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D Haas

Publications and source records attributed to D Haas.

At least 109 records · Page 6Linked to original sources
PubMed1989

ADP-ribosylation of actin causes increase in the rate of ATP exchange and inhibition of ATP hydrolysis.

ADP-ribosylation of skeletal muscle actin by Clostridium perfringens iota toxin increased the rate of exchange of actin-bound [gamma-32P]ATP by unlabelled ATP about twofold. Increased exchange rates were observed with ATP and ATP[gamma S], much less with ADP but not with AMP or NAD. ADP-ribosylation of skeletal muscle actin reduced "basal" and Mg2+ (1 mM)-induced ATP hydrolysis by about 80%. Similar inhibition of ATP hydrolysis was observed with liver actin ADP-ribosylated by Clostridium botulinum C2 toxin. The data indicate that ADP-ribosylation of actin at Arg-177 largely affects the ATP-binding and ATPase activity.

ADP Ribose Transferases↗
PubMed1989

Cyanide production by Pseudomonas fluorescens helps suppress black root rot of tobacco under gnotobiotic conditions.

Pseudomonas fluorescens CHA0 suppresses black root rot of tobacco, a disease caused by the fungus Thielaviopsis basicola. Strain CHA0 excretes several metabolites with antifungal properties. The importance of one such metabolite, hydrogen cyanide, was tested in a gnotobiotic system containing an artificial, iron-rich soil. A cyanidenegative (hcn) mutant, CHA5, constructed by a gene replacement technique, protected the tobacco plant less effectively than did the wild-type CHA0. Complementation of strain CHA5 by the cloned wild-type hcn genes restored the strain's ability to suppress disease. An artificial transposon carrying the hcn genes of strain CHA0 (Tnhcn) was constructed and inserted into the genome of another P.fluorescens strain, P3, which naturally does not produce cyanide and gives poor plant protection. The P3::Tnhcn derivative synthesized cyanide and exhibited an improved ability to suppress disease. All bacterial strains colonized the roots similarly and did not influence significantly the survival of T.basicola in soil. We conclude that bacterial cyanide is an important but not the only factor involved in suppression of black root rot.

Journal Article↗
PubMed1989

Genetic structure, function and regulation of the transposable element IS21.

The IncP plasmid R68.45 and other plasmids carrying tandem repeats of the insertion sequence IS21 [= (IS21)2] produce replicon fusions via transposition at high frequencies in Escherichia coli and other gram-negative bacteria, whereas plasmids with a single IS21 copy, e.g. R68, give replicon fusions rarely. The 2131 bp nucleotide sequence of IS21 was determined; at the ends there were 11 bp inverted repeats with one mismatch. Two adjacent open reading frames, istA and istB, were located on one DNA strand of IS21. In E. coli maxicells, polypeptides of 46 kDa (the istA gene product) and 30 kDa (the istB gene product) were expressed by (IS21)2 plasmids, but not by IS21 plasmids. Genetic analysis of (IS21)2 plasmids indicates that the IS21-IS21 junctions form a promoter, which initiates transcription of the istAB operon in one of the two IS21 elements. A single IS21 element fused to an inducible external tac promoter expressed both proteins after induction, but did not promote effective replicon fusion, unless an IS21-IS21 junction (the preferred site for IS21 transposase action) was also present on the plasmid carrying the tac-IS21 construct. The sequences located between the IS21 elements in (IS21)2, 3 bp in R68.45 or 2 bp in pME28, were not recovered in the replicon fusion products. Homologous recombination between the directly oriented IS21 elements in the fusion products led to plasmids with a single IS21 insertion. Analysis of the latter showed that IS21 had a low, but not totally random specificity of insertion and created target duplications of 4 bp (occasionally 5 bp).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

[Results of the surgical-radiologic treatment strategy used in laryngeal cancer in Jena patient material from 1964 to 1981].

The retrospective analysis of a radiotherapeutical material includes 452 patients with laryngeal carcinoma of the University Jena during the period from 1964 to 1981. The primary radiotherapy was done according to therapeutic protocol exclusively in glottal tumors of stage T1 and T2, exceptionally refusers of operation or inoperables. The portion of total laryngectomy is accordingly high with 47.6% of all patients. The 5-years-survival of all patients is 48% (217 of 452 patients). The results are compared with results of other therapeutic statements.

Aged↗
PubMed1988

Complete pathologic maturation and regression of stage IVS neuroblastoma without treatment.

Spontaneous maturation of Stage IVS neuroblastoma has been postulated as a mechanism for its favorable prognosis, but this has rarely been documented pathologically. We report on a patient with congenital Stage IVS neuroblastoma who had extensive subcutaneous and bone-marrow involvement. Serial photographs, biopsies, and vanillomandelic acid determinations documented the tumor's initial progression which was followed by spontaneous maturation and involution of the patient's disease over a 6-year period. No cytotoxic therapy was administered. Favorable biologic prognostic factors were documented, including tumor DNA and protein analyses for N-myc amplification or overexpression and analysis for serum neuron-specific enolase and ferritin. Implications for management and therapy of Stage IVS neuroblastoma are discussed with reference to this case and the recent literature.

Biomarkers, Tumor↗
PubMed1988

N2-succinylornithine in ornithine catabolism of Pseudomonas aeruginosa.

Most Pseudomonas aeruginosa PAO mutants which were unable to utilize L-arginine as the sole carbon and nitrogen source (aru mutants) under aerobic conditions were also affected in L-ornithine utilization. These aru mutants were impaired in one or several enzymes involved in the conversion of N2-succinylornithine to glutamate and succinate, indicating that the latter steps of the arginine succinyltransferase pathway can be used for ornithine catabolism. Addition of aminooxyacetate, an inhibitor of the N2-succinylornithine 5-aminotransferase, to resting cells of P. aeruginosa in ornithine medium led to the accumulation of N2-succinylornithine. In crude extracts of P. aeruginosa an ornithine succinyltransferase (L-ornithine:succinyl-CoA N2-succinyltransferase) activity could be detected. An aru mutant having reduced arginine succinyltransferase activity also had correspondingly low levels of ornithine succinyltransferase. Thus, in P. aeruginosa, these two activities might be due to the same enzyme, which initiates aerobic arginine and ornithine catabolism.

Acyltransferases↗
PubMed1988

Low- and intermediate-copy-number cloning vectors based on the Pseudomonas plasmid pVS1.

The cloning vector pME290 (6.8 kb), which is derived from the Pseudomonas plasmid pVS1 and has about 7 copies, was mutagenized in vitro to provide derivatives with altered copy numbers. Thus, pME292 (about 1-3 copies) and pME294 (about 15-20 copies) were isolated. These vectors were used in the characterization of the P. aeruginosa argF gene encoding ornithine carbamoyltransferase.

Cloning, Molecular↗
PubMed1988

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.

Amino Acid Oxidoreductases↗
PubMed1988

Integration of replication-defective R68.45-like plasmids into the Pseudomonas aeruginosa chromosome.

R68.45 and other similar broad-host-range (IncP) plasmids carrying a tandem repeat of the 2.1 kb insertion element IS21 mobilize the chromosome of many different Gram-negative bacteria. To analyse the structure of R68.45-chromosome cointegrates, whose involvement in the mobilization process had been postulated previously, we selected for the stable integration of R68.45-like plasmids into the Pseudomonas aeruginosa chromosome. Two plasmids were chosen: pME28, a transfer-deficient, mobilizable RP1 derivative with an inactive replication control (trfA) gene, and pME487, an R68.45 derivative with a trfA(ts) mutation causing temperature-sensitive replication. Chromosomally integrated pME28 and pME487 were found to be flanked by single IS21 elements. This structure is in agreement with a 'cut-and-paste' mode of R68.45 transposition. pME28 and pME487 showed a low specificity of insertion but rarely (less than 0.1%) induced auxotrophic mutations. Hfr (high-frequency-of-recombination) donors of P. aeruginosa could be obtained by chromosomal integration of pME487 or pME28; in the latter case, the transfer functions lacking from pME28 had to be provided in trans on an autonomous plasmid. Hfr donors gave higher conjugational linkage and transferred longer stretches of the P. aeruginosa chromosome than did R68.45 donors. This suggests that the integration of R68.45 into the donor chromosome is short-lived in P. aeruginosa.

Chromosomes, Bacterial↗
PubMed1988

Anabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa: nucleotide sequence and transcriptional control of the argF structural gene.

In Pseudomonas aeruginosa PAO the anabolic ornithine carbamoyltransferase (OTCase, EC 2.1.3.3) is the product of the argF gene and the only arginine biosynthetic enzyme whose synthesis is repressible by arginine. We have determined the complete nucleotide sequence of the argF gene including its promoter-control region. The deduced amino acid sequence of the anabolic OTCase consists of 305 residues (Mr 33,924), and this was confirmed by the N-terminal amino acid sequence, the total amino acid composition, and the subunit Mr of the purified enzyme. The native anabolic OTCase (Mr 110,000 to 125,000) was found to be a trimer by cross-linking experiments. P. aeruginosa also has a catabolic OTCase (the arcB gene product), which catalyzes the reverse reaction of the anabolic conversion. At the nucleotide sequence level, the P. aeruginosa argF gene had 52.4% identity with the arcB gene. The Escherichia coli argF and argI genes, which code for anabolic OTCase isoenzymes, had 47.3 and 44.9% identity, respectively, with the P. aeruginosa argF sequence. This suggests that these four genes have evolved from a common ancestral gene. The arcB gene appears to be more closely related to the E. coli argF gene than to the P. aeruginosa argF gene. Two transcripts (mRNA-1, mRNA-2) of the P. aeruginosa argF gene were identified by S1 mapping. The transcription initiation site for mRNA-1 was preceded by sequences having partial homology with the E. coli -35 and -10 consensus promoter sequences. No sequence similar to consensus promoters of enteric bacteria was found upstream of the 5' end of mRNA-2. E. coli carrying a P. aeruginosa argF+ recombinant plasmid produced mRNA-1 with low efficiency but no (or very little) mRNA-2. Arginine repressed argF transcription in P. aeruginosa. In the argF promoter region no sequence homologous to the "arg box" (arginine operator module) of E. coli was found. The mechanism of arginine repression in P. aeruginosa thus appears to be different from that in E. coli.

Amino Acid Sequence↗
PubMed1987

Primary and quaternary structure of the catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa. Extensive sequence homology with the anabolic ornithine carbamoyltransferases of Escherichia coli.

We have determined the complete nucleotide sequence of the arcB gene from Pseudomonas aeruginosa strain PAO and we have purified the arcB product, the catabolic ornithine carbamoyltransferase (EC 2.1.3.3), to apparent homogeneity from the same strain. The N-terminal amino acid sequence, the total amino acid composition and the subunit size of the purified enzyme were in agreement with nucleotide sequencing results, which predict a polypeptide of 336 amino acids (Mr 38,108). Crosslinking experiments suggest that the native enzyme (apparent Mr approx. 420,000) basically consists of a trimer aggregating to form nonamers or dodecamers. The arcB gene of P. aeruginosa had strong homology with the argF and argI genes which code for the anabolic ornithine carbamoyltransferase isoenzymes in Escherichia coli; 63% of the nucleotides and 57% of the amino acids were absolutely conserved in arcB and argF. This indicates a close evolutionary relationship between these genes although their products have different physiological functions in the cell. Under conditions of induction (energy depletion) the catabolic ornithine carbamoyltransferase represented greater than or equal to 10% of the total cellular protein. Like other highly expressed Pseudomonas genes, the arcB gene was found not to use seven codons which correspond to minor or weakly interacting tRNA species in E. coli.

Amino Acid Sequence↗
PubMed1987

Mode of replicon fusion mediated by the duplicated insertion sequence IS21 in Escherichia coli.

The insertion sequence IS21 (2.1 kb) originating from the broad-host-range IncP plasmid R68 transposes infrequently; by contrast, the IS21 tandem repeat found on the derivative R68.45 is highly active in transpositional mobilization of other replicons in a variety of Gram-negative bacteria. The mobilized plasmids are joined to R68.45 by single IS21 copies in direct orientation. The formation of IS21 tandem duplications was observed in cointegrates between R68.45 and pBR325::IS21 and also in an RP1::IS21 plasmid derivative in which a segment located between two directly repeated copies of IS21 was deleted spontaneously. We speculate that IS21 tandem repeats can arise when the termini of two IS21 elements are specifically joined in a transposition or deletion event. A resistance gene flanked by two IS21 elements in direct orientation did not behave as a transposon. The omega fragment carrying transcription and translation stop signals was inserted into various sites of the IS21 tandem repeat; in this way it could be shown that the left IS21 element (which is next to the kanamycin resistance gene in R68.45) was 100 times more active in cointegrate formation than was the right-hand element. Cointegrates between the conjugative plasmid R751 and pBR325 derivatives carrying IS21 and IS21::omega in tandem contained a single IS21 at one replicon junction and a single IS21::omega at the other. In the IS21 duplications the inner IS21 ends were preferentially recognized (presumably by IS21 transposase), whereas the outer termini were not required for cointegrate formation. Based on these findings a conservative (simple) pathway of transposition is proposed for R68.45 and other plasmids with an IS21 tandem repeat.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Deletion↗
PubMed1986

Expression of biosynthetic genes from Pseudomonas aeruginosa and Escherichia coli in the heterologous host.

We examine the expression of constitutive or repressible, monocistronic genes from Pseudomonas aeruginosa and Escherichia coli after their transfer to the heterologous host. To this end, chromosomal DNA from P. aeruginosa was cloned into the mobilizable broad-host-range vector pKT240; recombinant plasmids carrying the argA, argF, or proC genes were identified by complementation of the corresponding auxotrophic mutations. The isofunctional E. coli genes and the E. coli proB gene were subcloned into pKT240 from existing recombinant plasmids. The enzyme expression specified by the Pseudomonas genes in E. coli, calculated per gene copy, ranged from 0.3%-5% of the levels observed in Pseudomonas. Fusion of the P. aeruginosa proC gene to the E. coli consensus tac promoter resulted in very high proC enzyme production in E. coli, indicating that, at least in this case, the expression barrier is essentially at the level of transcriptional initiation. The E. coli argA and argF enzymes, which are controlled by repression in their native host, were synthesized constitutively in P. aeruginosa at 5% of the levels measured in E. coli under derepressed conditions. The constitutive E. coli proB and proC genes were expressed at high levels (ca. 50%) in the heterologous host. These results support the idea that P. aeruginosa may be a more permissive host than E. coli for the heterologous expression of genes from gram-negative bacteria.

Chromosomes, Bacterial↗
PubMed1986

Heterologous expression and regulation of the lysA genes of Pseudomonas aeruginosa and Escherichia coli.

The Pseudomonas aeruginosa lysA gene encoding diaminopimelate decarboxylase (DAP-decarboxylase) was cloned into a broad host range vector. This gene complemented a lys mutation at the lys-12 locus of P. aeruginosa and a lysA defect in Escherichia coli. The P. aeruginosa DAP-decarboxylase was synthesized constitutively in P. aeruginosa as well as in E. coli, where the Pseudomonas lysA gene was poorly expressed. By contrast, the E. coli lysA gene was expressed well in P. aeruginosa and subject to lysine regulation when the E. coli LysR activator protein was provided. This indicates that the mechanism of transcriptional activation for the E. coli lysA gene is effective in the heterologous host.

Bacterial Proteins↗
PubMed1986

IS21 insertion in the trfA replication control gene of chromosomally integrated plasmid RP1: a property of stable Pseudomonas aeruginosa Hfr strains.

Broad host range IncP-1 plasmids are able to integrate into the chromosome of gram-negative bacteria. Strains carrying an integrated plasmid can be obtained when the markers of a temperature-sensitive (ts) plasmid derivative are selected at non-permissive temperature; in this way Hfr (high frequency) donor strains can be formed. The integrated plasmids, however, tend to be unstable in the absence of continuous selective pressure. In order to obtain stable Hfr donor strains of Pseudomonas aeruginosa PAO, we constructed a derivative of an RP1 (ts) plasmid, pME134, which was defective in the resolvase gene (tnpR) of transposon Tn801. Chromosomal integration of pME134 was selected in a recombination-deficient (rec-102) PAO strain at 43 degrees C. Plasmid integration occurred at different sites resulting in a useful set of Hfr strains that transferred chromosomal markers unidirectionally. The tnpR and rec-102 mutations prevented plasmid excision from the chromosome. In several (but not all) Hfr strains that grew well and retained the integrated plasmid at temperatures below 43 degrees C, the insertion element IS21 of RP1 was found to be inserted into the trfA locus (specifying an essential trans-acting replication function) of the integrated plasmid. One such Hfr strain was rendered rec+; from its chromosome the pME134::IS21 plasmid (= pME14) was excised and transferred by conjugation to Escherichia coli where pME14 could replicate autonomously only when a helper plasmid provided the trfA+ function in trans. Thus, it appears that trfA inactivation favours the stability of chromosomally integrated RP1 in P. aeruginosa.

Chromosomes, Bacterial↗
PubMed1986

N-Succinylated intermediates in an arginine catabolic pathway of Pseudomonas aeruginosa.

Arginine-nonutilizing (aru) mutants of Pseudomonas aeruginosa strain PAO converted L-arginine to N(2)-succinylarginine or N-succinylglutamate, which were identified by high-voltage electrophoresis and HPLC. Addition of aminooxyacetate, an inhibitor of pyridoxal phosphate-dependent enzymes, to resting cells of the wild-type PAO1 in arginine medium led to the accumulation of N(2)-succinylornithine. Enzyme assays with crude P. aeruginosa extracts established the following pathway: L-arginine + succinyl-CoA --> N(2)-succinylarginine --> N(2)-succinylornithine --> N_succinylglutamate 5-semialdehyde --> N-succinylglutamate --> succinate + glutamate. Succinyl-CoA may be regenerated from glutamate via 2-ketoglutarate. L-Arginine induced the enzymes of the pathway, and succinate caused catabolite repression. Purified N(2)-acetylornithine 5-aminotransferase (N(2)-acetyl-L-ornithine: 2-oxoglutarate aminotransferase, EC 2.6.1.11), an arginine biosynthetic enzyme, efficiently transaminated N(2)-succinylornithine; this explains the enzyme's dual role in arginine biosynthesis and catabolism. The succinylarginine pathway enables P. aeruginosa to utilize arginine efficiently as a carbon source under aerobic conditions, whereas the other three arginine catabolic pathways previously established in P. aeruginosa fulfill different functions.

Journal Article↗
PubMed1986

The arcABC operon required for fermentative growth of Pseudomonas aeruginosa on arginine: Tn5-751-assisted cloning and localization of structural genes.

Pseudomonas aeruginosa is able to utilize L-arginine as the energy source for growth under anaerobic, nitrate-free conditions. Mutations in the chromosomal arcABC gene cluster specifying the inducible arginine deiminase pathway enzymes abolish fermentative growth on arginine. From two different arc::Tn5-751 insertion mutants of P. aeruginosa recombinant plasmids have been derived which carry a resistance marker of transposon Tn5-751 plus flanking parts of the arc region. These recombinant plasmids served to reconstruct in vitro the functional arcABC cluster on a 5.6 kb fragment, which was inserted into the broad-host-range vector pKT240. In P. aeruginosa this 5.6 kb segment complemented arcABC mutations in trans and contained the control region necessary in cis for arc enzyme induction by oxygen limitation and arginine. The results of subcloning experiments and transcriptional lacZ fusions, the polarity of transposon insertions and the effect of external promoters led to the conclusion that the structural genes arcA (for arginine deiminase), arcB (for catabolic ornithine carbamoyltransferase) and arcC (for carbamate kinase) are contiguous and transcribed in the same direction. Thus, the arcABC cluster appears to have the characteristics of an operon. In Escherichia coli the cloned arcABC genes were expressed at low, non-inducible levels; strong vector promoters enhanced arc expression up to 100-fold. This indicates that transcriptional initiation at the arc promoter(s) is poor in E. coli.

Arginine↗