In Pseudomonas syringae pv. phaseolicola, expression of the argK gene, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase, is regulated indirectly by temperature and directly by a precursor resembling carbamoylphosphate

Pseudomonas syringae pv. phaseolicola synthesizes a non-host-specific toxin, phaseolotoxin, and also synthesizes a phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) to protect itself from its own toxin. ROCT is encoded by argK, which is expressed coordinately with phaseolotoxin synthesis at 18 degrees C. To investigate the regulatory mechanisms of this system, null mutants were constructed for argK, argF (encoding the phaseolotoxin-sensitive OCTase [SOCT]), and amtA (encoding an amidinotransferase involved in phaseolotoxin synthesis). The argF mutant did not exhibit arginine auxotrophy when grown in M9 medium at 28 degrees C, because under this condition SOCT was replaced by ROCT. This loss of thermoregulation of argK was apparently caused by accumulation of carbamoylphosphate, one of the substrates of SOCT. Carbamoylphosphate, which has a structure similar to that of the inorganic moiety of phaseolotoxin, was used in induction assays with wild-type P. syringae pv. phaseolicola and was shown to be able to induce argK expression in M9 medium at 28 degrees C. These results indicate that argK expression is independent of temperature and is regulated directly by a compound resembling the inorganic moiety of phaseolotoxin.     

Ornithine carbamoyltransferase genes and phaseolotoxin immunity in Pseudomonas syringae pv. phaseolicola

Two different DNA fragments encoding ornithine carbamoyltransferase (OCTase) were cloned from Pseudomonas syringae pv. phaseolicola NPS3121. These fragments did not cross-hybridize and encoded OCTases which differed with respect to their sensitivity to purified phaseolotoxin, an OCTase inhibitor produced by this phytopathogenic bacterium. Recombinant plasmids carrying these DNA fragments complemented OCTase-deficient strains of Escherichia coli and Pseudomonas aeruginosa. Extracts of the complemented E. coli strain contained OCTase enzyme activities with similar degrees of sensitivity to purified phaseolotoxin as extracts of P.s.phaseolicola grown at either 20 or 30°C. The OCTase activity detectable in extracts of P.s.phaseolicola grown at 20°C is insensitive to phaseolotoxin while that detectable in extracts of cells grown at 30°C is sensitive to the toxin. E.coli HB101 harboring recombinant plasmids carrying the gene(s) encoding the phaseolotoxin-insensitive enzyme activity exhibited resistance to purified phaseolotoxin. The results of Tn5 mutagenesis and Southern blotting and the pattern of complementation of OCTase-deficient and Tox^- mutant strains suggest that the gene(s) encoding the phaseolotoxin-insensitive OCTase is part of a gene cluster involved in phaseolotoxin production.     

Molecular analysis of thermoregulation of phaseolotoxin-resistant ornithine carbamoyltransferase (argK) from Pseudomonas syringae pv. phaseolicola

The phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) and phaseolotoxin are produced by Pseudomonas syringae pv. phaseolicola at 18 degrees C but not at 28 degrees C. At 28 degrees C, the pathogen produces a protein(s) that binds (in vitro) to a 485-bp fragment (thermoregulatory region, TRR) from a heterologous clone from the pathogen genomic library, which in multiple copies overrides thermoregulation of phaseolotoxin production in wild-type cells (K. B. Rowley, D. E. Clements, M. Mandel, T. Humphreys, and S. S. Patil, Mol. Microbiol. 8:625-635, 1993). We report here that DNase I protection analysis of the 485-bp fragment shows that a single site is protected from cleavage by the protein in the 28 degrees C extract and that this site contains two repeats of a core motif G/C AAAG separated by a 5-bp spacer. Partially purified binding protein forms specific complexes with a synthetic oligonucleotide containing four tandem repeats of this motif. A 492-bp upstream fragment from argK encoding ROCT also forms specific complexes with the protein in the 28 degrees C crude extract, and a 260-bp subfragment from the TRR containing the binding site cross competes with the argk fragment, indicating that the same protein binds to nucleotides in both fragments. DNase I protection analysis of the fragment from argK revealed four separate protected sequence elements, with element III containing half of the core motif sequence (CTTTG), and the other elements containing similar sequences. Gel shift assays were done with DNA fragments from which one or all of the sites were removed as competitor DNAs against the argK probe. The results of these experiments confirmed that the binding sites (in argK) are necessary for the protein to bind to the argK fragment in a specific manner. Taken together, the results of studies presented here suggest that in cells of P. syringae pv. phaseolicola grown at high temperature argK may be negatively regulated by the protein produced at this temperature.     

Isolation and characterization of the gene from Pseudomonas syringae pv. phaseolicola encoding the phaseolotoxin-insensitive ornithine carbamoyltransferase

Summary The gene coding for the phaseolotoxin-insensitive ornithine carbamoyltransferase (OCTase) fromPseudomonas syringae pv.phaseolicola has been cloned and sequenced. The gene has a deduced coding capacity for a polypeptide with a calculated M, of 36520 daltons. Comparison of the amino acid sequence of the OCTase enzymes encoded by theP. aeruginosa argF and theEscherichia coli argI andargF genes with the deduced sequence of the newly identified gene shows that 79 amino acid residues are strictly conserved in all four polypeptides; among these 7 out of 9 residues are involved in enzyme function. Of three amino acid regions that have been implicated in substrate binding or catalysis, two are strictly conserved, and the third involved in carbamoylphosphate binding differs. This correlates well with published data showing that phaseolotoxin competes for the carbamoylphosphate binding site in the phaseolotoxin-sensitive OCTases. We propose that the gene be namedargK.     

Genetic and physiological characterization of Pseudomonas aeruginosa mutants affected in the catabolic ornithine carbamoyltransferase.

In Pseudomonas aeruginosa arginine can be degraded by the arginine "dihydrolase" system, consisting of arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase. Mutants of P. aeruginosa strain PAO affected in the structural gene (arcB) of the catabolic ornithine carbamoyltransferase were isolated. Firt, and argF mutation (i.e., a block in the anabolic ornithine carbamoyltransferase) was suppressed specifically by a mutationally altered catabolic ornithine carbamoyltransferase capable of functioning in the anabolic direction. The suppressor locus arcB (Su) was mapped by transduction between hisII and argA. Second, mutants having lost suppressor activity were obtained. The Su- mutations were very closely linked to arcB (Su) and caused strongly reduced ornithine carbamoyltransferase activities in vitro. Under aerobic conditions, a mutant (PA0630) which had less than 1% of the wild-type catabolic ornithine carbamoyltransferase activity grew on arginine as the only carbon and nitrogen source, at the wild-type growth rate. When oxygen was limiting, strain PA0630 grown on arginine excreted citrulline in the stationary growth phase. These observations suggest that during aerobic growth arginine is not degraded exclusively via the dihydrolase pathway.     

Effect of phaseolotoxin on the synthesis of arginine and protein

Mesophyll cells in discs cut from primary leaves of Phaseolus vulgaris L. were exposed to a concentration of phaseolotoxin that inhibited ornithine carbamoyltransferase (OCTase) measured in an extract of the tissue. This treatment also blocked incorporation of exogenous [¹⁴C] ornithine into protein-arginine of the mesophyll cells. By contrast more than 80% of the [¹⁴C]ornithine supplied to untreated tissue was incorporated into protein-arginine in 565 minutes. Protein synthesis in mesophyll cells was unaffected by phaseolotoxin because treated tissue continued to incorporate [¹⁴C]leucine into protein at the same rate as the untreated control. The phaseolotoxin-treated tissue should therefore remain metabolically competent and this prediction was reinforced by the finding that the rate of photosynthetic O₂ evolution per unit chlorophyll was similar for tissue from the phaseolotoxin-induced chlorosis and from green healthy tissue. Phaseolotoxin also blocked OCTase but not protein synthesis in exponentially growing cell suspension cultures. Phaseolotoxin rapidly inhibited growth of Escherichia coli and this effect was rapidly reversed by arginine. Thus, the toxic effects of phaseolotoxin may be attributed to the inhibition of OCTase which, in turn, blocks arginine synthesis. Protein accumulation is blocked as a consequence, but protein synthesis is unaffected. Chlorosis is due to reduced chlorophyll synthesis and this is presumably a consequence of the lower protein level in affected tissue.     

Isolation and characterization of the gene coding for the amidinotransferase involved in the biosynthesis of phaseolotoxin in Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola is the causal agent of the "halo blight" disease of beans. A key component in the development of the disease is a nonhost-specific toxin, Ndelta-(N'-sulphodiaminophosphinyl)-ornithyl-alanyl-homoarginine, known as phaseolotoxin. The homoarginine residue in this molecule has been suggested to be the product of L-arginine:lysine amidinotransferase activity, previously detected in extracts of P. syringae pv. phaseolicola grown under conditions of phaseolotoxin production. We report the isolation and characterization of an amidinotransferase gene (amtA) from P. syringae pv. phaseolicola coding for a polypeptide of 362 residues (41.36 kDa) and showing approximately 40% sequence similarity to L-arginine:inosamine-phosphate amidinotransferase from three species of Streptomyces spp. and 50.4% with an L-arginine:glycine amidinotransferase from human mitochondria. The cysteine, histidine, and aspartic acid residues involved in substrate binding are conserved. Furthermore, expression of the amtA and argK genes and phaseolotoxin production occurs at 18 degrees C but not at 28 degrees C. An amidinotransferase insertion mutant was obtained that lost the capacity to synthesize homoarginine and phaseolotoxin. These results show that the amtA gene isolated is responsible for the amidinotransferase activity detected previously and that phaseolotoxin production depends upon the activity of this gene.     

The global arginine regulator ArgR controls expression of argF in Pseudomonas syringae pv. phaseolicola but is not required for the synthesis of phaseolotoxin or for the regulated expression of argK

In Pseudomonas syringae pv. phaseolicola the enzyme ornithine carbamoyltransferase (OCTase), encoded by argF, is negatively regulated by argR, similar to what has been reported for Pseudomonas aeruginosa. However, production of the phaseolotoxin-resistant OCTase encoded by argK, synthesis of phaseolotoxin, and infectivity for bean pods occur independently of the ArgR protein.     

Citrulline synthesis in rat tissues and liver content of carbamoyl phosphate and ornithine

Rat liver ornithine carbamoyltransferase appears to be located exclusively in the mitochondria; the activity that is found in the soluble fraction is indistinguishable from mitochondrial ornithine carbamoyltransferase by simple kinetic criteria, and seems to result from breakage of mitochondria during homogenization. Of several rat tissues studied, only the liver and the mucosa of small intestine contain significant amounts of ornithine carbamoyltransferase; the activity in intestinal mucosa is less than one thousandth of that in liver. Qualitatively, this distribution coincides with that of carbamoyl phosphate synthetase I and its cofactor, acetylglutamate. The rat liver contents of carbamoyl phosphate and ornithine were 0.1 and 0.15mumol/g wet wt. of tissue respectively. On the basis of these values, it is proposed that in vivo the ornithine carbamoyltransferase activity of liver may be much lower than its maximal activity in vitro might suggest.     

A specific requirement for biotin in the synthesis of ornithine carbamoyltransferase by yeast

1. Growth of a biotin-requiring strain of Saccharomyces cerevisiae in a medium containing a suboptimum concentration of biotin for growth caused a decreased synthesis of ornithine carbamoyltransferase as compared with yeast grown in a medium containing an optimum concentration of biotin. Inclusion of the biotin homologues norbiotin or homobiotin, but not bishomobiotin, in the biotin-deficient medium caused an appreciable increase in ornithine carbamoyltransferase synthesis without affecting growth or synthesis of total RNA and protein. The addition of norbiotin to biotin-deficient medium had no effect on the respiratory activity of the yeast or on the synthesis of aspartate carbamoyltransferase, acid phosphatase, beta-fructofuranosidase or malate dehydrogenase. 2. Synthesis of acetylornithine deacetylase and acetylornithine acetyltransferase was slightly diminished by the imposition of biotin deficiency, but the effect was not as great as on ornithine carbamoyltransferase synthesis. Incorporation of norbiotin in the biotin-deficient medium had no marked effect on the synthesis of any other arginine-pathway enzyme except ornithine carbamoyltransferase. 3. l-Ornithine induced synthesis of ornithine carbamoyltransferase in yeast grown in biotin-deficient medium, but in yeast grown in this medium supplemented with norbiotin it repressed synthesis of the enzyme. l-Arginine had no detectable effect on ornithine carbamoyltransferase synthesis by the yeast grown in biotin-deficient medium with or without norbiotin. l-Aspartate repressed synthesis of ornithine carbamoyltransferase in biotin-deficient yeast and completely nullified the stimulatory effect of norbiotin on synthesis of the enzyme in this yeast. 4. There was no increase in ornithine carbamoyltransferase synthesis in biotin-deficient yeast incubated in phosphate buffer, pH4.5, containing glucose and biotin or norbiotin. In biotin-deficient yeast suspended in complete medium containing an optimum concentration of biotin, there was an increase in ornithine carbamoyltransferase synthesis only after the onset of growth.     

Phaseolotoxin transport in Escherichia coli and Salmonella typhimurium via the oligopeptide permease.

Phaseolotoxin [(N delta-phosphosulfamyl)ornithylalanylhomoarginine], a phytotoxic tripeptide produced by Pseudomonas syringae pv. phaseolicola that inhibits ornithine carbamoyltransferase, is transported into Escherichia coli and Salmonella typhimurium via the oligopeptide transport system (Opp). Mutants defective in oligopeptide permease (Opp-) were resistant to phaseolotoxin. Spontaneous phaseolotoxin-resistant mutants (Toxr) lacked the Opp function as evidenced by their cross-resistance to triornithine and failure to utilize glycylhistidylglycine as a source of histidine. Growth inhibition by phaseolotoxin was prevented by peptides known to be transported via the Opp system and by treatment of the toxin with L-aminopeptidase. In both E. coli and S. typhimurium, Toxr mutations were cotransducible with trp, suggesting that the opp locus occupies similar positions in genetic maps of these bacteria.     

In vitro transport of ornithine carbamoyltransferase precursor into rat liver mitochondria using a more homologous medium

The precursor of ornithine carbamoyltransferase can be transported in vitro into rat liver mitochondria using the postmitochondrial supernatant from rat liver, a more homologous medium than the commonly used rabbit reticulocyte lysate. The transport of the precursor in the case of reticulocyte lysate requires a standard translation mixture. In the presence of the postmitochondrial supernatant the same is true. However, when the components of the translation mixture were added individually to the postmitochondrial supernatant, it was found that spermidine or spermine, at physiological concentrations, sufficed for the transport of the precursor of ornithine carbamoyltransferase. The activity of the postmitochondrial supernatant was inactivated by trypsin and slightly decreased by RNase treatment; it was not lost by dialysis or by heating at 100 degrees C.     

Experimental evolution of enzyme temperature activity profile: selection in vivo and characterization of low-temperature-adapted mutants of Pyrococcus furiosus ornithine carbamoyltransferase

We have obtained mutants of Pyrococcus furiosus ornithine carbamoyltransferase active at low temperatures by selecting for complementation of an appropriate yeast mutant after in vivo mutagenesis. The mutants were double ones, still complementing at 15 degrees C, a temperature already in the psychrophilic range. Their kinetic analysis is reported.     

Metabolic enzymes from psychrophilic bacteria: challenge of adaptation to low temperatures in ornithine carbamoyltransferase from Moritella abyssi

The enzyme ornithine carbamoyltransferase (OTCase) of Moritella abyssi (OTCase(Mab)), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCase(Mab) displays maximal activity at rather low temperatures (23 to 25 degrees C) compared to other cold-active enzymes and is much less thermoresistant than its homologues from Escherichia coli or thermophilic procaryotes. In vitro the enzyme is in equilibrium between a trimeric state and a dodecameric, more stable state. The melting point and denaturation enthalpy changes for the two forms are considerably lower than the corresponding values for the dodecameric Pyrococcus furiosus OTCase and for a thermolabile trimeric mutant thereof. OTCase(Mab) displays higher K(m) values for ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue delta-N-phosphonoacetyl-L-ornithine (PALO). OTCase(Mab) differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high K(m) values and the lower sensitivity to PALO. The K(m) for ornithine, however, is substantially lower at low temperatures. A survey of the catalytic efficiencies (k(cat)/K(m)) of OTCases adapted to different temperatures showed that OTCase(Mab) activity remains suboptimal at low temperature despite the 4.5-fold decrease in the K(m) value for ornithine observed when the temperature is brought from 20 to 5 degrees C. OTCase(Mab) adaptation to cold indicates a trade-off between affinity and catalytic velocity, suggesting that optimization of key metabolic enzymes at low temperatures may be constrained by natural limits.     

Pseudomonas syringae phytotoxins: mode of action, regulation, and biosynthesis by peptide and polyketide synthetases.

Coronatine, syringomycin, syringopeptin, tabtoxin, and phaseolotoxin are the most intensively studied phytotoxins of Pseudomonas syringae, and each contributes significantly to bacterial virulence in plants. Coronatine functions partly as a mimic of methyl jasmonate, a hormone synthesized by plants undergoing biological stress. Syringomycin and syringopeptin form pores in plasma membranes, a process that leads to electrolyte leakage. Tabtoxin and phaseolotoxin are strongly antimicrobial and function by inhibiting glutamine synthetase and ornithine carbamoyltransferase, respectively. Genetic analysis has revealed the mechanisms responsible for toxin biosynthesis. Coronatine biosynthesis requires the cooperation of polyketide and peptide synthetases for the assembly of the coronafacic and coronamic acid moieties, respectively. Tabtoxin is derived from the lysine biosynthetic pathway, whereas syringomycin, syringopeptin, and phaseolotoxin biosynthesis requires peptide synthetases. Activation of phytotoxin synthesis is controlled by diverse environmental factors including plant signal molecules and temperature. Genes involved in the regulation of phytotoxin synthesis have been located within the coronatine and syringomycin gene clusters; however, additional regulatory genes are required for the synthesis of these and other phytotoxins. Global regulatory genes such as gacS modulate phytotoxin production in certain pathovars, indicating the complexity of the regulatory circuits controlling phytotoxin synthesis. The coronatine and syringomycin gene clusters have been intensively characterized and show potential for constructing modified polyketides and peptides. Genetic reprogramming of peptide and polyketide synthetases has been successful, and portions of the coronatine and syringomycin gene clusters could be valuable resources in developing new antimicrobial agents.     

The inhibition of ornithine transcarbamoylase from Escherichia coli W by phaseolotoxin.

The mechanism of inhibition of ornithine transcarbamoylase by the bacterial toxin phaseolotoxin [N-delta-(phosphosulphamyl)ornithylalanylhomoarginine] was investigated. Ornithine transcarbamoylase was purified by affinity chromatography from Escherichia coli W argR- by using N-delta-(phosphonoacetyl)ornithine as the ligand. Under steady-state conditions phaseolotoxin inhibition was reversible and exhibited mixed kinetics with respect to carbamoyl phosphate. The apparent Ki and apparent K'i were 0.2 microM and 10 microM respectively. Inhibition with respect to ornithine was noncompetitive, with an apparent Ki of 0.9 microM. These data are consistent with competitive binding of phaseolotoxin to the carbamoyl phosphate-binding site of the enzyme. The toxin also appears to be able to bind to the enzyme-carbamoyl phosphate complex, although, since K'i is 50 times greater than Ki, this event is kinetically much less significant. In the presence of phaseolotoxin ornithine transcarbamoylase exhibited a transient phase of activity before a steady state. This is consistent with low rates of association and dissociation for the toxin with enzyme and the enzyme-toxin complex. Rate constants of 2.5 X 10(4)M-1 X s-1 and 5 X 10(-3)s-1 were estimated for the association and dissociation constants respectively.     

Identification and characterization of two temperature-induced surface-associated proteins of Streptococcus suis with high homologies to members of the Arginine Deiminase system of Streptococcus pyogenes

The present study was performed to identify stress-induced putative virulence proteins of Streptococcus suis. For this, protein expression patterns of streptococci grown at 32, 37, and 42 degrees C were compared by one- and two-dimensional gel electrophoresis. Temperature shifts from 32 and 37 to 42 degrees C induced expression of two cell wall-associated proteins with apparent molecular masses of approximately 47 and 53 kDa. Amino-terminal sequence analysis of the two proteins indicated homologies of the 47-kDa protein with an ornithine carbamoyltransferase (OCT) from Streptococcus pyogenes and of the 53-kDa protein with the streptococcal acid glycoprotein (SAGP) from S. pyogenes, an arginine deiminase (AD) recently proposed as a putative virulence factor. Cloning and sequencing the genes encoding the putative OCT and AD of S. suis, octS and adiS, respectively, revealed that they had 81.2 (octS) and 80.2% (adiS) identity with the respective genes of S. pyogenes. Both genes belong to the AD system, also found in other bacteria. Southern hybridization analysis demonstrated the presence of the adiS gene in all 42 serotype 2 and 9 S. suis strains tested. In 9 of these 42 strains, selected randomly, we confirmed expression of the AdiS protein, homologous to SAGP, by immunoblot analysis using a specific antiserum against the SAGP of S. pyogenes. In all strains AD activity was detected. Furthermore, by immunoelectron microscopy using the anti-S. pyogenes SAGP antiserum we were able to demonstrate that the AdiS protein is expressed on the streptococcal surface in association with the capsular polysaccharides but is not coexpressed with them.