Biomedical subjects

L A Sayavedra-Soto

Publications and source records attributed to L A Sayavedra-Soto.

At least 19 recordsLinked to original sources

PubMed2006

Product repression of alkane monooxygenase expression in Pseudomonas butanovora.

Physiological and regulatory mechanisms that allow the alkane-oxidizing bacterium Pseudomonas butanovora to consume C2 to C8 alkane substrates via butane monooxygenase (BMO) were examined. Striking differences were observed in response to even- versus odd-chain-length alkanes. Propionate, the downstream product of propane oxidation and of the oxidation of other odd-chain-length alkanes following beta-oxidation, was a potent repressor of BMO expression. The transcriptional activity of the BMO promoter was reduced with as little as 10 microM propionate, even in the presence of appropriate inducers. Propionate accumulated stoichiometrically when 1-propanol and propionaldehyde were added to butane- and ethane-grown cells, indicating that propionate catabolism was inactive during growth on even-chain-length alkanes. In contrast, propionate consumption was induced (about 80 nmol propionate consumed.min(-1).mg protein(-1)) following growth on the odd-chain-length alkanes, propane and pentane. The induction of propionate consumption could be brought on by the addition of propionate or pentanoate to the growth medium. In a reporter strain of P. butanovora in which the BMO promoter controls beta-galactosidase expression, only even-chain-length alcohols (C2 to C8) induced beta-galactosidase following growth on acetate or butyrate. In contrast, both even- and odd-chain-length alcohols (C3 to C7) were able to induce beta-galactosidase following the induction of propionate consumption by propionate or pentanoate.

Alcohols↗

PubMed2005

Effects of dichloroethene isomers on the induction and activity of butane monooxygenase in the alkane-oxidizing bacterium "Pseudomonas butanovora".

We examined cooxidation of three different dichloroethenes (1,1-DCE, 1,2-trans DCE, and 1,2-cis DCE) by butane monooxygenase (BMO) in the butane-utilizing bacterium "Pseudomonas butanovora." Different organic acids were tested as exogenous reductant sources for this process. In addition, we determined if DCEs could serve as surrogate inducers of BMO gene expression. Lactic acid supported greater rates of oxidation of the three DCEs than the other organic acids tested. The impacts of lactic acid-supported DCE oxidation on BMO activity differed among the isomers. In intact cells, 50% of BMO activity was irreversibly lost after consumption of approximately 20 nmol mg protein(-1) of 1,1-DCE and 1,2-trans DCE in 0.5 and 5 min, respectively. In contrast, a comparable loss of activity required the oxidation of 120 nmol 1,2-cis DCE mg protein(-1). Oxidation of similar amounts of each DCE isomer ( approximately 20 nmol mg protein(-1)) produced different negative effects on lactic acid-dependent respiration. Despite 1,1-DCE being consumed 10 times faster than 1,2,-trans DCE, respiration declined at similar rates, suggesting that the product(s) of oxidation of 1,2-trans DCE was more toxic to respiration than 1,1-DCE. Lactate-grown "P. butanovora" did not express BMO activity but gained activity after exposure to butane, ethene, 1,2-cis DCE, or 1,2-trans DCE. The products of BMO activity, ethene oxide and 1-butanol, induced lacZ in a reporter strain containing lacZ fused to the BMO promoter, whereas butane, ethene, and 1,2-cis DCE did not. 1,2-trans DCE was unique among the BMO substrates tested in its ability to induce lacZ expression.

Alkanes↗

PubMed2001

Induction of butane consumption in Pseudomonas butanovora.

The induction of the enzyme activities involved in butane metabolism in Pseudomonas butanovora was characterized. P. butanovora was grown on butane or its metabolites, both singly and in mixtures with other growth substrates. Cells grown in each of the butane metabolites readily consumed the growth substrate and downstream metabolites, but consumed the upstream butane metabolites more slowly. Upstream activities in the butane metabolism could be induced by downstream metabolites, but to much lower levels than with the primary substrate. The induction of butane oxidation was not repressed when P. butanovora was grown or incubated in a mixture of butane and 1-butanol, butyraldehyde or butyrate. However, no induction of butane consumption was observed in a mixture of butane and lactate, which is indicative of catabolite repression. In lactate-grown cells that were rid of the growth substrate and incubated with butane and acetylene (to inactivate newly formed butane monooxygenase), the consumption of butane, 1-butanol and butyraldehyde consumption was not induced. The overall results suggest an independent regulatory mechanism for each of the enzyme activities in butane metabolism. In addition, a low, constitutive butane oxidation was observed in cells grown on substrates other than butane metabolites.

1-Butanol↗

PubMed2001

Bioaugmentation of butane-utilizing microorganisms to promote cometabolism of 1,1,1-trichloroethane in groundwater microcosms.

The transformation of 1,1,1-trichloroethane (1,1,1-TCA) in bioaugmented and non-augmented microcosms was evaluated. The microcosms contained groundwater and aquifer materials from a test site at Moffett Field, Sunnyvale, CA. The initial inoculum for bioaugmentation was a butane-utilizing enrichment from the subsurface of the Hanford DOE site. The non-augmented microcosm required 80 days of incubation before butane-utilization was observed while the augmented microcosms required 3 days. Initially the augmented microcosms were effective in transforming 1,1,1-TCA, but their transformation ability decreased after prolonged incubation. The non-augmented microcosms initially showed limited 1,1,1-TCA transformation but improved with time. After 440 days, both the non-augmented and augmented microcosms had similar transformation yields (0.04 mg 1,1,1-TCA/mg butane) and had similar microbial composition (DNA fingerprints). Subsequent microcosms, when bioaugmented with a Hanford enrichment that was repeatedly grown in 100% mineral media, did not effectively grow or transform 1,1,1-TCA under groundwater nutrient conditions. Microcosm tests to study the effect of mineral media on transformation ability were performed with the Hanford enrichment. Microcosms with 50% mineral media in groundwater most effectively utilized butane and transformed 1,1,1-TCA, while microcosms with groundwater only and microcosms with 5% mineral media in groundwater lost their 1,1,1-TCA transformation ability. DNA fingerprinting indicated shifts in the microbial composition with the different mineral media combinations. Successful bioaugmentation was achieved by enriching butane-utilizers from Moffett Field microcosms that were effective in groundwater with no mineral media added. The results suggest that successful in-situ bioaugmentation might be achieved through the addition of enriched cultures that perform well under subsurface nutrient conditions.

Biodegradation, Environmental↗

PubMed2001

Transcript analysis of multiple copies of amo (encoding ammonia monooxygenase) and hao (encoding hydroxylamine oxidoreductase) in Nitrosomonas europaea.

The genes encoding ammonia monooxygenase (amoCAB), hydroxylamine oxidoreductase (hao), and the c-type cytochrome c-554 (hcy) are present in multiple copies in the genome of Nitrosomonas europaea. The upstream regions of the two copies of amoC, the three copies of hao, and one copy of hcy were cloned and sequenced. Primer extension reactions were done to identify transcription start sites for these genes, as well as for amoA. Putative sigma(70) promoter sequences were found associated with all but one of the mapped transcription start sites. Primer extensions were done with amoC primers using RNA harvested from cells incubated with and without ammonium. The experiments suggested that N. europaea cells may be able to use different promoters in the presence and absence of ammonium.

Ammonia↗

PubMed2000

Differential regulation of amoA and amoB gene copies in Nitrosomonas europaea.

Nitrosomonas europaea contains two nearly identical copies of the operon, amoCAB, which encodes the ammonia monooxygenase (AMO) enzyme. Cells of N. europaea containing single mutations in either amoA or amoB gene copies were incubated in ammonium both prior to and after exposure to acetylene or light. For each strain, the O(2) consumption rates and amounts of AmoA polypeptide, the active site-containing subunit of AMO, produced in each strain were determined. Strains carrying a mutation in either the amoA(2) or amoB(2) genes responded similarly to wild-type cells, but the strains carrying mutations in the amoA(1) or amoB(1) genes responded differently from the wild-type, or from each other. These results suggest that the copies of amoA and amoB are differentially regulated upon exposure to different external stimuli.

Acetylene↗

PubMed1998

Transcription of the amoC, amoA and amoB genes in Nitrosomonas europaea and Nitrosospira sp. NpAV.

Nitrifying bacteria such as Nitrosomonas europaea and Nitrosospira sp. NpAV use ammonia monooxygenase (AMO) for oxidation of their primary growth substrate, ammonia. Two polypeptides of AMO are coded for by contiguous genes, amoA and amoB, which are preceded by a third gene, amoC. The amoCAB clusters are present in multiple copies in nitrifying bacteria of the beta subdivision. These bacteria also have one amoC copy that is not adjacent to a copy of amoAB. The seven known amoC genes in different nitrifiers code for similar polypeptides (> 68%). Reverse transcriptase-polymerase chain reactions and Northern blots indicated that amoC from the amoCAB cluster is contained on a transcript with amoAB. Two other transcripts were detected with amo probes and may be a product of processing of the amoCAB mRNA or independent transcripts.

Amino Acid Sequence↗

PubMed1998

Mutagenesis and expression of amo, which codes for ammonia monooxygenase in Nitrosomonas europaea.

Nitrosomonas europaea has two copies of the operon encoding ammonia monooxygenase (AMO). The nucleotide sequences of the two copies of amoA were obtained, and they were found to differ by one nucleotide. To determine if both copies of amoA were functional, insertional mutagenesis was performed to inactivate either copy of amoA alone. A DNA cassette containing the lacZ and kan genes inserted into amoA was constructed. Mutagenesis was done by using transformation and homologous recombination to mobilize the cassette into the chromosomal copies of amoA. Mutations were obtained in both copies of amoA. Either copy of amoA was sufficient to support growth when the other copy was disrupted. However, inactivation of one copy of amoA, but not the other, resulted in slower growth. Measurements of ammonia-dependent O2 consumption, which depends on AMO, confirmed that the slower-growing mutant had lower activity while the faster-growing mutant had near wild-type levels of activity. Similarly, as measured by [14C]acetylene label incorporation, there was less active AMO present in the slower-growing mutant than in the faster-growing mutant or in the wild type. Northern blot analysis of transcription likewise showed that the slower-growing mutant had less full-sized AMO mRNA.

Acetylene↗

PubMed1996

Comparison of isotope exchange, H2 evolution, and H2 oxidation activities of Azotobacter vinelandii hydrogenase.

Azotobacter vinelandii hydrogenase was purified aerobically with a 35% yield. The purified enzyme catalyzed H2 oxidation at much greater velocity than H2 evolution. There was a large difference in activation energy for the two reactions. EA was 10 kcal/mol for H2 oxidation and 22 kcal/mol for evolution. This difference in activation energies between the two reactions means that the ratio of oxidation velocity to evolution velocity drops from 70 at 33 degrees C to 8 at 48 degrees C. With D2 and H2O as substrates, both membranes and purified enzyme produced only H2 and no HD in the isotope exchange reaction. The velocity of isotope exchange was equal to the velocity of H2 evolution from reduced methyl viologen, indicating that the two reactions share the same rate-limiting step. D2 and H2 inhibited H2 evolution, but D2 did not inhibit isotope exchange. We conclude that H2 and D2 do not inhibit H2 evolution by competing with H+ for the active site of the reduced enzyme. The Km for D2 in isotope exchange is 40-times greater than its Km in D2 oxidation. The difference in Km cannot be accounted for by differences in kcat. We propose that redox environment regulates hydrogenase's affinity for D2 (and likely H2 as well).

Aerobiosis↗

PubMed1996

Induction of ammonia monooxygenase and hydroxylamine oxidoreductase mRNAs by ammonium in Nitrosomonas europaea.

In Nitrosomonas europaea, ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) catalyse the oxidation of ammonia (NH3) to nitrite (NO2-). A transcript of 3500 bases hybridizes to probes for amoA and amoB (genes that code for AMO proteins). A transcript of 2100 bases hybridizes to probes for hao (the gene that codes for HAO). Induction of the mRNAs detected by amo and hao probes required the presence of ammonium (NH4+). To correlate new levels of mRNA with de novo activity, existent mRNA pools and AMO activity were depleted prior to induction by NH4+. The mRNAs of AMO and HAO were depleted by depriving the cells of energy for at least 8 h; AMO activity was inactivated with acetylene (C2H2) after mRNA depletion. In cells treated this way, levels of new AMO mRNA and de novo AMO enzyme activity were correlated with increased NH4+ concentrations up to 1 mM after 3 h of incubation. HAO mRNA also increased in the NH4(+)-treated cells. Other proteins and RNAs induced by NH4+ were detected in 14CO2-labelling experiments. The AMO and HAO mRNAs were preferentially synthesized during energy-limiting conditions.

Ammonium Sulfate↗

PubMed1996

Mutagenesis of hydroxylamine oxidoreductase in Nitrosomonas europaea by transformation and recombination.

Mutagenesis of Nitrosomonas europaea was achieved by electroporation and recombination. To demonstrate this, an aminoglycoside 3'-phosphotransferase (kan) gene was specifically inserted into each of the three gene copies of hao individually. Southern hybridizations and PCR analysis showed the incorporation of the kan gene at the chosen genetic loci. The isolation of mutant strains was achieved in 7 to 14 days when the strains were grown on solid medium. The induced mutations were stable even in the absence of kanamycin-selective pressure for periods of up to 45 days in culture. The mutant strains did not show an observable phenotype different from that of the wild type when grown under the same conditions.

Base Sequence↗

PubMed1995

Generation of polymerase chain reaction-specific probes for library screening using single degenerate primers.

Degenerate oligonucleotide primers were made to peptide sequences from hydroxylamine oxidoreductase (HAO) from Nitrosomonas europaea. The primers were used singly in PCR reactions to amplify portions of the gene for HAO from genomic DNA. Southern hybridizations using fragments amplified with each primer showed that they labeled the same genomic DNA fragments. The PCR-amplified fragments were successfully used to screen a gene library for clones containing the HAO gene. The method of isolating genes by PCR with single primers has general utility.

Base Sequence↗

PubMed1995

Substitution of Azotobacter vinelandii hydrogenase small-subunit cysteines by serines can create insensitivity to inhibition by O2 and preferentially damages H2 oxidation over H2 evolution.

Mutants in which conserved cysteines 294, 297 or 64 and 65 of the Azotobacter vinelandii hydrogenase small subunit were replaced by serines were studied. Cysteines 294 and 297 are homologous to cysteines 246 and 249 of the Desulfovibrio gigas hydrogenase, and these cysteines are ligands to the [3Fe-4S] clusters (A. Volbeda, M.-H. Charon, C. Piras, E. C. Hatchikian, M. Frey, and J. C. Fontecilla-Camps, Nature (London) 373:580-587, 1995). Cysteine 65 is homologous to cysteine 20 of the D. gigas hydrogenase, and this cysteine is a ligand to the proximal [4Fe-4S] cluster. All three mutants retained some hydrogenase activity. All three mutants studied had H2 oxidation-to-H2 evolution activity ratios with whole cells of approximately 1.5, compared with 46 for the wild type. The changes preferentially deplete H2 oxidation activity, while having less effect on evolution. The K64,65C-->S hydrogenase was partially purified and had a specific activity for the evolution reaction that was 22% that of the wild type, while the oxidation-specific activity was 2% that of the wild type. Because cysteine 65 provides a ligand to the proximal [4Fe-4S] cluster, this cluster can be altered without entirely eliminating enzyme activity. Likewise, the detection of H2 evolution and H2 oxidation activities with whole cells and membranes of the K294C-->S and K297C-->S mutants indicates that the [3Fe-4S] cluster can also be altered or possibly eliminated without entirely eliminating enzyme activity. Membranes with K294C-->S or K297C-->S hydrogenase were uninhibited by O2 in H2 oxidation and uninhibited by H2 in H2 evolution. Wild-type membranes and membranes with K64,65C-->S hydrogenase were both sensitive to these inhibitors. These data indicate that the [3Fe-4S] cluster controls the reversible inhibition of hydrogenase activity by O2 or H2.

Azotobacter vinelandii↗

PubMed1994

Sequence of hcy, a gene encoding cytochrome c-554 from Nitrosomonas europaea.

Cytochrome c-554 (Cyt c-554) was purified from Nitrosomonas europaea. The N-terminal and internal amino acid sequences were determined. A synthetic oligodeoxyribonucleotide primer based on the N-terminal sequence was used to construct a PCR clone. This clone was used to identify genomic DNA fragments containing the gene encoding Cyt c-554. We determined the nucleotide sequence of this gene and named it hcy for hydroxylamine oxidoreductase-linked cytochrome.

Amino Acid Sequence↗

PubMed1994

Characterization of the gene encoding hydroxylamine oxidoreductase in Nitrosomonas europaea.

Hydroxylamine oxidoreductase (HAO) catalyzes the oxidation of hydroxylamine to nitrite in Nitrosomonas europaea. The electrons released in the reaction are partitioned to ammonium monooxygenase and to the respiratory chain. The immediate acceptor of electrons from HAO is believed to be cytochrome c-554 (Cyt c-554). We have isolated a genomic DNA fragment containing the structural gene encoding HAO (hao) and a part of the gene for Cyt c-554. The nucleotide sequence of hao was determined, and its transcription was analyzed. The open reading frame (ORF) encodes amino acid sequences matching the purified peptides of HAO. A 64.28-kDa protein is encoded in this ORF, in close agreement with the empirically determined molecular mass of 63 kDa. The N terminus was located 24 amino acids from the start codon, suggesting the presence of a leader sequence. The putative eight heme-binding peptides were localized in this ORF. The gene for Cyt c-554 was located 1,200 bp downstream from the 3' end of hao. An ORF was identified in the upstream region from hao and may encode a protein of unknown function. Data bank searches did not reveal proteins with substantial similarities to HAO, but they did reveal similarities between Cyt c-554 and other c-type cytochromes.

Amino Acid Sequence↗

PubMed1993

In Azotobacter vinelandii hydrogenase, substitution of serine for the cysteine residues at positions 62, 65, 294, and 297 in the small (HoxK) subunit affects H2 oxidation [corrected].

The essential role of the small (HoxK) subunit of hydrogenase of Azotobacter vinelandii in H2 oxidation was established. This was achieved by modification of the two Cys-X2-Cys amino acid motifs at the N and C termini of the HoxK subunit (Cys-62, -65, -294, and -297). The Cys codons were individually mutated to Ser codons. Modifications in these two motifs resulted in loss of hydrogenase activity. At the N terminus, the mutations of the codons for the motif Cys-62-Thr-Cys-64-Cys-65 decreased the activity of hydrogenase to levels no higher than 30% of those of the parental strain. H2 oxidation with the alternate electron acceptors methylene blue and benzyl viologen was decreased. H2 evolution and exchange activities were also affected. Cys-64 possibly substitutes for either Cys-62 or Cys-65, allowing for partial activity. Mutation of the codons for Cys-294 and Cys-297 to Ser codons resulted in no hydrogenase activity. The results are consistent with alterations of the ligands of FeS clusters in the HoxK subunit of hydrogenase [corrected].

Amino Acid Sequence↗

PubMed1992

The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase.

The roles of the product of the hoxZ gene immediately downstream of the hydrogenase gene (hoxKG) in Azotobacter vinelandii were investigated by constructing and characterizing a mutant with the center of the hoxZ gene deleted. The strain lacking the functional hoxZ gene product exhibited a low rate of H2 oxidation with O2 as the electron acceptor relative to that of the wild-type strain. Nevertheless, when the enzyme was exogenously activated and methylene blue was used as the electron acceptor from hydrogenase, rates of H2 oxidation comparable to those in the wild-type strain were observed. These results suggest that the gene product of hoxZ plays a role in activating and maintaining hydrogenase in a reduced active state. The product of hoxZ could also be the linkage necessary for transfer of electrons from H2 to the electron transport chain.

Azotobacter vinelandii↗

PubMed1992

Taq DNA polymerase for labeling DNA using random primers.

The efficiency of labeling DNA with Taq DNA polymerase for probing nucleic acid blots was evaluated as an alternative to the more common procedure of using the Klenow fragment. The DNA was labeled with Taq DNA polymerase using random primers. The DNA was labeled specifically and efficiently. Synthesized DNA showed fragments of sizes smaller than those produced by the Klenow fragment and could be performed with as little as 0.5 pg of DNA. The use of Taq DNA polymerase appeared to be limited by the amount of radiolabeled nucleotide used and was more sensitive to non-optimized conditions in the reaction mixture than the Klenow fragment. The relative amounts of incorporated nucleotide in comparable conditions were, on occasions, 10% to 25% lower with Taq DNA polymerase than when using the Klenow fragment; nevertheless, the use of the Taq DNA polymerase to label DNA with random primers offers a very good alternative to the Klenow fragment as shown by this report.

Blotting, Southern↗