The Yersinia pestis V antigen is a regulatory protein necessary for Ca2(+)-dependent growth and maximal expression of low-Ca2+ response virulence genes.

The low-Ca2+ response is a multicomponent virulence regulon of the human-pathogenic yersiniae in which 12 known virulence genes are coordinately regulated in response to environmental cues of temperature, Ca2+, and nucleotides such as ATP. Yersinial growth also is regulated, with full growth yield being permitted at 37 degrees C only if Ca2+ or a nucleotide is present. In this study, we constructed and characterized a mutant Yersinia pestis specifically defective in the gene encoding the V antigen, one of the virulence genes of the low-Ca2+ response. An in-frame internal deletion-insertion mutation was made by removing bases 51 through 645 of lcrV and inserting 61 new bases. The altered lcrV was introduced into the low-Ca2+ response plasmid in Y. pestis by allelic exchange, and the resulting mutant was characterized for its two-dimensional protein profiles, growth, expression of an operon fusion to another low-Ca2+ response virulence operon, and virulence in mice. The mutant had lost its Ca2+ and nucleotide requirement for growth, showed diminished expression of Ca2(+)-and nucleotide-regulated virulence genes, and was avirulent in mice. The mutation could be complemented with respect to the growth property by supplying native V antigen operon sequences in trans in high copy number (on pBR322). Partial complementation of the growth defect and almost complete complementation of the virulence defect were seen with a lower-copy-number complementing replicon (a pACYC184 derivative). The data are consistent with the interpretation that V antigen is bifunctional, with a role in regulating growth and expression of low-Ca2+ response virulence genes in addition to its putative role as a secreted virulence protein.     

Transport of Ca2+ by Yersinia pestis.

Low-calcium-response, or Lcr, plasmids of yersiniae are known to promote an in vitro nutritional requirement for 2.5 mM Ca2+ at 37 degrees C which, if not fulfilled, results in cessation of growth with induction of virulence functions (Lcr+). The mechanism whereby Ca2+ regulates this metabolic shift is unknown. Radioactive Ca2+ was not actively accumulated by yersiniae but was excluded by an exit reaction analogous to those described for other bacteria. Nevertheless, cultivation at 37 degrees C with 0.1 mM Ca2+, a level insufficient to prevent restriction of cell division, promoted significantly more binding of the cation by Lcr+ organisms than by plasmid-deficient Lcr- mutants. According, Lcr+ yersiniae may possess unique ligands capable of recognizing Ca2+.     

Ca2(+)-dependent glucose transport in chicken intestine

Transport of glucose was measured in the intestine of white leghorn layers in vivo using ligated upper small intestinal segment in the presence of Ca2+ and other ions either singly or in combination. Transport of glucose across the intestine was very significantly increased with Ca2+ than Na+, K+ and Po4(3-) individually, but when Ca2+ was combined with Na+, K+ and PO4(3-), the glucose absorption increased significantly over that achieved by Na+ ions alone. These data revealed that Ca2+ ions might be exerting the major influence on glucose transport processes of the chicken intestine.     

Biliary glycoprotein, a member of the immunoglobulin supergene family, functions in vitro as a Ca2(+)-dependent intercellular adhesion molecule

Intercellular adhesion molecules can be classified as Ca2+ dependent or Ca2+ independent. This classification has significant functional implications regarding cellular interactions. The best characterized Ca2(+)-dependent adhesion molecules, such as L-CAM or E-cadherin, belong to the family of closely related cell surface molecules called cadherins. On the other hand, those immunoglobulin supergene family members which function as adhesion molecules, such as neural cell adhesion molecule, have been found to be Ca2+ independent. In agreement with this generalization, we have recently shown that carcinoembryonic antigen (CEA) and nonspecific cross-reacting antigen (NCA), two closely related members of the CEA family, a subset of the immunoglobulin supergene family, function in vitro as Ca2(+)-independent adhesion molecules. In contrast, we show here that transfectants of a third member of the CEA family, biliary glycoprotein (BGP), also aggregate homotypically in suspension but require Ca2+ for aggregation. In addition, like the cadherins and unlike CEA or NCA or other adhesion molecules of the immunoglobulin supergene family, BGP transfectant aggregation requires physiological temperatures. Two forms of BGP, with three and two immunoglobulin C2-set domains, show Ca2(+)- and temperature-dependent adhesion, so that these properties do not reside in the third C2-set domain. The significance of this expression in the range of functional properties of the immunoglobulin supergene family and its CEA subset is discussed.     

Genetic dissection of Ca2(+)-dependent ion channel function in Paramecium.

The ciliated protozoan, Paramecium, broadcasts the activity of its individual ion channel classes through its swimming behaviour. This fact has made it possible to isolate mutants with defective ion currents, simply by selecting individuals with abnormal swimming patterns. At least four of Paramecium's ion currents are activated by rising intracellular calcium concentration, including two K+ currents and a Na+ current. A variety of cell lines with defects in these Ca2(+)-dependent currents have been isolated: in several cases, the defects have been traced to mutations in the structural gene for calmodulin. Sequence analysis of calmodulins from these and other Ca2(+)-dependent ion-current mutants may enable a detailed mapping of putative channel interaction domains on the surface of the calmodulin molecule.     

Identification of a Ca2(+)-dependent cell-cell adhesion molecule in endothelial cells

Confluent cultures of aortic endothelial cells contain two different cell-cell adhesion mechanisms distinguished by their requirement for calcium during trypsinization and adhesion. A hybridoma clone was isolated producing a monoclonal antibody Ec6C10, which inhibits Ca2(+)-dependent adhesion of endothelial cells. There was no inhibition of Ca2(+)-independent adhesion of endothelial cells and only a minor effect on Ca2(+)-dependent adhesion of smooth muscle cells. Immunoblotting analysis shows that the antibody Ec6C10 recognizes a protein in endothelial but not epithelial cells with an apparent molecular weight of 135,000 in reducing conditions and 130,000 in non-reducing conditions. Monoclonal antibody Ec6C10 reacts with an antigen at the cell surface as shown by indirect immunofluorescence of confluent endothelial cells in a junctional pattern outlining the cobblestone morphology of the monolayer. Removal of extracellular calcium increased the susceptibility of the antigen recognized by antibody Ec6C10 to proteolysis by trypsin. The role of the Ca2(+)-dependent cell adhesion molecule in organization of the dense peripheral microfilament band in confluent endothelial cells was examined by adjusting the level of extracellular calcium to modulate cell-cell contact. Addition of the monoclonal antibody Ec6C10 at the time of the calcium switch inhibited the extent of formation of the peripheral F-actin band. These results suggest an association between cell-cell contact and the peripheral F-actin band potentially through the Ca2(+)-dependent CAM.     

A Ca2(+)-activated, Mg2(+)-dependent ATPase with high affinities for both Ca2+ and Mg2+ in vascular smooth muscle microsomes: comparison with plasma membrane Ca2(+)-pump ATPase

A Ca2(+)-ATPase with a high affinity for free Ca2+ (apparent Km of 0.13 microM) was found and characterized in membrane fractions from porcine aortic and coronary artery smooth muscles in comparison with the plasma membrane Ca2(+)-pump ATPase purified from porcine aorta by calmodulin affinity chromatography. The activity of the high-affinity Ca2(+)-ATPase became enriched in a plasma membrane-enriched fraction, suggesting its localization in the plasma membrane. The enzyme was fully active in the absence of exogenously added Mg2+, but required a minute amount of Mg2+ for its activity as evidenced by the findings that it was fully active in the presence of 0.1 microM free Mg2+ but lost the activity in a reaction mixture containing trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid as a divalent cation chelator which has, unlike EGTA, high affinities for both Ca2+ and Mg2+. It was able to utilize a variety of nucleoside di- and triphosphates as substrates, such as ADP, GDP, ATP, GTP, CTP, and UTP, showing a broad substrate specificity. The activity of the enzyme was not modified by calmodulin (5, 10 micrograms/ml). Trifluoperazine, a calmodulin antagonist, had a partial inhibitory effect on the activity at 30 to 240 microM, but this inhibition could not be reproduced by a more specific calmodulin antagonist, W-7, indicating that this inhibition by trifluoperazine was not specific. Furthermore, the high-affinity Ca2(+)-ATPase activity was not modified either by low concentrations (0.5-9 microM) of vanadate or by 1-100 microM p-chloromercuribenzoic acid. Cyclic GMP, nitroglycerin, and nicorandil did not have any effect on the enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Translocation of protein kinase C in human polymorphonuclear neutrophils. Regulation by cytosolic Ca2(+)-independent and Ca2(+)-dependent mechanisms

[3H]Phorbol dibutyrate [( 3H]PDB) rapidly and reversibly binds to human polymorphonuclear neutrophils (PMN). Ca2+/diacylglycerol/phospholipid-dependent protein kinase C appeared to be the receptor for this binding because: a diacylglycerol, dioctanoylglycerol, competed with [3H]PDB for PMN binding sites; a blocker of protein kinase C-phospholipid interactions, sphinganine, inhibited PMN binding of [3H]PDB; and changes in cytosolic Ca2+ apparently regulated PMN binding of the label. Relevant to the last point, disrupted PMN contained 9 X 10(5) phorbol diester receptors/cell, whereas intact PMN had only 1.6 X 10(5) such receptors that were accessed by the ligand. This number fell to 1.0 X 10(5) in Ca2(+)-depleted PMN and rose to 2.5 X 10(5) in cells stimulated with the Ca2+ ionophore, ionomycin. This ionomycin effect lasted for greater than 16 min, correlated temporally with changes in cytosolic Ca2+, did not occur in Ca2(+)-depleted PMN, and was blocked by sphinganine. A second ionophore, A23187, likewise induced Ca2(+)-dependent rises in [3H]PDB binding. These results fit the standard model, wherein rises in cytosolic Ca2+ cause protein kinase C to translocate from cytosol to plasmalemma and thereby become more available to [3H]PDB. In contrast, two humoral agonists, N-formyl-Met-Leu-Phe (fMLP) and leukotriene (LT)B4, had actions that did not fit this model. They stimulated PMN to increase the availability of PDB binding sites by a sphinganine-sensitive mechanism, but their actions differed from those of ionophores. They induced biphasic (t = 15 and 60 s) increases in [3H]PDB binding while eliciting monophasic (t = 15 s), short-lived (t less than 1 min) rises in cytosolic Ca2+. In Ca2(+)-depleted PMN, moreover, fMLP and LTB4 stimulated slow (t greater than or equal to 30 s), monophasic, prominent rises in [3H]PDB binding and binding site number without appreciably altering cytosolic Ca2+. We suggest, therefore, that fMLP and LTB4 translocate protein kinase C using two sequential mechanisms. The first involves Ca2+ transients and thus produces abrupt (t = 15 s), rapidly reversing responses. The second mechanism uses an unrelated signal to effect a more slowly evolving (t = 60 s) movement of protein kinase C to plasmalemma. Hence, the standard model does not explain all instances of protein kinase C translocation, and a cytosolic Ca2(+)-independent signal contributes to the regulation of protein kinase C as well as those responses elicited by the effector enzyme.     

Methionine transport in Yersinia pestis.

Yersinia pestis TJW, an avirulent wild-type strain, requires phenylalanine and methionine for growth. It was of interest to examine and define the methionine transport system because of this requirement. The methionine system showed saturation kinetics with a Km for transport of approximately 9 times 10(-7) M. After 8 s of methionine transport, essentially all of the methionine label appeared in S-adenosyl-L-methionine (SAM) as detected in ethanol extracts. Small amounts of free methionine was detected intracellularly after 1 min of transport. Addition of glucose increased significantly the amount of intracellular methionine at 1 min. A series of SAM metabolic products was detected after 90 s to 5 min of transport including: 5'-thiomethyladenosine, homoserine lactone, S-adenosyl homoserine, and a fluorescent methyl receptor compound. Results from assays for SAM synthetase in spheroplast fractions showed a small (16%) but significant portion of synthetase associated with the membrane. However, most of the enzyme activity was associated with the cytoplasmic fraction. Methionine transport was characterized by a high degree of stereospecificity. No competition occurred from structurally unrelated amino acids. Although uptake was inhibited by uncoupling and sulfhydryl reagents, no efflux was observed. Results using energy inhibitors on unstarved and starved cells showed that respiratory inhibitors such as potassium cyanide (KCN) and amytal were most effective, and that arsenate was least effective. KCN plus arsenate completely blocked utilization of energy derived from glucose, and KCN completely blocked utilization of energy deived from D-lactate. The data indicate that methionine transport in Y. pestis is linked to the trapping of methionine in SAM. The results further suggest that this transport system can be classified as a permease-bound system where transport is coupled to an energized membrane state and to respiration.     

Effect of substrate on Ca2(+)-concentration required for activity of the Ca2(+)-dependent proteinases, mu- and m-calpain

The Ca2+ concentrations required for half-maximal activity of mu- and m-calpain purified from bovine skeletal muscle were tested using four different protein substrates and three different synthetic peptide substrates. Hammersten casein, the commonly used substrate for measuring mu- and m-calpain activity, required 2.5 microM Ca2+ for half-maximal activity of mu-calpain and 290 microM Ca2+ for half-maximal activity of m-calpain. When Hammersten casein was dialyzed against 8 M urea and 10 mM EDTA to remove all endogenous Ca2+, it required 1.9 and 290 microM Ca2+ for half-maximal activity of mu- and m-calpain, respectively. Rabbit skeletal muscle myofibrils and rabbit skeletal muscle troponin required 65 microM and 24 microM Ca2+ for half-maximal activity of mu-calpain and 380 microM and 580 microM Ca2+ for half-maximal activity of m-calpain, respectively. The three synthetic substrates tested, Suc-Leu-Tyr-MCA, Boc-Leu-Thr-Arg-MCA, and Suc-Leu-Leu-Val-Tyr-MCA, required 1.6 microM to 3.7 microM Ca2+ for half-maximal activity of mu-calpain and 200 to 560 microM Ca2+ for half-maximal activity of m-calpain.     

Expression of Yersinia pestis antigens encoded by the Ca2+-dependence plasmid

Antigens coded by the Ca2(+)-dependance plasmid were found in the cultural medium, cytoplasm and outer membranes of the three monoplasmid (pCadV) strains of Yersinia pestis with the different basic properties. The presence of 20 mM of Mg2+ at least in the medium is necessary for optimal expression of these proteins. The existence of strain differences in the bacterial cells reaction to temperature, cultivation medium has been demonstrated. No difference in the pCad-dependent proteins was found in Yersinia pestis and the causative agents of pseudotuberculosis, enterocolitis.     

Consequences of Ca2+ deficiency on macromolecular synthesis and adenylate energy charge in Yersinia pestis.

A 37 but not 26 degrees C virulent Yersinia pestis is known to require at least 2.5 mM Ca2+ for growth; this requirement is potentiated by Mg2+. After shift of log-phase cells (doubling time of 2 h) from 26 to 37 degrees C in Ca2+-deficient medium, shutoff of net ribonucleic acid synthesis preceded that of protein and cell mass. With 2.5 mM Mg2+, about two doublings in cell mass and number occurred before restriction with synthesis of sufficient deoxyribonucleic acid to account for initiation and termination of two postshift rounds of chromosome replication. Temperature shift with 20 mMMg2+ resulted in a single doubling of cell mass and number with one round of chromosome replication. Subsequent to shutoff of ribonucleic acid accumulation, ribonucleoside but not deoxyribonucleoside triphosphate pools became reduced to about 50% of normal values and the adenylate energy change fell from about 0.8, typical of growing cells, to about 0.6. Excretion of significant concentrations of adenine nucleotides under both permissive and restrictive conditions was observed. Only trace levels (less than 0.01 microM ol/g [dry weight]) of guaninosine 5'-diphosphate 3'-diphosphate accumulated under restrictive or permissive conditions; guanosine 5'-triphosphate 3'-diphosphate was not detected. Return of fully restricted cells from 37 to 26 degrees C with Ca2+ resulted in prompt growth, whereas addition of Ca2+ at 37 degrees C was ineffective. This finding indicates that the observed temperature-sensitive lesion in ribonucleic acid synthesis that results in restriction can be prevented but not reversed by cultivation with Ca2+.     

Identification and cloning of a hemin storage locus involved in the pigmentation phenotype of Yersinia pestis.

The temperature-dependent absorption of sufficient exogenous hemin or Congo red to form pigmented colonies of Yersinia pestis has been termed the pigmentation phenotype (Pgm+). Spontaneous mutation to a Pgm- phenotype results in the loss of a number of divergent physiological characteristics, including the ability to store hemin and to bind Congo red at 26 degrees C. In this study, we generated and isolated transposon insertion mutants that are hemin storage negative (Hms-) and therefore unable to form pigmented colonies. These mutations are due to single mini-kan insertions within a 19.5-kilobase (kb) SalI fragment of chromosomal DNA. Restriction site analysis of eight mutants identified a minimum of six potentially different insertion sites spanning an approximately 10-kb hemin storage (hms) locus. The 19.5-kb SalI fragment (containing approximately 18 kb of Y. pestis DNA and the mini-kan insert) was cloned from one of these mutants, KIM6-2012. By using this cloned fragment as a DNA probe, the mechanism of spontaneous mutation to a Pgm- phenotype was identified as a massive deletion event. The deletion spans at least 18 kb of genomic DNA in spontaneous Pgm- mutants from nine separate strains of Y. pestis. DNA adjacent to the mini-kan insert was used to identify a clone containing a wild-type hms locus. A spontaneous Pgm- mutant of Y pestis KIM containing this clone exhibits an Hms+ phenotype. The hms::mini-kan mutations and cloned wild-type hms locus generated in this study will greatly aid in identifying the function of hemin storage in Y. pestis.     

cDNA structure and expression of calpactin, a peptide involved in Ca2(+)-dependent cell aggregation in sponges.

Aggregation of cells of the marine sponge Geodia cydonium is mediated by an aggregation factor (AF) particle of Mr 1.3 X 10(8). It is now reported that the AF particle is associated with calpactin, which was ascribed a role in the cell-adhesion process. In order to identify the sequence similarity to other members of the lipocortin family, the cDNA of sponge calpactin was cloned and found to display an 80% sequence similarity to vertebrate calpactin II but only a 47% similarity to calpactin I. The calpactin gene, which contains the consensus sequence coding for the amino acids G-T-D-E, was expressed in Escherichia coli and subsequently purified to a 37000-Mr polypeptide. Both the p32 and the p37 are provided with approximately two Ca2+ ions/molecule and the property to bind to phospholipids. The dissociation constant (calpactin-Ca2+) was in the absence of phospholipids in the range 500-700 microM-Ca2+ but in their presence about 20-30 microM-Ca2+. On the basis of (i) inhibition studies with antibodies to calelectrin and (ii) competition experiments with soluble phospholipids (both chemically defined as well as total homologous membrane lipids) we conclude that the AF-associated calpactin and plasma-membrane-bound phospholipid(s) are involved in cell-cell aggregation in sponges.     

Genetic variations in the pgm locus among natural isolates of Yersinia pestis

A PCR-based screening method was used to study the genetic variations of the pgm locus among natural isolates of Yersinia pestis from China. Our results indicate that genetic variations in the pgm locus are well correlated with biovars of Y. pestis and plague foci, suggesting that the pgm locus plays a role in Y. pestis adaptation to its environment. The gene encoding two-component regulatory system sensor kinase became a pseudogene in all strains of biovar Orientalis due to a thymidine deletion, while it is intact in all the strains of the other biovars. Only strains from Foci H and L are the same as Yersinia pseudotuberculosis in that they have an intact transmembrane helix in the sensor kinase protein, which is lost in all the other strains because of the 18 bp in-frame deletion. The IS100 element that flanks the 39 terminus of the pgm locus was inserted into the chromosome during the within-species microevolution of Y. pestis, which is absent in strains from Foci G, H and L and also in Y. pseudotuberculosis. This fact indicates that the strains from these three foci are of an older lineage of Chinese Y. pestis. It is this IS100 element's absence that maintained high stability of the pgm locus in the Y. pestis strains from these three foci. The IS285 element insertion in the pigmentation segment and the IS100 element insertion in the downstream flanking region of the pgm locus are only present in strains from Foci H and L. The flanking region outside the 59 terminus of the upstream IS100 element is identical in the strains from these two foci, which is different in the other strains. All of these unique characteristics suggest that they are of a special lineage of Chinese Y. pestis.     

A novel dual Ca2+ sensor system regulates Ca2+-dependent neurotransmitter release

Ca²⁺-dependent neurotransmitter release requires synaptotagmins as Ca²⁺ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains—C2A and C2B—to Ca²⁺. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca²⁺ sensor in Caenorhabditis elegans. Here, we report a new Ca²⁺ sensor, SNT-3, which triggers delayed Ca²⁺-dependent neurotransmitter release. snt-1;snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca²⁺ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca²⁺ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca²⁺ entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked amplitude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini–mediated membrane tethering. Our findings therefore reveal a novel dual Ca²⁺ sensor system in C. elegans and provide significant insights into Ca²⁺-regulated exocytosis.