Biomedical subjects

Eugene L Madsen

Publications and source records attributed to Eugene L Madsen.

12 recordsLinked to original sources

PubMed2006

Expression analysis of the fpr (ferredoxin-NADP+ reductase) gene in Pseudomonas putida KT2440.

The ferredoxin-NADP+ reductase (fpr) participates in cellular defense against oxidative damage. The fpr expression in Pseudomonas putida KT2440 is induced by oxidative and osmotic stresses. FinR, a LysR-type transcriptional factor near the fpr gene in the P. putida KT2440 genome, is required for induction of the fpr under both conditions. We have shown that the fpr and finR gene products can counteract the effects of oxidative and osmotic stresses. Interestingly, FinR-independent expression occurs either during a long period of incubation with paraquat or with high concentrations of oxidative stress agent. This result indicates that there may be additional regulators present in the P. putida KT2440 genome. In contrast to in vivo expression kinetics of fpr from the plant pathogen, Pseudomonas syringae, the fpr gene from P. putida KT2440 exhibited unusually prolonged expression after oxidative stress. Transcriptional fusion and Northern blot analysis studies indicated that the FinR is negatively autoregulated. Expression of the fpr promoter was higher in minimal media than in rich media during exponential phase growth. Consistent with this result, the fpr and finR mutants had a long lag phase in minimal media in contrast to wild-type growth characteristics. Antioxidants such as ascorbate could increase the growth rate of all tested strains in minimal media. This result confirmed that P. putida KT2440 experienced more oxidative stress during exponential growth in minimal media than in rich media. Endogenous promoter activity of the fpr gene is much higher during exponential growth than during stationary growth. These findings demonstrate new relationships between fpr, finR, and the physiology of oxidative stress in P. putida KT2440.

Cell Proliferation↗

PubMed2006

The naphthalene catabolic (nag) genes of Polaromonas naphthalenivorans CJ2: evolutionary implications for two gene clusters and novel regulatory control.

Polaromonas naphthalenivorans CJ2, found to be responsible for the degradation of naphthalene in situ at a coal tar waste-contaminated site (C.-O. Jeon et al., Proc. Natl. Acad. Sci. USA 100:13591-13596, 2003), is able to grow on mineral salts agar media with naphthalene as the sole carbon source. Beginning from a 484-bp nagAc-like region, we used a genome walking strategy to sequence genes encoding the entire naphthalene degradation pathway andadditional flanking regions. We found that the naphthalene catabolic genes in P. naphthalenivorans CJ2 were divided into one large and one small gene cluster, separated by an unknown distance. The large gene cluster (nagRAaGHAbAcAdBFCQEDJI'ORF1tnpA) is bounded by a LysR-type regulator (nagR). The small cluster (nagR2ORF2I"KL) is bounded by a MarR-type regulator (nagR2). The catabolic genes of P. naphthalenivorans CJ2 were homologous to many of those of Ralstonia U2, which uses the gentisate pathway to convert naphthalene to central metabolites. However, three open reading frames (nagY, nagM, and nagN), present in Ralstonia U2, were absent. Also, P. naphthalenivorans carries two copies of gentisate dioxygenase (nagI) with 77.4% DNA sequence identity to one another and 82% amino acid identity to their homologue in Ralstonia sp. strain U2. Investigation of the operons using reverse transcription PCR showed that each cluster was controlled independently by its respective promoter. Insertional inactivation and lacZ reporter assays showed that nagR2 is a negative regulator and that expression of the small cluster is not induced by naphthalene, salicylate, or gentisate. Association of two putative Azoarcus-related transposases with the large cluster and one Azoarcus-related putative salicylate 5-hydroxylase gene (ORF2) in the small cluster suggests that mobile genetic elements were likely involved in creating the novel arrangement of catabolic and regulatory genes in P. naphthalenivorans.

Azoarcus↗

PubMed2005

The use of stable isotope probing techniques in bioreactor and field studies on bioremediation.

Stable isotope probing (SIP) is a molecular technique that allows investigators to follow the flow of atoms in isotopically enriched molecules through complex microbial communities into metabolically active microorganisms. Thus, SIP has immense promise for discovering microorganisms responsible for ecologically important biogeochemical reactions in nature. Applications of SIP to biodegradation and bioremediation processes are still in their infancy. In the past few years, approximately a dozen biodegradation studies using SIP based on the analysis of labeled DNA, RNA or phospholipid fatty acids have been completed. Results have begun to link biomarkers (especially sequences of 16S ribosomal RNA and functional genes) to biodegradation reactions in naturally occurring microbial communities. As extensive compilations of ecologically important genotypes and phenotypes accrue, predictive abilities for contaminant metabolism in particular habitats may be achieved.

Bacteria↗

PubMed2005

Identifying microorganisms responsible for ecologically significant biogeochemical processes.

Throughout evolutionary time, and each day in every habitat throughout the globe, microorganisms have been responsible for maintaining the biosphere. Despite the crucial part that they play in the cycling of nutrients in habitats such as soils, sediments and waters, only rarely have the microorganisms actually responsible for key processes been identified. Obstacles that have traditionally impeded fundamental microbial ecology inquiries are now yielding to technical advancements that have important parallels in medical microbiology. The pace of new discoveries that document ecological processes and their causative agents will no doubt accelerate in the near future, and might assist in ecosystem management.

Anaerobiosis↗

PubMed2005

Use of field-based stable isotope probing to identify adapted populations and track carbon flow through a phenol-degrading soil microbial community.

The goal of this field study was to provide insight into three distinct populations of microorganisms involved in in situ metabolism of phenol. Our approach measured 13CO2 respired from [13C]phenol and stable isotope probing (SIP) of soil DNA at an agricultural field site. Traditionally, SIP-based investigations have been subject to the uncertainties posed by carbon cross-feeding. By altering our field-based, substrate-dosing methodologies, experiments were designed to look beyond primary degraders to detect trophically related populations in the food chain. Using gas chromatography-mass spectrometry (GC/MS), it was shown that (13)C-labeled biomass, derived from primary phenol degraders in soil, was a suitable growth substrate for other members of the soil microbial community. Next, three dosing regimes were designed to examine active members of the microbial community involved in phenol metabolism in situ: (i) 1 dose of [13C]phenol, (ii) 11 daily doses of unlabeled phenol followed by 1 dose of [13C]phenol, and (iii) 12 daily doses of [13C]phenol. GC/MS analysis demonstrated that prior exposure to phenol boosted 13CO2 evolution by a factor of 10. Furthermore, imaging of 13C-treated soil using secondary ion mass spectrometry (SIMS) verified that individual bacteria incorporated 13C into their biomass. PCR amplification and 16S rRNA gene sequencing of 13C-labeled soil DNA from the 3 dosing regimes revealed three distinct clone libraries: (i) unenriched, primary phenol degraders were most diverse, consisting of alpha-, beta-, and gamma-proteobacteria and high-G+C-content gram-positive bacteria, (ii) enriched primary phenol degraders were dominated by members of the genera Kocuria and Staphylococcus, and (iii) trophically related (carbon cross-feeders) were dominated by members of the genus Pseudomonas. These data show that SIP has the potential to document population shifts caused by substrate preexposure and to follow the flow of carbon through terrestrial microbial food chains.

Alphaproteobacteria↗

PubMed2004

Development and application of pyrolysis gas chromatography/mass spectrometry for the analysis of bound trinitrotoluene residues in soil.

TNT (trinitrotoluene) is a contaminant of global environmental significance, yet determining its environmental fate has posed longstanding challenges. To date, only differential extraction-based approaches have been able to determine the presence of covalently bound, reduced forms of TNT in field soils. Here, we employed thermal elution, pyrolysis, and gas chromatography/mass spectrometry (GC/MS) to distinguish between covalently bound and noncovalently bound reduced forms of TNT in soil. Model soil organic matter-based matrixes were used to develop an assay in which noncovalently bound (monomeric) aminodinitrotoluene (ADNT) and diaminonitrotoluene (DANT) were desorbed from the matrix and analyzed at a lower temperature than covalently bound forms of these same compounds. A thermal desorption technique, evolved gas analysis, was initially employed to differentiate between covalently bound and added 15N-labeled monomeric compounds. A refined thermal elution procedure, termed "double-shot analysis" (DSA), allowed a sample to be sequentially analyzed in two phases. In phase 1, all of an added 15N-labeled monomeric contaminant was eluted from the sample at relatively low temperature. In phase 2 during high-temperature pyrolysis, the remaining covalently bound contaminants were detected. DSA analysis of soil from the Louisiana Army Ammunition Plant (LAAP; approximately 5000 ppm TNT) revealed the presence of DANT, ADNT, and TNT. After scrutinizing the DSA data and comparing them to results from solvent-extracted and base/ acid-hydrolyzed LAAP soil, we concluded that the TNT was a noncovalently bound "carryover" from phase 1. Thus, the pyrolysis-GC/MS technique successfully defined covalently bound pools of ADNT and DANT in the field soil sample.

Gas Chromatography-Mass Spectrometry↗

PubMed2004

Polaromonas naphthalenivorans sp. nov., a naphthalene-degrading bacterium from naphthalene-contaminated sediment.

Strain CJ2T, capable of growth on naphthalene as a sole carbon and energy source, was isolated from coal-tar-contaminated freshwater sediment. The Gram reaction of strain CJ2T was negative. The cells were non-spore-forming, non-motile cocci (without flagella). The isolate was found to be an aerobic heterotroph capable of utilizing glucose and other simple sugars. Growth was observed between 4 and 25 degrees C (optimum, 20 degrees C) and between pH 6.0 and 9.0 (optimum, pH 7.0-7.5). The G+C content of the genomic DNA was 61.5 mol% and the major quinone was ubiquinone-8. The peptidoglycan of strain CJ2T was determined as belonging to type A1-gamma, meso-diaminopimelic acid. The major fatty acids of strain CJ2T were 16:1omega7c (67.0%), 16:0 (19.6%), 18:1omega7c (approximately 7.9%) and 10:0 3-OH (approximately 2.5%). The polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Mycolic acid and glycolipids could not be detected. Comparative 16S rDNA analysis indicated that strain CJ2T is related to the family Comamonadaceae and that the nearest phylogenetic relative was Polaromonas vacuolata 34-PT (97.1% similarity). On the basis of the physiological and molecular properties, the naphthalene-degrading isolate was designated Polaromonas naphthalenivorans sp. nov. The type strain is CJ2T (=ATCC BAA-779T=DSM 15660T).

Biodegradation, Environmental↗

PubMed2003

Horizontal transfer of phnAc dioxygenase genes within one of two phenotypically and genotypically distinctive naphthalene-degrading guilds from adjacent soil environments.

Several distinct naphthalene dioxygenases have been characterized to date, which provides the opportunity to investigate the ecological significance, relative distribution, and transmission modes of the different analogs. In this study, we showed that a group of naphthalene-degrading isolates from a polycyclic aromatic hydrocarbon (PAH)-contaminated hillside soil were phenotypically and genotypically distinct from naphthalene-degrading organisms isolated from adjacent, more highly contaminated seep sediments. Mineralization of (14)C-labeled naphthalene by soil slurries suggested that the in situ seep community was more acclimated to PAHs than was the in situ hillside community. phnAc-like genes were present in diverse naphthalene-degrading isolates cultured from the hillside soil, while nahAc-like genes were found only among isolates cultured from the seep sediments. The presence of a highly conserved nahAc allele among gram-negative isolates from the coal tar-contaminated seep area provided evidence for in situ horizontal gene transfer and was reported previously (J. B. Herrick, K. G. Stuart-Keil, W. C. Ghiorse, and E. L. Madsen, Appl. Environ. Microbiol. 63:2330-2337, 1997). Natural horizontal transfer of the phnAc sequence was also suggested by a comparison of the phnAc and 16S ribosomal DNA sequences of the hillside isolates. Analysis of metabolites produced by cell suspensions and patterns of amplicons produced by PCR analysis suggested both genetic and metabolic diversity among the naphthalene-degrading isolates of the contaminated hillside. These results provide new insights into the distribution, diversity, and transfer of phnAc alleles and increase our understanding of the acclimation of microbial communities to pollutants.

Bacteria↗

PubMed2003

Identification and characterization of the conjugal transfer region of the pCg1 plasmid from naphthalene-degrading Pseudomonas putida Cg1.

Hybridization and restriction fragment length polymorphism data (K. G. Stuart-Keil, A. M. Hohnstock, K. P. Drees, J. B. Herrick, and E. L. Madsen, Appl. Environ. Microbiol. 64:3633-3640, 1998) have shown that pCg1, a naphthalene catabolic plasmid carried by Pseudomonas putida Cg1, is homologous to the archetypal naphthalene catabolic plasmid, pDTG1, in P. putida NCIB 9816-4. Sequencing of the latter plasmid allowed PCR primers to be designed for amplifying and sequencing the conjugal transfer region in pCg1. The mating pair formation (mpf) gene, mpfA encoding the putative precursor of the conjugative pilin subunit from pCg1, was identified along with other trb-like mpf genes. Sequence comparison revealed that the 10 mpf genes in pCg1 and pDTG1 are closely related (61 to 84% identity) in sequence and operon structure to the putative mpf genes of catabolic plasmid pWW0 (TOL plasmid of P. putida) and pM3 (antibiotic resistance plasmid of Pseudomonas. spp). A polar mutation caused by insertional inactivation in mpfA of pCg1 and reverse transcriptase PCR analysis of mRNA showed that this mpf region was involved in conjugation and was transcribed from a promoter located upstream of an open reading frame adjacent to mpfA. lacZ transcriptional fusions revealed that mpf genes of pCg1 were expressed constitutively both in liquid and on solid media. This expression did not respond to host exposure to naphthalene. Conjugation frequency on semisolid media was consistently 10- to 100-fold higher than that in liquid media. Thus, conjugation of pCg1 in P. putida Cg1 was enhanced by expression of genes in the mpf region and by surfaces where conditions fostering stable, high-density cell-to-cell contact are manifest.

Bacterial Proteins↗

PubMed2002

Interaction of NahR, a LysR-type transcriptional regulator, with the alpha subunit of RNA polymerase in the naphthalene degrading bacterium, Pseudomonas putida NCIB 9816-4.

NahR, a LysR-type transcriptional regulator, is required for expression of naphthalene catabolic operons. However, detailed mechanisms of transcriptional activation by NahR are poorly understood. Many transcriptional activators make direct contact with RNA polymerase (RNAP) to initiate transcription. We investigated the hypothesis that direct contact between NahR and the alpha subunit of RNAP (alphaRNAP) may be involved in expression of the naphthalene catabolic operons in Pseudomonas putida NCIB 9816-4. Interactions between the NahR and alphaRNAP in P. putida NCIB 9816-4 were analyzed using the yeast two-hybrid system. The results obtained indicate that protein-protein interactions occur between alphaRNAP and the NahR. Gene activation by NahR is consistent with the general transcriptional mechanism of class I transcription factors, which function by contacting alphaRNAP.

Amino Acid Sequence↗

PubMed2002

Detection in coal tar waste-contaminated groundwater of mRNA transcripts related to naphthalene dioxygenase by fluorescent in situ hybridization with tyramide signal amplification.

The ideal ecological metabolic activity assay would be applied to naturally occurring microbial populations immediately fixed in the field, and the assay would focus upon intracellular parameters indicative of a dynamic biogeochemical process. In this study, fluorescent in situ hybridization (FISH) with tyramide signal amplification (TSA) detected intracellular mRNA in bacteria. Detection sensitivity was enhanced by using a Hamamatsu color chilled CCD camera and extended exposure times. Pseudomonas putida NCIB 9816-4, a model naphthalene degrading bacterium, was used to refine the protocol. Probe Ac627BR was developed for detecting naphthalene dioxygenase (nahAc) mRNA transcripts. Only induced cells showed positive hybridization to probe Ac627BR. Results were verified by RNase A or DNase I digestion of samples prior to hybridization. When applied to field-fixed groundwater samples, the naphthalene dioxygenase mRNA probe conferred fluorescence on a subset (approximately 1%) of the cells present in the contaminated groundwater. This methodology represents progress towards achieving one of the longstanding goals of environmental microbiology: to simultaneously ascertain the identity, activity, and biogeochemical impact of individual microorganisms in situ-in soil, water, or sediment where they dwell.

Bacteria↗

PubMed2002

nahR, encoding a LysR-type transcriptional regulator, is highly conserved among naphthalene-degrading bacteria isolated from a coal tar waste-contaminated site and in extracted community DNA.

In Pseudomonas putida strain G7, a LysR-type positive transcriptional activator protein encoded by nahR is necessary for activation of two operons involved in naphthalene catabolism [Schell, M. A. & Poser, E. F. (1989). J Bacteriol 171, 837-846]. The role of an nahR homologue, NCIB-nahR, in another naphthalene-metabolizing bacterium, P. putida NCIB 9816-4 was verified. Targeted disruption of NCIB-nahR by homologous recombination resulted in a growth defect in the presence of naphthalene or salicylate as sole carbon and energy source. The nahR homologues and intergenic regions between nahR-like and nahG-like genes from P. putida NCIB 9816-4 and seven bacteria native to a naphthalene-rich coal tar contaminated site were amplified by PCR using degenerate primers. The amplified nahR homologues and the intergenic regions were cloned and sequenced. Alignment of the deduced amino acid sequences from NahR homologues revealed that NahR-like proteins showed only minor variations in all investigated naphthalene-degrading isolates. The intergenic regions, together with known NahR-binding sites showed the consensus NahR-protein-binding sites (5'-ATTCACGCTN(2)TGAT-3'). Surprisingly, amplified intergenic regions from naphthalene-degrading micro-organisms native to this study site were 100% identical to that of the pDTG1 plasmid (an archetypal naphthalene-catabolic plasmid from Pseudomonas putida NCIB 9816-4), but the nahR coding regions were not. DNA representing the uncultured microbial community was extracted from six sediment samples with varying coal tar exposure histories. PCR amplification of nahR from sediment DNA was observed in contaminated samples, but in uncontaminated samples only following laboratory incubation with naphthalene. The sediment-derived PCR products were sequenced and also found to be almost identical to known nahR genes. Thus, the structure and function of nahR-nahG regulatory genes appear to be highly conserved.

Amino Acid Sequence↗