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F Junker

Publications and source records attributed to F Junker.

12 recordsLinked to original sources
PubMed2001

A WbpL mutant of Pseudomonas putida DOT-T1E strain, which lacks the O-antigenic side chain of lipopolysaccharides, is tolerant to organic solvent shocks.

Lipopolysaccharides (LPS) are major components of the outer membrane of gram-negative bacteria and are considered a defense barrier. To determine if LPS play a role in resistance to solvents in the solvent-tolerant Pseudomonas putida DOT-T1E strain, we have generated mutants unable to synthesize the O-antigen side chain of LPS. The wbpL gene, encoding the enzyme that begins the synthesis of the O-antigen side chain of LPS of the solvent-tolerant strain, was cloned, sequenced, and knocked out in vitro with a cassette encoding kanamycin resistance, and a mutant called WbpL0 of the DOT-T1E strain was generated in vivo by site-directed mutagenesis. The WbpL mutant was compared with the wild-type strain with regard to tolerance to a number of toxic compounds, including chelating agents, organic acids, detergents, and aromatic hydrocarbons. It was found that the mutant was as tolerant as the wild-type strain to organic acids and aromatic hydrocarbons and more sensitive to ethylenediaminetetraacetic acid and deoxycholate.

Amino Acid Sequence↗
PubMed1999

Involvement of the cis/trans isomerase Cti in solvent resistance of Pseudomonas putida DOT-T1E.

Pseudomonas putida DOT-T1E is a solvent-resistant strain that is able to grow in the presence of high concentrations of toluene. We have cloned and sequenced the cti gene of this strain, which encodes the cis/trans isomerase, termed Cti, that catalyzes the cis-trans isomerization of esterified fatty acids in phospholipids, mainly cis-oleic acid (C(16:1,9)) and cis-vaccenic acid (C(18:1,11)), in response to solvents. To determine the importance of this cis/trans isomerase for solvent resistance a Cti-null mutant was generated and characterized. This mutant showed a longer lag phase when grown with toluene in the vapor phase; however, after the lag phase the growth rate of the mutant strain was similar to that of the wild type. The mutant also showed a significantly lower survival rate when shocked with 0.08% (vol/vol) toluene. In contrast to the wild-type strain, which grew in liquid culture medium at temperatures up to 38.5 degrees C, the Cti-null mutant strain grew significantly slower at temperatures above 37 degrees C. An in-frame fusion of the Cti protein with the periplasmic alkaline phosphatase suggests that this constitutively expressed enzyme is located in the periplasm. Primer extension studies confirmed the constitutive expression of Cti. Southern blot analysis of total DNA from various pseudomonads showed that the cti gene is present in all the tested P. putida strains, including non-solvent-resistant ones, and in some other Pseudomonas species.

Bacterial Proteins↗
PubMed1998

Microbial desulfonation.

Organosulfonates are widespread compounds, be they natural products of low or high molecular weight, or xenobiotics. Many commonly found compounds are subject to desulfonation, even if it is not certain whether all the corresponding enzymes are widely expressed in nature. Sulfonates require transport systems to cross the cell membrane, but few physiological data and no biochemical data on this topic are available, though the sequences of some of the appropriate genes are known. Desulfonative enzymes in aerobic bacteria are generally regulated by induction, if the sulfonate is serving as a carbon and energy source, or by a global network for sulfur scavenging (sulfate-starvation-induced (SSI) stimulon) if the sulfonate is serving as a source of sulfur. It is unclear whether an SSI regulation is found in anaerobes. The anaerobic bacteria examined can express the degradative enzymes constitutively, if the sulfonate is being utilized as a carbon source, but enzyme induction has also been observed. At least three general mechanisms of desulfonation are recognisable or postulated in the aerobic catabolism of sulfonates: (1) activate the carbon neighboring the C-SO3- bond and release of sulfite assisted by a thiamine pyrophosphate cofactor; (2) destabilize the C-SO3- bond by addition of an oxygen atom to the same carbon, usually directly by oxygenation, and loss of the good leaving group, sulfite; (3) an unidentified, formally reductive reaction. Under SSIS control, different variants of mechanism (2) can be seen. Catabolism of sulfonates by anaerobes was discovered recently, and the degradation of taurine involves mechanism (1). When anaerobes assimilate sulfonate sulfur, there is one common, unknown mechanism to desulfonate the inert aromatic compounds and another to desulfonate inert aliphatic compounds; taurine seems to be desulfonated by mechanism (1).

Alkanesulfonates↗
PubMed1997

Conjugative plasmids and the degradation of arylsulfonates in Comamonas testosteroni.

Comamonas testosteroni T-2 degrades p-toluenesulfonate (TSA) via p-sulfobenzoate (PSB) and protocatechuate and degrades toluenecarboxylate via terephthalate (TER) and protocatechuate. The appropriate genes are expressed in at least five regulatory units, some of which are also found in C. testosteroni PSB-4 (F. Junker, R. Kiewitz, and A. M. Cook, J. Bacteriol. 179:919-927, 1997). C. testosteroni T-2 was found to contain two plasmids, pTSA (85 kbp) and pT2T (50 kbp); a TSA- mutant (strain TER-1) contained only plasmid pT2T. C. testosteroni PSB-4, which does not degrade TSA, contained one plasmid, pPSB (85 kbp). The type strain contained no plasmids. Conjugation experiments showed that plasmid pTSA (possibly in conjunction with pT2T) was conjugative, and the single copy of the TSA operon (tsaMBCD) with its putative regulator gene (tsaR) in strain T-2 was found on plasmid pTSA, which also carried the PSB genes (psbAC) and presumably transport for both substrates. Plasmid pTSA was assigned to the IncP1 beta group and was found to carry two copies of insertion element IS1071. Plasmid pPSB (of strain PSB-4), which could be maintained in strains with plasmid pTSA or pT2T, was also conjugative and was found to carry the PSB genes as well as to contain two copies of IS1071. In attempted conjugations with the type strain, no plasmid was recovered, but the PSB+ transconjugant carried two copies of IS1071 in the chromosome. We presume the PSB genes to be located in a composite transposon. The genes encoding the putative TER operon and degradation of protocatechuate, with the meta cleavage pathway, were attributed a chromosomal location in strains T-2 and PSB-4.

Amino Acid Sequence↗
PubMed1997

Characterization of the p-toluenesulfonate operon tsaMBCD and tsaR in Comamonas testosteroni T-2.

Comamonas testosteroni T-2 uses a standard, if seldom examined, attack on an aromatic compound and oxygenates the side chain of p-toluenesulfonate (TS) (or p-toluenecarboxylate) to p-sulfobenzoate (or terephthalate) prior to complete oxidation. The expression of the first three catabolic enzymes in the pathway, the TS methyl-monooxygenase system (comprising reductase B and oxygenase M; TsaMB), p-sulfobenzyl alcohol dehydrogenase (TsaC), and p-sulfobenzaldehyde dehydrogenase (TsaD), is coregulated as regulatory unit R1 (H. R. Schlafli Oppenberg, G. Chen, T. Leisinger, and A. M. Cook, Microbiology [Reading] 141:1891-1899, 1995). The components of the oxygenase system were repurified, and the N-terminal amino acid sequences were confirmed and extended. An internal sequence of TsaM was obtained, and the identity of the [2Fe-2S] Rieske center was confirmed by electron paramagnetic resonance spectroscopy. We purified both dehydrogenases (TsaC and TsaD) and determined their molecular weights and N-terminal amino acid sequences. Oligonucleotides derived from the partial sequences of TsaM were used to identify cloned DNA from strain T-2, and about 6 kb of contiguous cloned DNA was sequenced. Regulatory unit R1 was presumed to represent a four-gene operon (tsaMBCD) which was regulated by the LysR-type regulator, TsaR, encoded by a deduced one-gene transcriptional unit. The genes for the inducible TS transport system were not at this locus. The oxygenase system was confirmed to be a class IA mononuclear iron oxygenase, and class IA can now be seen to have two evolutionary groups, the monooxygenases and the dioxygenases, though the divergence is limited to the oxygenase components. The alcohol dehydrogenase TsaC was confirmed to belong to the short-chain, zinc-independent dehydrogenases, and the aldehyde dehydrogenase TsaD was found to resemble several other aldehyde dehydrogenases. The operon and its putative regulator are compared with units of the TOL plasmid.

Alcohol Dehydrogenase↗
PubMed1996

Degradative pathways for p-toluenecarboxylate and p-toluenesulfonate and their multicomponent oxygenases in Comamonas testosteroni strains PSB-4 and T-2.

Three multicomponent oxygenases involved in the degradation of p-toluenesulfonate and p-toluenecarboxylate and the regulation of their synthesis have been examined in three strains (T-2, PSB-4 and TER-1) of Comamonas testosteroni. Strain T-2 utilizes p-toluenesulfonate as a source of carbon and energy for growth via p-sulfobenzoate and protocatechuate, and p-toluenecarboxylate via terephthalate and protocatechuate, and has the unusual property of requiring the reductase (TsaB) of the toluenesulfonate methyl monooxygenase system (TsaMB) in an incompletely expressed sulfobenzoate dioxygenase system (PsbAC) [Schläfli Oppenberg, H.R., Chen, G., Leisinger, T. & Cook, A. M. (1995). Microbiology 141, 1891-1899]. The independently isolated C. testosteroni PSB-4 utilized only sulfobenzoate and terephthalate via protocatechuate. Mutant TER-1, derived from strain T-2, utilized only terephthalate via protocatechuate. We detected no enzymes of the pathway from toluenesulfonate to sulfobenzoate in strains PSB-4 and TER-1, and confirmed by PCR and Southern blot analysis that the genes (tsaMB) encoding toluenesulfonate monooxygenase were absent. We concluded that, in strain PSB-4, the regulatory unit encoding the genes for the conversion of toluenesulfonate to sulfobenzoate was missing, and that generation of mutant TER-1 involved deletion of this regulatory unit and of the regulatory unit encoding desulfonation of sulfobenzoate. The degradation of sulfobenzoate in strain PSB-4 was catalysed by a fully inducible sulfobenzoate dioxygenase system (PsbACPSB-4), which, after purification of the oxygenase component (PsbAPSB-4), turned out to be indistinguishable from the corresponding component from strain T-2 (PsbAT-2). Reductase PsbCPSB-4, which we could separate but not purify, was active with oxygenase PsbAPSB-4 and PsbAT-2. Oxygenase PsbAPSB-4 was shown by electron paramagnetic resonance spectroscopy to contain a Rieske [2Fe-2S] centre. The enzyme system oxygenating terephthalate was examined and the oxygenase component purified and characterized. The oxygenase component in strains T-2 (and mutant TER-1) and PSB-4 were indistinguishable. The reductase component, which we separated but failed to purify, was active with the oxygenase from all strains. Gains and losses of blocks of genes in evolution is discussed.

Benzenesulfonates↗
PubMed1994

Oxygenation and spontaneous deamination of 2-aminobenzenesulphonic acid in Alcaligenes sp. strain O-1 with subsequent meta ring cleavage and spontaneous desulphonation to 2-hydroxymuconic acid.

2-Aminobenzenesulphonic acid (2AS) is degraded by Alcaligenes sp. strain O-1 via a previously detected but unidentified intermediate. A mutant of strain O-1 was found to excrete this intermediate, which was isolated and identified by m.s., 1H- and 13C-n.m.r. as 3-sulphocatechol (3SC). Proteins from cell extracts of strain O-1 were separated by anion-exchange chromatography. A multicomponent oxygenase was observed to convert 1 mol each of NADH, O2 and 2AS into 1 mol each of 3SC, NH3 and NAD+. The enzyme presumably catalysed formation of the ring of a 2-amino-2,3-diol moiety, and elimination in the amino group led to a rearomatization. 3SC was further degraded via meta ring cleavage, which could be prevented by inactivation of the 3-sulphocatechol-2,3-dioxygenase (3SC23O) with 3-chlorocatechol. In Tris buffer, the separated 3SC23O catalysed the reaction of 1 mol each of 3SC and O2 involving a transient yellow intermediate, and release of 1 mol of sulphite and two organic products. The major product was identified by n.m.r. and by g.c./m.s. as 5-carboxypenta-2,4-dien-5-olide (CPDO), an indicator of formation of 2-hydroxymuconic acid (2HM). The second product was identified as the Z,E isomer of 2HM by comparison with authentic material. When the CPDO in the product mixture was chemically hydrolysed to (Z,E)-2HM, 1 mol of (Z,E)-2HM/mol of 3SC was observed. If oxygenation of 3SC by 3SC23O was carried out in phosphate buffer, only a single product was detected, a keto form of 2HM. This dioate was also formed from authentic (Z,E)-2HM in phosphate buffer. Formation of the natural product (Z,E)-2HM from the xenobiotic, 3SC, seems to involve oxygenation to the unstable 2-hydroxy-6-sulphonomuconic acid semialdehyde, which hydrolyses spontaneously to 2HM. There would appear to be at least one spontaneous reaction per enzyme reaction in this pathway.

Alcaligenes↗
PubMed1994

3-Sulphocatechol 2,3-dioxygenase and other dioxygenases (EC 1.13.11.2 and EC 1.14.12.-) in the degradative pathways of 2-aminobenzenesulphonic, benzenesulphonic and 4-toluenesulphonic acids in Alcaligenes sp. strain O-1.

Alcaligenes sp. strain O-1 utilizes three sulphonated aromatic compounds as sole sources of carbon and energy for growth in minimal salts medium-benzenesulphonate (BS), 4-toluenesulphonate (TS) and 2-aminobenzenesulphonate (2AS). The degradative pathway(s) in 2AS-grown cells are initiated with membrane transport, NADH-dependent dioxygenation and meta ring cleavage. The specific activity of the NADH-dependent dioxygenation(s) varied with the growth phase and was maximal near the end of exponential growth for each growth substrate. Cells were harvested at this point from BS-, TS- and 2AS-salts medium. Cells grown with each sulphonated substrate could oxygenate all three compounds, but only 2AS-grown cells consumed 2 mol O2 per mol 2AS or BS or TS. BS- and TS-grown cells consumed 2 mol O2 per mol BS or TS but failed to oxygenate the product of oxygenation of 2AS, 3-sulphocatechol (3SC). These observations were repeated with cell extracts and we concluded that there were two sets of desulphonative pathways in the organism, one for 2AS and one for BS and TS. We confirmed this hypothesis by separating the degradative enzymes from 2AS-, BS- or TS-grown cells. A 2AS dioxygenase system and a 3SC-2,3-dioxygenase (3SC23O) were detected in 2AS-grown cells only. In both BS- and TS-grown cells a dioxygenase system for BS and TS was observed as well as a principal catechol 2,3-dioxygenase (C23O-III), neither of which was present in 2AS-grown cells. The 3SC23O was purified to near homogeneity, found to be monomeric (M(r) 42,000), and to catalyse 2,3-dioxygenation to a product that decayed spontaneously to sulphite and 2-hydroxymuconate. The 2AS dioxygenase system could cause not only deamination of 2AS but also desulphonation of BS and TS. The BS dioxygenase could desulphonate BS and apparently either desulphonate or deaminate 2AS. Strain O-1 thus seems to contain two putative, independently regulated operons involving oxygenation and spontaneous desulphonation(s). One operon encodes at least the 2AS dioxygenase system and 3SC23O whereas the other encodes at least the BS/TS dioxygenase system and C23O-III.

Alcaligenes↗
PubMed1992

Characterization of tertiary interactions in a folded protein by NMR methods: studies of pH-induced structural changes in human growth hormone.

The pH-induced conformational changes in human growth hormone (hGH) have been studied, using a new quantitative NMR approach that combines 13C labeling of specific backbone carbonyl carbons with a complete spectral analysis of the corresponding 13C resonances. Thus, a complete analysis of the carbonyl resonances of the 26 Leu residues of hGH and their variation with pH provided detailed information about the equilibrium folding processes of the protein, including information about the kinetics of the folding. By combining this information with the pH dependence of readily identifiable 1H resonances, the pH-induced changes observed in the carbonyl carbon spectra can be associated with specific regions in the protein and can be ascribed to a series of localized adjustments in the tertiary structure, brought about by changes in the hydrogen bond interactions or electrostatic interactions between different residues in the globular folded protein. The preexchange lifetimes of these adjustments range from a fraction of a millisecond to a few milliseconds.

Amino Acid Sequence↗
PubMed1992

Specific carbon-13 labelling of leucine residues in human growth hormone.

Biosynthetic human growth hormone specifically 13C-labelled in the carbonyl positions of all 26 leucine residues has been obtained by recombinant DNA techniques using 13C-labelled leucine and an E. coli strain that requires leucine. It is shown that, on the whole, the labelling is specific with no significant mislabelling as would have been the case had the 13C-labelled leucine been metabolized.

Carbon Isotopes↗
PubMed1989

Crystallization and X-ray data collection on human growth hormone.

Single crystals of natural sequence human growth hormone have been grown from media containing ethanol, acetone or paraldehyde. Recombinant growth hormone in its native and desamidated form and pituitary hormone have been crystallized. A full native set of diffraction data extending to 3.5 A resolution has been obtained with synchrotron radiation for crystals of recombinant human growth hormone grown from ethanol. The identity of the material in these crystals has been established by anion-exchange chromatography.

Chromatography, Ion Exchange↗