Mutations affecting the synthesis of NADP-dependent glutamate dehydrogenase in Pseudomonas aeruginosa

NADP-dependent glutamate dehydrogenase (NADP-GDH) was purified to homogeneity from Pseudomonas aeruginosa strain 8602 (PAC 1). The Mr determined by Sephadex gel filtration was 280,000; the subunit Mr determined by SDS-PAGE was 45,000. Mutant strains lacking NADP-GDH and glutamate synthase (Gdh-Glt-) required glutamate for growth. Transductants that lacked only NADP-GDH were indistinguishable from the wild-type strain in growth properties. It was concluded that NADP-GDH is not essential for growth of the wild-type organism and that glutamate formation via NAD-dependent glutamate dehydrogenase does not occur to a significant extent. A mutant strain, 39, producing high NADP-GDH activity, synthesized normal NADP-GDH and had the same intracellular glutamate concentrations as its parent. The mutation responsible for the synthesis of high levels of NADP-GDH was shown, by transduction, to be closely linked to the NADP-GDH structural gene (gdhA).     

The importance of arginine residues in the catalytic and regulatory functions of bovine-liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase reacts rapidly with 2,3-butanedione to yield modified enzyme with 29% of its original maximum activity, but no change in its Michaelis constants for substrates and coenzymes. No significant reduction in the inactivation rate is produced by the addition of the allosteric activator ADP or inhibitor GTP, while partial protection against inactivation is provided by the coenzyme NAD+ or substrate 2-oxoglutarate when added separately. The most marked decrease in the rate of inactivation (about 10-fold) is provided by the combined addition of NAD+ and 2-oxoglutarate, suggesting that modification takes place in the region of the active site. Reaction with 2,3-butanedione also results in loss of the ability of the enzyme to be activated by ADP. Addition of ADP (but not NAD+, 2-oxoglutarate or GTP) to the incubation mixture protects markedly against the loss of activatability of ADP. It is concluded that 2,3-butanedione produces two distinguishable effects on glutamate dehydrogenase: a relatively specific modification of the regulatory ADP site and a distinct modification in the active center. Reaction of two arginyl residues per peptide chain appears to be responsible for disruption of the ADP activation property of the enzyme, while alteration of a maximum of five arginyl residues can be related to the reduction of maximum catalytic activity. Electrostatic interactions between the positively charged arginine groups and the negatively charged substrate, coenzyme and allosteric purine nucleotide may be important for the normal function of glutamate dehydrogenase.     

Growth of Pseudomonas aeruginosa mutants lacking glutamate synthase activity.

Mutant strains SU1, SU4, and US1 lacking glutamate synthase (GOGAT) activity were isolated from strains of P. aeruginosa for which histidine is a growth rate-limiting source of nitrogen. Strains SU1 and SU4 were unable to grow when a low concentration of ammonia and a variety of compounds, including histidine, were supplied as sole sources of nitrogen. A revertant of strain SU1, strain 39, produced no GOGAT but high levels of nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase and had restored ability to grow on a limited number of nitrogen sources. Strain US1 grew at the same rate in histidine medium as did its parent; it was derepressed for glutamine synthase synthesis, and histidase was less sensitive to repression by ammonia than in the parent strain. We conclude that GOGAT is not essential for growth on histidine but high levels of glutamine synthase are required nd high levels of nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase can sustain growth at low concentrations of ammonia in the absence of GOGAT.     

Occurrence of thermolabile and regulatory NAD-linked glutamate dehydrogenase in Pseudomonas fluorescens.

NAD-linked glutamate dehydrogeanse [EC 1.4.1.2] was detected together with NADP-linked glutamate dehydrogenase [EC 1.4.1.4] and aspartase [EC 4.3.1.1] in Pseudomonas fluorescens cells. The three enzymes were distinctly separated by DEAE-Sephadex column chromatography. The NAD-linked enzyme was extremely thermolabile and was rapidly inactivated even at temperatures as low as 35--40 degrees C. The combined addition of NAD+ and glutamate, however, effectively stabilized the enzyme. The glutamate saturation profile of the NAD-linked enzyme exhibited cooperativity with a Hill coefficient (n) of 1.4. ATP inhibited the enzyme in an allosteric manner, increasing the n value to 2.2. These results suggest a novel type of metabolic regulation shared by the three enzymes in the biosynthesis and catabolism of amino acids.     

Mutants of Pseudomonas aeruginosa unable to inactivate allantoinase and NADP-dependent glutamate dehydrogenase

Both allantoinase and NADP-GDH in Pseudomonas aeruginosa were inactivated when cells reached the stationary phase of growth. Mutants unable to inactivate these enzymes were isolated. Results with recombinants showed that the mutation is not located in the structural genes of these enzymes but in an independent gene involved in the inactivation.Abbreviations NADP-GDH NADP-dependent glutamate dehydrogenase - Ani^- mutant allantoinase non-inactivating mutant - GOGAT glutamate synthase     

Purification and characterization of an arginine regulatory protein, ArgR, from Pseudomonas aeruginosa and its interactions with the control regions for the car, argF, and aru operons.

Pseudomonas aeruginosa ArgR, a regulatory protein that plays a major role in the control of certain biosynthetic and catabolic arginine genes, was purified to homogeneity. ArgR was shown to be a dimer of two equal subunits, each with a molecular mass of 37,000 Da. Determination of the amino-terminal amino acid sequence showed it to be identical to that predicted from the derived sequence for the argR gene. DNase I footprinting showed that ArgR protects a region of 45 to 47 bp that overlaps the promoters for the biosynthetic car and argF operons, indicating that ArgR exerts its negative control on the expression of these operons by steric hindrance. Studies were also carried out with the aru operon, which encodes enzymes of the catabolic arginine succinyl-transferase pathway. Quantitative S1 nuclease experiments showed that expression of the first gene in this operon, aruC, is initiated from an arginine-inducible promoter. Studies with an aruC::lacZ fusion showed that this promoter is under the control of ArgR. DNase I experiments indicated that ArgR protects two 45-bp binding sites upstream of aruC; the 3' terminus for the downstream binding site overlaps the -35 region for the identified promoter. Gel retardation experiments yielded apparent dissociation constants of 2.5 x 10(-11), 4.2 x 10(-12), and 7.2 x 10(-11) M for carA, argF, and aruC operators, respectively. Premethylation interference and depurination experiments with the car and argF operators identified a common sequence, 5'-TGTCGC-3', which may be important for ArgR binding. Alignment of ArgR binding sites reveals that the ArgR binding site consists of two half-sites, in a direct repeat arrangement, with the consensus sequence TGTCGCN8AAN5.     

The fourth arginine catabolic pathway of Pseudomonas aeruginosa

D-Arginine dehydrogenase activity was discovered in Pseudomonas aeruginosa. This enzyme was inducible by its substrate, D-arginine, as well as by its product, 2-ketoarginine, but not by L-arginine. The enzyme activity was measured in vitro, in the presence of artificial electron acceptore (phenazine methosulphate and iodonitrotetrazolium chloride). 2-ketoarginine was catabolized further to 4-guanidinobutyraldehyde, 4-guanidinobutyrate and 4-aminobutyrate. Two enzymes involved, 4-guanidinobutyraldehyde dehydrogenase and guanidinobutyrase, were inducible by 2-ketoarginine; the latter enzyme was also strongly induced by 4-guanidinobutyrate. An arginine racemase activity was detected by an invivo test. E-Arginine had the potential to be catabolized via the D-arginine dehydrogenase pathway and, after racemization, via the three L-arginine catabolic pathyways previously demonstrated in P. aeruginosa. In mutants blocked in the L-arginine succinyltransferase pathway, but no in the wild-type, L-arginine was channelled partially into the D-arginine dehydrogenase pathway. Mutations in the kauB locus abolished growth of P. aeruginosa on 2-ketoarginine, agmatine and putrescine, and led to loss of 4-guanidinobutyraldehyde dehydrogenase and 4-aminobutyaldehyde dehydrogenase activites. Thus, these two activites appear to be due to one enzyme in P. aeruginosa. The kauB locus was mapped on the chromosome between lysA and argB and was not linked to known genes involved in the three L-arginine catabolic pathways. The existence of four arginine catabolic pathways illustrates the metabolic versatility of P. aeruginosa.     

铜绿假单胞菌的双组分系统

双组分系统是存在于原核和少部分真核生物细胞中的信号转导系统,主要由组氨酸蛋白激酶和反应调节蛋白组成,通过感应外界环境信号、信号输入、磷酸基团传递、信号输出等环节调节基因表达,使细胞能更加适应环境变化。铜绿假单胞菌为条件致病菌,其双组分系统构成多样、功能复杂且参与介导耐药性产生,因此铜绿假单胞菌的双组分系统日益引起人们关注。本文对铜绿假单胞菌双组分系统的组成、信号转导机制、种类、研究方法及其临床意义进行了综述。     

The arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa contains an additional gene, arcD, encoding a membrane protein

The arginine deiminase (ADI) pathway in Pseudomonas aeruginosa serves to generate ATP. The three enzymes involved, ADI, catabolic ornithine carbamoyltransferase and carbamate kinase, are induced by oxygen limitation and encoded by the contiguous arcABC genes. A 1.5-kb region upstream from arcABC was sequenced and found to contain an open reading frame, arcD, coding for a hydrophobic polypeptide of 52 kDa. The content and distribution of hydrophobic amino acids suggest that the arcD gene product may be a transmembrane protein. When arcD was fused to an Escherichia coli promoter, the ArcD protein was synthesized in E. coli maxicells and detected in the membrane fraction. In sodium dodecyl sulfate-polyacrylamide-gel electrophoresis the ArcD protein migrated like a 32-kDa protein; such anomalous electrophoretic mobility is known for other highly hydrophobic proteins. Mutations in arcD rendered the cells unable to utilize extracellular arginine as an energy source. Since anaerobic arginine consumption and ornithine release are coupled in P. aeruginosa, it is proposed that arcD specifies an arginine: ornithine antiporter or a part thereof. Insertions of IS21 or Tn1725 in arcD had a strong polar effect on the expression of the arcAB enzymes, indicating that the arc genes are organized as an arcDABC operon.     

Mapping of the arginine deiminase gene in Pseudomonas aeruginosa

A mutant of Pseudomonas aeruginosa PAO lacking arginine deiminase activity (arcA) was isolated by screening for a derivative of an arcB mutant (deficient in catabolic ornithine carbamoyltransferase) that did not excrete citrulline under conditions of limited aeration. The arcA mutation was highly cotransducible with arcB.     

Mutation of retS, encoding a putative hybrid two-component regulatory protein in Pseudomonas aeruginosa, attenuates multiple virulence mechanisms

Two-component regulatory systems play an important role in bacterial virulence. We report that mutation of a Pseudomonas aeruginosa gene designated retS (previously designated fimK; accession number PA4856) encoding a putative hybrid two-component regulator, attenuates multiple virulence mechanisms. The retS mutant was selected from a Tn5 transposon library of the cytotoxic P. aeruginosa strain PA103 based upon expression of a small-colony phenotype suggestive of reduced surface-associated "twitching" motility, a property dependent upon type IV pili. Subsequent analysis revealed that the mutant expressed pilin, albeit at lower levels than wild-type PA103. In a murine model of corneal infection, retS mutation was associated with delayed disease development and altered pathology. In vitro, retS mutants demonstrated loss of acute cytotoxic activity towards corneal epithelia as determined by trypan blue exclusion and by LDH release assays (P<0.0001). This coincided with loss of ExsA-regulated type III secretion. Mutation of retS also impaired ExsA-independent pathogenic mechanisms. When compared to the exsA mutant of PA103, retS mutants exhibited reduced epithelial adherence and invasion and reduced intracellular survival within the cells after invasion. Time-lapse video microscopy revealed that retS mutants, compared to exsA mutants, had a reduced capacity to access, and move along, the basal cell surfaces of corneal epithelial cell monolayers. Taken together, these data suggest that the protein encoded by retS regulates various properties of P. aeruginosa including both ExsA-dependent and ExsA-independent virulence mechanisms.