recA is required in the induction of pectin lyase and carotovoricin in Erwinia carotovora subsp. carotovora.

Pectin lyase (PNL) and the bacteriocin carotovoricin (CTV) were induced in Erwinia carotovora subsp. carotovora 71 by the DNA-damaging agents mitomycin C, nalidixic acid, and UV light. To determine whether the recA product was involved in the expression of these damage-inducible phenotypes, we cloned the E. carotovora subsp. carotovora recA+ gene, inactivated it by Tn5 insertion, and constructed an E. carotovora subsp. carotovora recA::Tn5 strain by gene replacement via homologous recombination. The RecA- strain was more sensitive to methyl methanesulfonate, nitroquinoline oxide, and UV light than its RecA+ parent. The recA mutation did not affect the production of pectate lyase, polygalacturonase, cellulase, and protease or the ability to cause soft rot of potato tubers. With this mutant, unlike with the RecA+ parent strain, PNL and CTV were not induced by mitomycin C or detected in potato tuber tissue. The RecA+ phenotype, including the inducibility of PNL and CTV, could, however, be restored in the mutant in trans by the recA+ gene from either E. carotovora subsp. carotovora or Escherichia coli. We conclude that, in E. carotovora subsp. carotovora, the recA product is required in the induction of PNL and CTV.     

Structural and functional insights into Erwinia carotovora L-asparaginase

Bacterial L-asparaginases are enzymes that catalyze the hydrolysis of l-asparagine to aspartic acid. For the past 30 years, these enzymes have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. Their intrinsic low-rate glutaminase activity, however, causes serious side-effects, including neurotoxicity, hepatitis, coagulopathy, and other dysfunctions. Erwinia carotovora asparaginase shows decreased glutaminase activity, so it is believed to have fewer side-effects in leukemia therapy. To gain detailed insights into the properties of E. carotovora asparaginase, combined crystallographic, thermal stability and cytotoxic experiments were performed. The crystal structure of E. carotovoral-asparaginase in the presence of L-Asp was determined at 2.5 A resolution and refined to an R cryst of 19.2 (R free = 26.6%) with good stereochemistry. Cytotoxicity measurements revealed that E. carotovora asparaginase is 30 times less toxic than the Escherichia coli enzyme against human leukemia cell lines. Moreover, denaturing experiments showed that E. carotovora asparaginase has decreased thermodynamic stability as compared to the E. coli enzyme and is rapidly inactivated in the presence of urea. On the basis of these results, we propose that E. carotovora asparaginase has limited potential as an antileukemic drug, despite its promising low glutaminase activity. Our analysis may be applicable to the therapeutic evaluation of other asparaginases as well.     

Characterization of transposon insertion out- mutants of Erwinia carotovora subsp. carotovora defective in enzyme export and of a DNA segment that complements out mutations in E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi.

Soft-rotting Erwinia spp. export degradative enzymes to the cell exterior (Out+), a process contributing to their ability to macerate plant tissues. Transposon (Tn5, Tn10, Tn10-lacZ) insertion Out- mutants were obtained in Erwinia carotovora subsp. carotovora 71 by using plasmid and bacteriophage lambda delivery systems. In these mutants, pectate lyases, polygalacturonase, and cellulase, which are normally excreted into the growth medium, accumulated in the periplasm. However, localization of the extracellular protease was not affected. The Out- mutants were impaired in their ability to macerate potato tuber tissue. Out+ clones were identified in a cosmid library of E. carotovora subsp. carotovora 71 by their ability to complement mutants. Localization of cyclic phosphodiesterase in the periplasm indicated that the Out+ plasmids did not cause lysis or a nonspecific protein release. The Out+ derivatives of the E. carotovora subsp. carotovora 71 mutants regained the ability to macerate potato tuber tissue. Our data indicate that a cluster of several genes is required for the Out+ phenotype. While one plasmid, pAKC260, restored the Out+ phenotype in each of the 31 mutants of E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi, it failed to render Escherichia coli export proficient. Homologs of E. carotovora subsp. carotovora 71 out DNA were detected by Southern hybridizations in subspecies of E. carotovora under high-stringency conditions. In contrast, E. chrysanthemi sequences bearing homology to the E. carotovora subsp. carotovora 71 out DNA were detectable only under low-stringency hybridization. Thus, although the out genes are functional in these two soft-rotting bacterial groups, the genes appear to have diverged.     

Survival of the phytopathogen Erwinia carotovora subsp. carotovora in sterile and non-sterile soil, sand and their admixture

Survival of phytopathogens in irrigation water and irrigated soil is of major concern to the agricultural community. In the present study, an Erwinia carotovora subsp. carotovora strain was tested for survival capability in three non-sterile and heat-sterilized soil matrices (soil, sand and soil + sand) over 35 d. In all non-sterile soil matrices, Erw. carotovora subsp. carotovora numbers declined below the detection limit over 35 d, the main variance being the decline rate related to nutrients available in the different matrices (soil, soil + sand and sand respectively). In heat-sterilized soil and soil + sand matrices Erw. carotovora subsp. carotovora revealed a regrowth, while in sterile sand matrix its decline was lower over the same time period. In previous published reports, when soil was sterilized by irradiation, such a regrowth was not observed. Application of an initial single load of sodium nitrate solution (70 mg l⁻¹) was found to extend bacteria survival rate in non-sterile and sterile soil columns. In sterile soil columns supplemented with sodium nitrate, Erw. carotovora subsp. carotovora did survive well for up to 60 d, with a major regrowth over the first 12 d and decline up to day 60, reaching initial loading numbers. The information on the potential survival of Erw. carotovora subsp. carotovora in soil for up to 35 d and regrowth in sterile soil should be of concern, especially when irrigation is performed with poor quality water.     

Synthesis and characterization of a peptide segment of (Ca2+ + Mg2+)-ATPase. A candidate for calcium transport site

The second tryptic digestion (TD2) of the (Ca2+ + Mg2+)-ATPase results in the decrease of Ca2+ transport due to uncoupling and the alteration of one of the two high affinity sites to a low affinity site. The eight amino acids adjacent to the tryptic digestion site form a torus with two carboxylic side chains of one aspartic and one glutamic acid for the fast twitch skeletal ATPase and two aspartic acids for the slow twitch/cardiac ATPase toward the inside. The eight amino acid peptides were synthesized for both forms of the ATPase and their binding characteristics were studied with luminescent Eu3+ as a Ca2+ analogue. The data indicate that the peptide binds Eu3+ with 1.0 Eu3+/peptide and strips off two water molecules. The peptide region is a candidate for the Ca2+ transport site of the (Ca2+ + Mg2+)-ATPase.     

Comparative study of regulatory mechanisms for pectinase production by Erwinia carotovora subsp. carotovora and Erwinia chrysanthemi

The production of pectinase, the major virulence determinant of soft-rot Erwinia species, is controlled by many regulatory factors. We focused on the major regulatory proteins, KdgR, CRP, Pir, and PecS, characterized mainly in E. chrysanthemi, and tested for their presence and function in the control of pectate lyase (Pel) and polygalacturonase (Peh) production in E. carotovora subsp. carotovora. Homologues of kdgR and crp but not of pir and pecS were detected by Southern blot analyses in E. carotovora subsp. carotovora. In fact, KdgR and CRP homologues of E. carotovora subsp. carotovora had high amino acid identities to those of E. chrysanthemi, including a complete match of the hypothetical helix-turn-helix DNA-binding motif. However, in Western blot analyses using anti-Pir (E. chrysanthemi) antibodies, a cross-reacting protein was present in both Erwinia species, although Pel production in E. carotovora subsp. carotovora was not further stimulated by adding plant extract into the medium containing PGA (polygalacturonic acid) in which hyperinduction by Pir has been reported in E. chrysanthemi EC16. When plasmids that contained each of these regulatory genes from E. chrysanthemi were introduced into E. carotovora subsp. carotovora, Pel production was controlled as predicted from their roles in E. chrysanthemi, except for PecS. PecS exerted a positive control in E. carotovora subsp. carotovora, in contrast to a negative control in E. chrysanthemi. DNA-binding assays demonstrated that KdgR, CRP, Pir, and PecS of E. chrysanthemi and KdgR and CRP homologues of E. carotovora subsp. carotovora could bind to the promoter regions of pel-1, pel-3, and peh of E. carotovora subsp. carotovora. Taken together, KdgR and CRP homologues of E. carotovora subsp. carotovora may regulate Pel and Peh production as in E. chrysanthemi. However, the presence of Pir and PecS homologues in E. carotovora subsp. carotovora was not identified in this study, though these proteins of E. chrysanthemi were functional on the promoter regions of the pectinase genes of E. carotovora subsp. carotovora.     

Mutation of the aspartic acid residues of the GDD sequence motif of poliovirus RNA-dependent RNA polymerase results in enzymes with altered metal ion requirements for activity.

The poliovirus RNA-dependent RNA polymerase, 3Dpol, is known to share a region of sequence homology with all RNA polymerases centered at the GDD amino acid motif. The two aspartic acids have been postulated to be involved in the catalytic activity and metal ion coordination of the enzyme. To test this hypothesis, we have utilized oligonucleotide site-directed mutagenesis to generate defined mutations in the aspartic acids of the GDD motif of the 3Dpol gene. The codon for the first aspartate (3D-D-328 [D refers to the single amino acid change, and the number refers to its position in the polymerase]) was changed to that for glutamic acid, histidine, asparagine, or glutamine; the codons for both aspartic acids were simultaneously changed to those for glutamic acids; and the codon for the second aspartic acid (3D-D-329) was changed to that for glutamic acid or asparagine. The mutant enzymes were expressed in Escherichia coli, and the in vitro poly(U) polymerase activity was characterized. All of the mutant 3Dpol enzymes were enzymatically inactive in vitro when tested over a range of Mg2+ concentrations. However, when Mn2+ was substituted for Mg2+ in the in vitro assays, the mutant that substituted the second aspartic acid for asparagine (3D-N-329) was active. To further substantiate this finding, a series of different transition metal ions were substituted for Mg2+ in the poly(U) polymerase assay. The wild-type enzyme was active with all metals except Ca2+, while the 3D-N-329 mutant was active only when FeC6H7O5 was used in the reaction. To determine the effects of the mutations on poliovirus replication, the mutant 3Dpol genes were subcloned into an infectious cDNA of poliovirus. The cDNAs containing the mutant 3Dpol genes did not produce infectious virus when transfected into tissue culture cells under standard conditions. Because of the activity of the 3D-N-329 mutant in the presence of Fe2+ and Mn2+, transfections were also performed in the presence of the different metal ions. Surprisingly, the transfection of the cDNA containing the 3D-N-329 mutation resulted in the production of virus at a low frequency in the presence of FeSO4 or CoCl2. The virus derived from transfection in the presence of FeSO4 grew slowly, while the viruses recovered from transfection in CoCl2 grew at a rate which was similar to that of the wild-type poliovirus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of fluoride action on the L-type calcium channel in cardiac ventricular myocytes

The modulatory actions of fluoride on the function of the dihydropyridine-sensitive (L-type) Ca2+ channel were studied in rabbit cardiac myocytes. In cell-attached voltage-clamp experiments, using barium as the charge carrier, fluoride increased the activity of the Ca2+ channel dose-dependently. Low concentrations (<10 mM) of fluoride increased the number of traces with channel activities, and decreased the number of traces without channel activities, resulting in a net increase in the open-channel probability. The effect of 5 mM fluoride on the Ca2+ channel was inhibited by the presence of non-hydrolyzable guanosine diphosphate analog in the cell. On the other hand, high concentrations (>10 mM) of fluoride increased the open-channel duration, resulting in a marked increase in open-channel probability. A pretreatment of myocytes with a phosphatase inhibitor, okadaic acid, virtually abolished the additional effect of fluoride on the open-channel duration or open probability. A concentration of up to 75 mM fluoride had no effect on the Ca2+-channel activity when the myocytes were pretreated with a potent inhibitor of protein kinases, indicating that fluoride increased the Ca2+- channel activity via modulation of the phosphorylation state of the myocyte or the channel protein alone.     

2-Keto-3-fluoroglutarate: a useful mechanistic probe of 2-keto-glutarate-dependent enzyme systems

2-Keto-3-fluoroglutaric acid prepared by acid hydrolysis of its diethyl ester is stable, as the free acid in aqueous solution at pH 2, and can be stored at -20 degrees C for several years. Both enantiomers are reduced by NADH in the presence of glutamate dehydrogenase (EC 1.4.1.2) to the two diastereomers of 3-fluoro-L-glutamate, which are stable at neutral pH and at high pH unless heated. 2-Keto-3-fluoroglutarate exists in solution almost entirely as a hydrate both at low and neutral pH. Both enantiomers of ketofluoroglutarate react with the pyridoxamine forms of aspartate, alanine and 4-aminobutyrate transaminases to give fluoride release. 2 mol of cosubstrate amino acid react for each mol of ketofluoroglutarate (KFG) when starting from the pyridoxamine form of the enzyme: 2 RCHNH2COOH + KFG + H2O----F- + NH4+ + glutamate + 2 RCOCOOH. Both diastereomers of fluoroglutamate are decarboxylated by glutamate decarboxylase (EC 4.1.1.15) with fluoride release: KFG + H2O----CO2 + F- + HCOCH2CH2COOH. By contrast, only one isomer of fluoroglutamate will react with the pyridoxal form of glutamate-oxalacetate transaminase to give fluoride release: HOOCCHNH2CHFCH2COOH + H2O----4F- + NH4+ + HOOCCOCH2CH2COOH. The enzymatic decarboxylation of 3-fluoroisocitrate produces only one enantiomer of ketofluoroglutarate, which is reduced to threo (2R,3R)-3-fluoroglutamate by NADH and glutamate dehydrogenase: [2R,3S]-HOOCCH(OH)CF(COOH)CH2COOH + NADP+----[3R]-KFG + CO2 + NADPH + H+. The proton, 13C, and 19F-NMR parameters of ketofluoroglutarate and the two fluoroglutamate diastereomers are presented. These molecules are useful probes of enzymatic mechanisms thought to involve carbanion intermediates.     

Phosphorylation of Caldesmon at Sites between Residues 627 and 642 Attenuates Inhibitory Activity and Contributes to a Reduction in Ca-Calmodulin Affinity

Caldesmon is an actin- and myosin-binding protein found in smooth muscle that inhibits actin activation of myosin ATPase activity. The activity of caldesmon is controlled by phosphorylation and by binding to Ca²⁺-calmodulin. We investigated the effects of phosphorylation by p²¹-activated kinase 3 (PAK) and calmodulin on the 22 kDa C-terminal fragment of caldesmon (CaD22). We substituted the major PAK sites, Ser-672 and Ser-702, with either alanine or aspartic acid to mimic nonphosphorylated and constitutively phosphorylated states of caldesmon, respectively. The aspartic acid mutation of CaD22 weakened Ca²⁺-calmodulin binding but had no effect on inhibition of ATPase activity. Phosphorylation of the aspartic acid mutant with PAK resulted in the slow phosphorylation of Thr-627, Ser-631, Ser-635, and Ser-642. Phosphorylation at these sites weakened Ca²⁺-calmodulin binding further and reduced the inhibitory activity of CaD22 in the absence of Ca²⁺-calmodulin. Phosphorylation of these sites of the alanine mutant of CaD22 had no effect on Ca²⁺-calmodulin binding but did reduce inhibition of ATPase activity. Thus, the region between residues 627 and 642 may contribute to the overall regulation of caldesmon's activity.     

Characterization of the Agrobacterium vitis pehA gene and comparison of the encoded polygalacturonase with the homologous enzymes from Erwinia carotovora and Ralstonia solanacearum.

DNA sequencing of the Agrobacterium vitis pehA gene revealed a predicted protein with an M(r) of 58,000 and significant similarity to the polygalacturonases of two other plant pathogens, Erwinia carotovora and Ralstonia (= Pseudomonas or Burkholderia) solanacearum. Sequencing of the N terminus of the PehA protein demonstrated cleavage of a 34-amino-acid signal peptide from pre-PehA. Mature PehA accumulated primarily in the periplasm of A. vitis and pehA+ Escherichia coli cells during exponential growth. A. vitis PehA released dimers, trimers, and monomers from polygalacturonic acid and caused less electrolyte leakage from potato tuber tissue than did the E. carotovora and R. solanacearum polygalacturonases.     

Native N-Terminus Nitrophorin 2 from the Kissing Bug: Similarities to and Differences from NP2(D1A)

The first amino acid of mature native nitrophorin 2 is aspartic acid, and when expressed in E. coli, the wild-type gene of the mature protein retains the methionine-0, which is produced by translation of the start codon. This form of NP2, (M0)NP2, has been found to have different properties from its D1A mutant, for which the Met0 is cleaved by the methionine aminopeptidase of E. coli (R.?E. Berry, T.?K. Shokhireva, I. Filippov, M.?N. Shokhirev, H. Zhang, F.?A. Walker, Biochemistry 2007, 46, 6830). Native N-terminus nitrophorin 2 ((ΔM0)NP2) has been prepared by employing periplasmic expression of NP2 in E. coli using the pelB leader sequence from Erwinia carotovora, which is present in the pET-26b expression plasmid (Novagen). This paper details the similarities and differences between the three different N-terminal forms of nitrophorin 2, (M0)NP2, NP2(D1A), and (ΔM0)NP2. It is found that the NMR spectra of high- and low-spin (ΔM0)NP2 are essentially identical to those of NP2(D1A), but the rate and equilibrium constants for histamine and NO dissociation/association of the two are different.     

Role of protein dissociation in the transport of acidic amino acids by the Ehrlich ascites tumor cell.

The pH profile for the uptake of L-glutamic acid by the Ehrlich ascites tumor cell arises largely as a sum of the decline with falling pH of a slow, Na+-dependent uptake by System A, and an increasing uptake by Na+-independent System L. The latter maximizes at about pH 4.5, following approximately the titration curve of the distal carboxyl group. This shift in route of uptake was verified by (a) a declining Na+-dependent component, (b) an almost corresponding decline in the 2-(methylamino)-isobutyric acid-inhibitable component, (c) a rising component inhibited by 2-aminonorbornane-2-carboxylic acid. Other amino acids recognized as principally reactive with Systems A or L yielded corresponding inhibitory effects with some conspicious exceptions: 2-Aminoisobutyric acid and even glycine become better substrates of System L as the pH is lowered; hence their inhibitory action on glutamic acid uptake is not lost. The above results were characterized by generally consistent relations among the half-saturation concentrations of the interacting amino acids with respect to: their own uptake, their inhibition of the uptake, one by another, and their trans stimulation of exodus, one by another. A small Na+-dependent component of uptake retained by L-glutamic acid but not by D-glutamic acid at pH 4.5 is inhibitable by methionine but by neither 2-(methylamino)-isobutyric acid nor the norbornane amino acid. We provisionally identified this component with System ASC, which transports L-glutamine throughout the pH range studied. No transport activity specific to the anionic amino acids was detected, and the unequivocally anionic cysteic acid showed neither significant mediated uptake nor inhibition of the uptake of glutamic aic or of the norbornane amino acid. The dicarboxylic amino acids take the sequence, aspartic acid less than glutamic acid less than alpha-aminoadipic acid less than S-carboxymethylcysteine, in their rate of mediated, Na+-independent uptake at low pH. Diiodotyrosine and two dissimilas isomers of nitrotyrosine also show acceleration of uptake as the phenolate group on the sidechain is protonated, a result indicating that the acidic group need not be a carboxyl group and need not take a specific position in space to be accepted at the receptor site L. The presence of the carboxyl group does not upset the normal stereospecificity of System L until it falls on the beta-carbon in aspartic acid; even then it is the presence of the carbonyl group and not of the intact carboxyl group nor of its hydroxyl group that cancels out the stereospecificity, as was shown by the absence of normal stereospecificity for aspartic acid and asparagine and its presence in glutamic acid, homoserine and glutamine. In agreement, the uptak of aspartic acid is peculiarly sensitive to the presence of an alpha-methyl group or of other structures that modify the orientation of the sidechain.