Manic-depressive tendencies and acceptance of self and others.
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Adolescent↗
Biomedical subjects
D Haas
Publications and source records attributed to D Haas.
Processing of the Pseudomonas arcDABC mRNA requires functional RNase E in Escherichia coli.
In Pseudomonas aeruginosa, arginine catabolism via the arginine deiminase pathway depends on the anaerobically inducible arcDABC operon, whose expression is further modulated by mRNA processing. Fusion of the cloned arc operon to an external tac promoter did not alter the processing pattern in P. aeruginosa and allowed heterologous expression in Escherichia coli. Processing within a specific region of the arcD mRNA was similar in P. aeruginosa and in E. coli. In E. coli, a conditional temperature-sensitive (ts) mutation in the gene specifying RNase E prevented cleavage of the arc mRNA at the non-permissive temperature, whereas mutations in the genes encoding RNase III or RNase P had no effect. We therefore speculate that in P. aeruginosa, an RNase E-like enzyme exists which is involved in the specific processing of the arc mRNA.
Amino Acid Transport Systems↗
Characterization of the arcD arginine:ornithine exchanger of Pseudomonas aeruginosa. Localization in the cytoplasmic membrane and a topological model.
The arcDABC operon of Pseudomonas aeruginosa encodes the enzymes of the arginine deiminase pathway and is induced by oxygen limitation. The arcD gene specifies a 53-kDa protein with arginine: ornithine exchange activity. The ArcD protein of P. aeruginosa, like the LysI lysine transporter of Corynebacterium glutamicum, has 13 hydrophobic regions which could span the cytoplasmic membrane. Fusion of a Caa (colicin A) epitope to the N-terminal part of ArcD permitted the localization, by immunoblotting, of the hybrid protein in the inner membrane of P. aeruginosa. Fusion of PhoA (alkaline phosphatase) to the very C terminus of ArcD produced another hybrid protein, which exhibited PhoA activity. Both ArcD hybrid proteins retained arginine transport activity and served to support a topological model which proposes that the N terminus is oriented toward the cytoplasm and the C terminus faces the periplasm. Further ArcD-PhoA fusions were consistent with this model. When the Caa epitope was fused to a C-terminal ArcD fragment consisting of only 5 hydrophobic domains, the resulting hybrid protein could be recovered intact from the inner membrane, suggesting that the C-terminal part of ArcD contains sufficient information for insertion into the membrane. This study illustrates the utility of the Caa epitope to tag membrane proteins.
Alkaline Phosphatase↗
Pseudomonas aeruginosa promoters which contain a conserved GG-N10-GC motif but appear to be RpoN-independent.
The proC gene of Pseudomonas aeruginosa encodes the constitutive delta 1-pyrroline 5-carboxylate reductase (the third enzyme of proline biosynthesis) and ranks among the numerous Pseudomonas genes which are poorly transcribed in Escherichia coli. The promoters of the proC gene were located by deletion mapping. The 5' ends of the proC transcripts originating from one promoter were determined by primer extension. This promoter has a GG-N10-GC motif with a 16 bp spacing between the GC doublet and the transcription start site. Such spacing is unusually long for sigma 54-dependent promoters. In rpoN mutants of P. aeruginosa and P. putida a proC'--'lacZ fusion was expressed at wild-type levels, suggesting that sigma 54 RNA polymerase is not involved in proC transcription. The expression of another P. aeruginosa gene, anr (for anaerobic regulation of nitrate respiration and anaerobic arginine degradation), also appeared to be independent of RpoN in Pseudomonas and occurred at a very low level in E. coli. The proC and anr promoters have sequence similarities in addition to the conserved GG--N10--GC motif and may also be related to some alg (alginate) promoters of P. aeruginosa. We propose that the proC and anr promoters are activated by proteins, including perhaps an alternative sigma factor, which are present in Pseudomonas but absent from E. coli.
Base Sequence↗
Multi-modal hemispatial deficits after left hemisphere stroke. A disorder of attention?
We report a patient who, after suffering infarcts involving the left temporo-parietal and left anterior cingulate regions, performed significantly worse on a variety of motor, language and sensory tasks when he acted in or attended to right hemispace. Performance on a number of tasks was better when acting in near as compared with far peri-personal space and with his head deviated to the left as compared with the right. Additionally, performance with the 'unaffected' left hand was often worse in right as compared with left hemispace. We propose that his deficits are attributable to a disruption of a left hemisphere-specific attentional system which normally serves to regulate the activation and articulation of left hemisphere processing modules.
Adult↗
RNA processing modulates the expression of the arcDABC operon in Pseudomonas aeruginosa.
Anaerobic growth of Pseudomonas aeruginosa on arginine depends on the arcDABC operon encoding the enzymes of the arginine deiminase pathway. The co-ordinate, anaerobic induction of these enzymes requires the FNR-like regulatory protein ANR, which activates the arc promoter lying upstream from arcD. By Northern hybridization experiments, three abundant arcA, arcAB and arcABC transcripts and three minor arcDA, arcDAB and arcDABC transcripts could be detected. The 5' ends of the arcA, arcAB and arcABC mRNAs were determined by S1 and primer extension mapping. These 5' ends appear to be generated by endonucleolytic cleavage (processing) in arcD mRNA rather than by a second promoter; this was concluded from the effects of insertion and deletion mutations in arcD. Intergenic inverted repeats between arcA and arcB as well as between arcB and arcC were shown to be involved in the formation of 3' ends of arc transcripts. Deletion of either intergenic region in the P. aeruginosa chromosome led to the loss of the arcA or arcAB transcript, respectively. Dot blot experiments revealed that arc mRNAs extracted from the wild-type strain had similar chemical half-lives in the arcA, arcB and arcC regions, ranging from 16 to 13 minutes. The half-life of arcD mRNA, by contrast, was significantly shorter, suggesting that this mRNA segment may be destabilized by the processing cuts within arcD. Deletion of the putative intergenic stem-loop structures did not result in a dramatic loss of arc mRNA stability. Thus, the intergenic hairpin structures do not contribute importantly to the overall mRNA stability; they might act primarily as partial transcription terminators and locally protect the 3' ends from exonuclease action. The expression levels of the four Arc proteins correlated approximately with the relative abundance of the corresponding mRNA segments. In conclusion, mRNA processing and, presumably, partial termination of transcription contribute to differential gene expression within the arc operon.
Amino Acid Transport Systems↗
Global control in Pseudomonas fluorescens mediating antibiotic synthesis and suppression of black root rot of tobacco.
Pseudomonas fluorescens CHA0 colonizes plant roots, produces several secondary metabolites in stationary growth phase, and suppresses a number of plant diseases, including Thielaviopsis basicola-induced black root rot of tobacco. We discovered that mutations in a P. fluorescens gene named gacA (for global antibiotic and cyanide control) pleiotropically block the production of the secondary metabolites 2,4-diacetylphloroglucinol (Phl), HCN, and pyoluteorin. The gacA mutants of strain CHA0 have a drastically reduced ability to suppress black root rot under gnotobiotic conditions, supporting the previous observations that the antibiotic Phl and HCN individually contribute to the suppression of black root rot. The gacA gene is directly followed by a uvrC gene. Double gacA-uvrC mutations render P. fluorescens sensitive to UV irradiation. The gacA-uvrC cluster is homologous to the orf-2 (= uvrY)-uvrC operon of Escherichia coli. The gacA gene specifies a trans-active 24-kDa protein. Sequence data indicate that the GacA protein is a response regulator in the FixJ/DegU family of two-component regulatory systems. Expression of the gacA gene itself was increased in stationary phase. We propose that GacA, perhaps activated by conditions of restricted growth, functions as a global regulator of secondary metabolism in P. fluorescens.
Amino Acid Sequence↗
arcD, the first gene of the arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa, encodes an arginine-ornithine exchanger.
In the absence of oxygen and nitrate, Pseudomonas aeruginosa metabolizes arginine via the arginine deiminase pathway, which allows slow growth on rich media. The conversion of arginine to ornithine, CO2, and NH3 is coupled to the production of ATP from ADP. The enzymes of the arginine deiminase pathway are organized in the arcDABC operon. The arcD gene encodes a hydrophobic polytopic membrane protein. Translocation of arginine and ornithine in membrane vesicles derived from an Escherichia coli strain harboring a recombinant plasmid carrying the arcD gene was studied. Arginine and ornithine uptake was coupled to the proton motive force with a bias toward the transmembrane electrical potential. Accumulated ornithine was readily exchangeable for external arginine or lysine. The exchange was several orders of magnitude faster than proton motive force-driven transport. The ArcD protein was reconstituted in proteoliposomes after detergent solubilization of membrane vesicles. These proteoliposomes mediate a stoichiometric exchange between arginine and ornithine. It is concluded that the ArcD protein is a transport system that catalyzes an electroneutral exchange between arginine and ornithine to allow high-efficiency energy conversion in the arginine deiminase pathway.
Amino Acid Transport Systems↗
[Thrombin-antithrombin III complex. Cause of venous vascular occlusion of the retina].
The influence of a disturbed hemostasis as one of the causes of retinal vein occlusions is still controversial. We investigated the functional state of the coagulation system in 16 patients, 7 with a nonischemic and 9 with an ischemic retinal vein occlusion, with an enzyme-linked immunosorbent assay for the determination of thrombin-antithrombin III complex (TAT). Patients with a history of thromboembolic disease, raised blood pressure and/or badly managed diabetes mellitus were excluded from the investigations. In healthy individuals the plasma concentration is 1.45 ng/ml +/- 0.4 (mean value +/- SD), ranging from 1.0 to 4.1 ng/ml. In our patients we measured TAT concentrations ranging from 2.0 to 48.0 ng/ml. In 2 of 7 plasma samples from patients with nonischemic retinal vein occlusion (2.1-6.3 ng/ml, mean = 3.3, SE +/- 0.6) and in 6 of 9 in ischemic retinal vein occlusion (2.0-48.0, mean = 13.2, SE +/- 5.1) TAT concentrations were found to be increased. These data indicate that disturbed hemostasis may be involved in retinal vein occlusion, especially that caused by ischemia. Furthermore, TAT may be useful in differentiating ischemic from nonischemic retinal vein occlusion.
Antithrombin III↗
Indole-3-acetic acid (IAA) synthesis in the biocontrol strain CHA0 of Pseudomonas fluorescens: role of tryptophan side chain oxidase.
Pseudomonas fluorescens strain CHA0 is an effective biocontrol agent against soil-borne fungal plant pathogens. In this study, indole-3-acetic acid (IAA) biosynthesis in strain CHA0 was investigated. Two key enzyme activities were found to be involved: tryptophan side chain oxidase (TSO) and tryptophan transaminase. TSO was induced in the stationary growth phase. By fractionation of a cell extract of strain CHA0 on DEAE-Sepharose, two distinct peaks of constitutive tryptophan transaminase activity were detected. A pathway leading from tryptophan to IAA via indole-3-acetamide, which occurs in Pseudomonas syringae subsp. savastanoi, was not present in strain CHA0. IAA synthesis accounted for less than or equal to 1.5% of exogenous tryptophan consumed by resting cells of strain CHA0, indicating that the bulk of tryptophan was catabolized via yet another pathway involving anthranilic acid as an intermediate. Strain CHA750, a mutant lacking TSO activity, was obtained after Tn5 mutagenesis of strain CHA0. In liquid cultures (pH 6.8) supplemented with 10 mM-L-tryptophan, growing cells of strains CHA0 and CHA750 synthesized the same amount of IAA, presumably using the tryptophan transaminase pathway. In contrast, resting cells of strain CHA750 produced five times less IAA in a buffer (pH 6.0) containing 1 mM-L-tryptophan than did resting cells of the wild-type, illustrating the major contribution of TSO to IAA synthesis under these conditions. In artificial soils at pH approximately 7 or pH approximately 6, both strains had similar abilities to suppress take-all disease of wheat or black root rot of tobacco. This suggests that TSO-dependent IAA synthesis is not essential for disease suppression.
Bacterial Proteins↗
Anaerobic growth and cyanide synthesis of Pseudomonas aeruginosa depend on anr, a regulatory gene homologous with fnr of Escherichia coli.
Anaerobic growth of Pseudomonas aeruginosa on nitrate or arginine requires the anr gene, which codes for a positive control element (ANR) capable of functionally complementing an fnr mutation in Escherichia coli. The anr gene was sequenced; it showed 51% identity with the fnr gene at the amino acid sequence level. Four cysteine residues known to be essential in the FNR protein are conserved in ANR. The anr gene product (deduced Mr 27,129) was visualized by the maxicell method and migrated like a 32 kDa protein in gel electrophoresis under denaturing conditions. An anr mutant of P. aeruginosa constructed by gene replacement was defective in nitrate respiration, arginine deiminase activity, and hydrogen cyanide biosynthesis, underscoring the diverse metabolic functions of ANR during oxygen limitation. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, and Pseudomonas mendocina all had a functional analogue of ANR, indicating that similar anaerobic control mechanisms exist in these bacteria.
Amino Acid Sequence↗
Anaerobic regulation of transcription initiation in the arcDABC operon of Pseudomonas aeruginosa.
The arcDABC operon of Pseudomonas aeruginosa encodes the enzymes of the arginine deiminase pathway, which is inducible under conditions of oxygen limitation and serves to generate ATP from arginine. The 5' end of arc mRNA extracted from anaerobically grown cells was determined by S1 and primer extension mapping. The transcription initiation site was located upstream of the arcD gene and 41.5 bp downstream of the center of the sequence TTGAC....ATCAG. This sequence, termed the ANR box, is similar to the consensus FNR recognition site of Escherichia coli. Transcription of the arc operon in P. aeruginosa was strongly decreased by a deletion of the TTGAC half site or by a mutation in the anr gene, which is known to code for the FNR-like regulatory protein ANR. During a transition from aerobic to anaerobic growth conditions, the concentrations of arc mRNAs and the levels of the ArcD and ArcA proteins rose in a parallel fashion. Mutational analysis of the arc promoter region led to the conclusion that the distance between the ANR box and the -10 promoter region is important for promoter strength, whereas the -35 region does not appear to be critical for arc promoter function. These findings and previous results indicate that anaerobic induction of the arc operon occurs at the level of transcription and requires the ANR box in cis and the ANR protein in trans.
Amino Acid Sequence↗
Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa.
A mutant of Pseudomonas aeruginosa was characterized which could not grow anaerobically with nitrate as the terminal electron acceptor or with arginine as the sole energy source. In this anr mutant, nitrate reductase and arginine deiminase were not induced by oxygen limitation. The anr mutation was mapped in the 60-min region of the P. aeruginosa chromosome. A 1.3-kb chromosomal fragment from P. aeruginosa complemented the anr mutation and also restored anaerobic growth of an Escherichia coli fnr deletion mutant on nitrate medium, indicating that the 1.3-kb fragment specifies an FNR-like regulatory protein. The arcDABC operon, which encodes the arginine deiminase pathway enzymes of P. aeruginosa, was rendered virtually noninducible by a deletion or an insertion in the -40 region of the arc promoter. This -40 sequence (TTGAC....ATCAG) strongly resembled the consensus FNR-binding site (TTGAT....ATCAA) of E. coli. The cloned arc operon was expressed at low levels in E. coli; nevertheless, some FNR-dependent anaerobic induction could be observed. An FNR-dependent E. coli promoter containing the consensus FNR-binding site was expressed well in P. aeruginosa and was regulated by oxygen limitation. These findings suggest that P. aeruginosa and E. coli have similar mechanisms of anaerobic control.
Anaerobiosis↗
Converting catabolic ornithine carbamoyltransferase to an anabolic enzyme.
Pseudomonas aeruginosa has an anabolic and a catabolic ornithine carbamoyltransferase (OTCase). In vitro, these homologous enzymes catalyze the same reaction (ornithine + carbamoyl phosphate (CP) in equilibrium citrulline + Pi), yet in vivo they function unidirectionally owing to specific kinetic properties. The catabolic OTC-ase cannot promote the anabolic reaction (citrulline formation) in vivo because of a sigmoidal CP saturation curve and a high CP concentration for half-maximal velocity. The structural basis for this kinetic specialization was examined. The catabolic OTCase lost most of its homotropic cooperativity and gained anabolic activity when an amino acid residue near the CP binding site, Glu-106, was replaced by alanine or glycine. In the anabolic OTCase of Escherichia coli the glutamine residue corresponding to Glu-106 was exchanged for glutamate; however, in this case no CP cooperativity was acquired. Thus, in catabolic OTCase, sequence features in addition to Glu-106 are important for sigmoidal CP saturation, and such a sequence was identified in the C-terminal part. By an in vivo gene fusion technique the 9 C-terminal amino acids of catabolic OTCase were replaced by the homologous 8 amino acids from anabolic OTCase of E. coli; the hybrid enzyme had a markedly reduced homotropic cooperativity. This gene fusion method should be generally useful for directed enzyme evolution.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
Comparison of proC and other housekeeping genes of Pseudomonas aeruginosa with their counterparts in Escherichia coli.
In a comparative study of housekeeping genes of Pseudomonas aeruginosa and Escherichia coli, the nucleotide sequence of a proline biosynthetic gene, proC, of P. aeruginosa has been determined. The subunit molecular mass (approximately 29 kDa) and the N-terminal amino acid sequence of purified delta 1-pyrroline 5-carboxylate reductase, the proC gene product, were in agreement with the proC nucleotide sequence. A survey of pairs of isofunctional genes from P. aeruginosa and E. coli reveals that within each pair, translated genes (including proC) have diverged more strongly than have untranslated genes specifying ribosomal or transfer RNAs. The translated genes, but not the untranslated ones, have a G + C content that is typical of the respective genomic G + C contents.
Amino Acid Sequence↗
The istA gene of insertion sequence IS21 is essential for cleavage at the inner 3' ends of tandemly repeated IS21 elements in vitro.
The bacterial 2.1 kb insertion sequence IS21 occurs as a tandem repeat [=(IS21)2] on the broad host range plasmid R68.45. In (IS21)2, the two IS21 elements are separated by 3 bp termed junction sequence. Plasmids carrying (IS21)2 form cointegrates with other replicons at high frequencies. The two IS21 genes, istA and istB, were found to be necessary for cointegrate formation in vivo. Since the outer ends of (IS21)2 are dispensable for cointegrate formation, we favor a transposition model according to which a plasmid carrying (IS21)2 is cleaved at the junction sequence; the opened plasmid is then inserted into a target replicon. Here we show that Escherichia coli cell extracts, which contained over-produced IstA protein, nicked a supercoiled (IS21)2 plasmid precisely at the inner 3' termini of IS21; the resulting staggered cut generated 5' protrusions. The istA gene, but not the istB gene, was required for in vitro cleavage of an IS21-IS21 junction. Because of this cleavage and our previous findings (generation of 4 bp target duplications and loss of the junction sequence after cointegrate formation in vivo) we propose that plasmids with (IS21)2 produce cointegrates by a mechanism which involves joining of the inner 3' ends of IS21 to the 5' ends of the target.
Base Sequence↗
Sequence analysis and expression of the arginine-deiminase and carbamate-kinase genes of Pseudomonas aeruginosa.
The arcABC operon of Pseudomonas aeruginosa encodes arginine deiminase, catabolic ornithine carbamoyltransferase and carbamate kinase, respectively. We have determined the nucleotide sequences of the arcA and arcC genes. The arcA open reading frame specifies a polypeptide of 46.3 kDa. The same molecular mass was obtained for the subunit of purified arginine deiminase after electrophoresis under denaturing conditions. The N-terminal amino acid sequence of arginine deiminase was in agreement with the corresponding nucleotide sequence. The native arginine deiminase had an estimated molecular mass of 175-180 kDa, suggesting a tetrametric structure. The enzyme was activated by Mg2+ or Mn2+ and strongly inhibited by Zn2+. The apparent Km for L-arginine was 0.04 mM in the presence of Mg2+ and 0.47 mM without Mg2+. The arcC open reading frame codes for a 33-kDa protein, confirming the molecular mass previously reported for the subunit of carbamate kinase. The translation-initiation site of arcC was determined by deletion mapping. Two regions of dyad symmetry found between arcA and arcC might stabilize the putative arcABC transcript in the upstream (arcA) region; this might contribute to the high level of arcA expression as compared to the moderate level of arcC expression. Carbamate kinase had 37% sequence similarity (and 13.5% identity) with the C-terminal part of carbamoyl-phosphate synthetase (large subunit) from Escherichia coli. Arginine deiminase had no apparent similarity with argininosuccinate lyase. Thus, the arcA and arcC genes do not appear to be closely related to arginine biosynthetic genes, whereas it had previously been shown that the arcB gene has a high degree of identity with the arginine biosynthetic argF genes of P. aeruginosa and E. coli.
Amino Acid Sequence↗