary carcinoma cells

Ca2+-ATPase was isolated from plasma membranes of Ehrlich ascites mammary carcinoma cells by means of calmodulin affinity chromatography. The purification procedure included removal of endogenous calmodulin from a Triton X-100 solubilizate of the membranes by DEAE ion-exchange chromatography as an essential step. With respect to its molecular mass, activation by calmodulin, Ca2+-dependent phosphorylation and highly sensitive inhibition by orthovanadate, the purified enzyme resembles the Ca2+-ATPase of erythrocyte membranes. In contrast to the strong calmodulin dependence of the isolated enzyme the Ca2+-ATPase in native Ehrlich ascites carcinoma cell membranes cannot be remarkably stimulated by added calmodulin. It is suggested that the membrane-bound Ca2+-ATPase in the presence of Ca2+ is activated by interaction with endogenously bound calmodulin.     

Cloning and characterization of human CADPS and CADPS2, new members of the Ca2+-dependent activator for secretion protein family

The recent identification of some of the components involved in regulated and constitutive exocytotic pathways has yielded important insights into the mechanisms of membrane trafficking and vesicle secretion. To understand precisely the molecular events taking place during vesicle exocytosis, we must identify all of the proteins implicated in these pathways. In this paper we describe the full-length cloning and characterization of human CADPS and CADPS2, two new homologs of the mouse Cadps protein involved in large dense-core vesicle (LDCV)-regulated exocytosis. We show that these two genes have disparate RNA expression patterns, with CADPS restricted to neural and endocrine tissues and CADPS2 expressed ubiquitously. We also identify a C2 domain, a known protein motif involved in calcium and phospholipid interactions, in both CADPS and CADPS2. We propose that CADPS functions as a calcium sensor in regulated exocytosis, whereas CADPS2 acts as a calcium sensor in constitutive vesicle trafficking and secretion. CADPS and CADPS2 were determined to span 475 kb and 561 kb on human chromosomes 3p21.1 and 7q31.3, respectively. The q31-q34 of human chromosome 7 has recently been identified to contain a putative susceptibility locus for autism (AUTS1). The function, expression profile, and location of CADPS2 make it a candidate gene for autism, and thus we conducted mutation screening for all 28 exons in 90 unrelated autistic individuals. We identified several nucleotide substitutions, including only one that would affect the amino acid sequence. No disease-specific variants were identified.     

Thrombin activation of the Na+/H+ exchanger in vascular smooth muscle cells. Evidence for a kinase C-independent pathway which is Ca2+-dependent and pertussis toxin-sensitive

The mechanism by which human alpha-thrombin activates the Na+/H+ exchanger was studied in cultured neonatal rat aortic smooth muscle cells. Thrombin (0.4 unit/ml) caused a rapid cell acidification followed by a slow, amiloride-inhibitable alkalinization (0.10-0.14 delta pHi above base line). In protein kinase C down-regulated cells (exposed to phorbol 12-myristate 13-acetate for 24 or 72 h), the delta pHi induced by thrombin was only partially attenuated. This protein kinase C-independent activation of the Na+/H+ exchanger was blocked by pertussis toxin (islet activating protein (IAP)), reducing delta pHi by 50%. IAP did not directly inhibit Na+/H+ exchange activity as assessed by the response to intracellular acid loading. Thrombin also stimulated arachidonic acid release by 2.5 fold and inositol trisphosphate release by 6.2 fold. IAP inhibited both of these activities by 50-60%. Intracellular Ca2+ chelation with 120 microM quin2 prevented the thrombin-induced Ca2+ spike, inhibited thrombin-induced arachidonic acid release by 75%, and inhibited thrombin-induced activation of the Na+/H+ exchanger in protein kinase C-deficient cells by 65%. Increased intracellular [Ca2+] alone was not sufficient to activate the Na+/H+ exchanger, since ionomycin (0.3-1.5 microM) failed to elevate cell pH significantly. 10 microM indomethacin inhibited thrombin-induced delta pHi in both control and protein kinase C down-regulated cells by 30-50%. Thus, thrombin can activate the Na+/H+ exchanger in vascular smooth muscle cells by a Ca2+-dependent, pertussis toxin-sensitive pathway which does not involve protein kinase C.     

Purification,cloning, functional expression and characterization of perakine reductase: the first example from the AKR enzyme family,extending the alkaloidal network of the plant <Emphasis Type="Italic">Rauvolfia</Emphasis>

Perakine reductase (PR) catalyzes an NADPH-dependent step in a side-branch of the 10-step biosynthetic pathway of the alkaloid ajmaline. The enzyme was cloned by a “reverse-genetic” approach from cell suspension cultures of the plant Rauvolfia serpentina (Apocynaceae) and functionally expressed in Escherichia coli as the N-terminal His₆-tagged protein. PR displays a broad substrate acceptance, converting 16 out of 28 tested compounds with reducible carbonyl function which belong to three substrate groups: benzaldehyde, cinnamic aldehyde derivatives and monoterpenoid indole alkaloids. The enzyme has an extraordinary selectivity in the group of alkaloids. Sequence alignments define PR as a new member of the aldo-keto reductase (AKR) super family, exhibiting the conserved catalytic tetrad Asp52, Tyr57, Lys84, His126. Site-directed mutagenesis of each of these functional residues to an alanine residue results in >97.8% loss of enzyme activity, in compounds of each substrate group. PR represents the first example of the large AKR-family which is involved in the biosynthesis of plant monoterpenoid indole alkaloids. In addition to a new esterase, PR significantly extends the Rauvolfia alkaloid network to the novel group of peraksine alkaloids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequences reported in this article have been submitted to the Gene Bank under Accession No: AY766462.