Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (19 results)

  • Thesis
  • Peer Reviewed
UC Davis Electronic Theses and Dissertations (2023)

AbstractA facultative pink-pigmented methylotroph, Methylorubrum populi PINKEL, was isolated from compost containing coffee grounds via a selective enrichment strategy using caffeine (1,3,7-trimethylxanthine) as the sole carbon, nitrogen, and energy source. In addition to caffeine, M. populi PINKEL was also able to grow on other methylxanthines, including theobromine (3,7-dimethylxanthine), paraxanthine (1,7-dimethylxanthine), 7-methylxanthine, and xanthine. Whole cell biodegradation assays in combination with HPLC analysis of spent media confirmed that M. populi PINKEL uses the N-demethylation pathway to degrade caffeine to a mixture of theobromine and paraxanthine. Both of these dimethylxanthines get converted to xanthine via two consecutive N-demethylation steps, with 7-methylxanthine as the intermediate. In silico comparison of the Pseudomonas putida CBB5 ndm genes and the genome of Methylorubrum populi PINKEL helped identify genes that are predicted to encode enzymes of the N-demethylation pathway (amino acid percent identities ranging from 41% to 65%). The ndm genes in M. populi PINKEL are organized in two separate clusters: the first cluster is approximately 3 kb in length and contains the ndmA gene (N1-demethylase; peg_1790); whereas the second cluster measures approximately 11.2 kb and contains the remaining four ndm genes: ndmB (N3-demethylase; peg_1782), ndmC (N7-demethylase; peg_1777), ndmD (reductase; peg_1781), and ndmE (peg_1776). Growth curve and HPLC analysis of each of the five Δndm mutants as well as the corresponding complemented strains helped determine not only which step of the pathway is affected by each individual ndm deletion, but that NdmA is responsible for the N1 and N3 demethylation of caffeine during the first step of the pathway. Transcriptional promoter fusion assays identified the three promoter regions that participate in the transcriptional regulatory system of the five ndm genes, each upstream of the following three genes: ndmA, ndmB, and ndmC. The inability of a ΔndmR (predicted to encode a LysR-type transcriptional regulator) mutant to upregulate expression from any of the three ndm promoter regions during inducing or un-inducing conditions indicates that the LysR protein positively regulates the N-demethylation pathway in M. populi PINKEL. Additionally, it was determined, that any of the intermediates of the N-demethylation pathway containing a methyl group can serve as an inducer of the pathway, although at different levels of induction. This report describes research on the caffeine-degrading bacterium Methylorubrum populi strain PINKEL as a model to study caffeine biodegradation and the characterization of the transcriptional regulatory system for caffeine-degrading enzymes.

    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2009)
    Widespread application of chloronitrobenzenes as feedstocks for the production of industrial chemicals and pharmaceuticals has resulted in extensive environmental contamination with these toxic compounds, where they pose significant risks to the health of humans and wildlife. While biotreatment in general is an attractive solution for remediation, its effectiveness is limited with chloronitrobenzenes due to the small number of strains that can effectively mineralize these compounds and their ability to degrade only select isomers. To address this need, we created engineered strains with a novel degradation pathway that reduces the total number of steps required to convert chloronitrobenzenes into compounds of central metabolism. We examined the ability of 2-nitrotoluene 2,3-dioxygenase from Acidovorax sp. strain JS42, nitrobenzene 1,2-dioxygenase (NBDO) from Comamonas sp. strain JS765, as well as active-site mutants of NBDO to generate chlorocatechols from chloronitrobenzenes, and identified the most efficient enzymes. Introduction of the wild-type NBDO and the F293Q variant into Ralstonia sp. strain JS705, a strain carrying the modified ortho pathway for chlorocatechol metabolism, resulted in bacterial strains that were able to sustainably grow on all three chloronitrobenzene isomers without addition of co-substrates or co-inducers. These first-generation engineered strains demonstrate the utility of nitroarene dioxygenases in expanding the metabolic capabilities of bacteria and provide new options for improved biotreatment of chloronitrobenzene-contaminated sites.
      Cover page: Application of nitroarene dioxygenases in the design of novel strains that degrade chloronitrobenzenes
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2017)
      Pseudomonas putida strain ASAD was isolated from compost because of its ability to utilize aspirin (acetylsalicylic acid) as a carbon and energy source. We report the draft genome sequence of strain ASAD, with an estimated length of 6.9 Mb. Study of this isolate will provide insight into the aspirin biodegradation pathway.
        Cover page: Genome Sequence of Pseudomonas putida Strain ASAD, an Acetylsalicylic Acid-Degrading Bacterium
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2017)
        Soil bacteria such as pseudomonads are widely studied due to their diverse metabolic capabilities, particularly the ability to degrade both naturally occurring and xenobiotic aromatic compounds. Chemotaxis, the directed movement of cells in response to chemical gradients, is common in motile soil bacteria and the wide range of chemicals detected often mirrors the metabolic diversity observed. Pseudomonas putida F1 is a soil isolate capable of chemotaxis toward, and degradation of, numerous aromatic compounds. We showed that P. putida F1 is capable of degrading members of a class of naturally occurring aromatic compounds known as hydroxycinnamic acids, which are components of lignin and are ubiquitous in the soil environment. We also demonstrated the ability of P. putida F1 to sense three hydroxycinnamic acids: p-coumaric, caffeic and ferulic acids. The chemotaxis response to hydroxycinnamic acids was induced during growth in the presence of hydroxycinnamic acids and was negatively regulated by HcaR, the repressor of the hydroxycinnamic acid catabolic genes. Chemotaxis to the three hydroxycinnamic acids was dependent on catabolism, as a mutant lacking the gene encoding feruloyl-CoA synthetase (Fcs), which catalyzes the first step in hydroxycinnamic acid degradation, was unable to respond chemotactically toward p-coumaric, caffeic, or ferulic acids. We tested whether an energy taxis mutant could detect hydroxycinnamic acids and determined that hydroxycinnamic acid sensing is mediated by the energy taxis receptor Aer2.
          Cover page: Pseudomonas putida F1 uses energy taxis to sense hydroxycinnamic acids
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2013)
          The bacterium Pseudonocardia dioxanivorans CB1190 grows on the cyclic ethers 1,4-dioxane (dioxane) and tetrahydrofuran (THF) as sole carbon and energy sources. Prior transcriptional studies indicated that an annotated THF monooxygenase (THF MO) gene cluster, thmADBC, located on a plasmid in CB1190 is upregulated during growth on dioxane. In this work, transcriptional analysis demonstrates that upregulation of thmADBC occurs during growth on the dioxane metabolite β-hydroxyethoxyacetic acid (HEAA) and on THF. Comparison of the transcriptomes of CB1190 grown on THF and succinate (an intermediate of THF degradation) permitted the identification of other genes involved in THF metabolism. Dioxane and THF oxidation activity of the THF MO was verified in Rhodococcus jostii RHA1 cells heterologously expressing the CB1190 thmADBC gene cluster. Interestingly, these thmADBC expression clones accumulated HEAA as a dead-end product of dioxane transformation, indicating that despite its genes being transcriptionally upregulated during growth on HEAA, the THF MO enzyme is not responsible for degradation of HEAA in CB1190. Similar activities were also observed in RHA1 cells heterologously expressing the thmADBC gene cluster from Pseudonocardia tetrahydrofuranoxydans K1.
            Cover page: Oxidation of the Cyclic Ethers 1,4-Dioxane and Tetrahydrofuran by a Monooxygenase in Two Pseudonocardia Species
            • Article
            • Peer Reviewed
            UC Davis Previously Published Works (2014)
            Soil bacteria are generally capable of growth on a wide range of organic chemicals, and pseudomonads are particularly adept at utilizing aromatic compounds. Pseudomonads are motile bacteria that are capable of sensing a wide range of chemicals, using both energy taxis and chemotaxis. Whilst the identification of specific chemicals detected by the ≥26 chemoreceptors encoded in Pseudomonas genomes is ongoing, the functions of only a limited number of Pseudomonas chemoreceptors have been revealed to date. We report here that McpC, a methyl-accepting chemotaxis protein in Pseudomonas putida F1 that was previously shown to function as a receptor for cytosine, was also responsible for the chemotactic response to the carboxylated pyridine nicotinic acid.
              Cover page: Cytosine chemoreceptor McpC in Pseudomonas putida F1 also detects nicotinic acid
              • Article
              • Peer Reviewed
              UC Davis Previously Published Works (2019)
              Although alcohols are toxic to many microorganisms, they are good carbon and energy sources for some bacteria, including many pseudomonads. However, most studies that have examined chemosensory responses to alcohols have reported that alcohols are sensed as repellents, which is consistent with their toxic properties. In this study, we examined the chemotaxis of Pseudomonas putida strain F1 to n-alcohols with chain lengths of 1 to 12 carbons. P. putida F1 was attracted to all n-alcohols that served as growth substrates (C2 to C12) for the strain, and the responses were induced when cells were grown in the presence of alcohols. By assaying mutant strains lacking single or multiple methyl-accepting chemotaxis proteins, the receptor mediating the response to C2 to C12 alcohols was identified as McfP, the ortholog of the P. putida strain KT2440 receptor for C2 and C3 carboxylic acids. Besides being a requirement for the response to n-alcohols, McfP was required for the response of P. putida F1 to pyruvate, l-lactate, acetate, and propionate, which are detected by the KT2440 receptor, and the medium- and long-chain carboxylic acids hexanoic acid and dodecanoic acid. β-Galactosidase assays of P. putida F1 carrying an mcfP-lacZ transcriptional fusion showed that the mcfP gene is not induced in response to alcohols. Together, our results are consistent with the idea that the carboxylic acids generated from the oxidation of alcohols are the actual attractants sensed by McfP in P. putida F1, rather than the alcohols themselves.IMPORTANCE Alcohols, released as fermentation products and produced as intermediates in the catabolism of many organic compounds, including hydrocarbons and fatty acids, are common components of the microbial food web in soil and sediments. Although they serve as good carbon and energy sources for many soil bacteria, alcohols have primarily been reported to be repellents rather than attractants for motile bacteria. Little is known about how alcohols are sensed by microbes in the environment. We report here that catabolizable n-alcohols with linear chains of up to 12 carbons serve as attractants for the soil bacterium Pseudomonas putida, and rather than being detected directly, alcohols appear to be catabolized to acetate, which is then sensed by a specific cell-surface chemoreceptor protein.
                Cover page: Chemotaxis of Pseudomonas putida F1 to Alcohols Is Mediated by the Carboxylic Acid Receptor McfP
                • Article
                • Peer Reviewed
                UC Davis Previously Published Works (2007)

                Background

                The initial step involved in oxidative hydroxylation of monoaromatic and polyaromatic compounds by the microorganism Sphingobium yanoikuyae strain B1 (B1), previously known as Sphingomonas yanoikuyae strain B1 and Beijerinckia sp. strain B1, is performed by a set of multiple terminal Rieske non-heme iron oxygenases. These enzymes share a single electron donor system consisting of a reductase and a ferredoxin (BPDO-FB1). One of the terminal Rieske oxygenases, biphenyl 2,3-dioxygenase (BPDO-OB1), is responsible for B1's ability to dihydroxylate large aromatic compounds, such as chrysene and benzo[a]pyrene.

                Results

                In this study, crystal structures of BPDO-OB1 in both native and biphenyl bound forms are described. Sequence and structural comparisons to other Rieske oxygenases show this enzyme to be most similar, with 43.5 % sequence identity, to naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. While structurally similar to naphthalene 1,2-dioxygenase, the active site entrance is significantly larger than the entrance for naphthalene 1,2-dioxygenase. Differences in active site residues also allow the binding of large aromatic substrates. There are no major structural changes observed upon binding of the substrate. BPDO-FB1 has large sequence identity to other bacterial Rieske ferredoxins whose structures are known and demonstrates a high structural homology; however, differences in side chain composition and conformation around the Rieske cluster binding site are noted.

                Conclusion

                This is the first structure of a Rieske oxygenase that oxidizes substrates with five aromatic rings to be reported. This ability to catalyze the oxidation of larger substrates is a result of both a larger entrance to the active site as well as the ability of the active site to accommodate larger substrates. While the biphenyl ferredoxin is structurally similar to other Rieske ferredoxins, there are distinct changes in the amino acids near the iron-sulfur cluster. Because this ferredoxin is used by multiple oxygenases present in the B1 organism, this ferredoxin-oxygenase system provides the structural platform to dissect the balance between promiscuity and selectivity in protein-protein electron transport systems.
                  Cover page: Structural investigations of the ferredoxin and terminal oxygenase components of the biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1
                  • Article
                  • Peer Reviewed
                  UC Davis Previously Published Works (2022)
                  In this study, the Cutaneotrichosporon dermatis strain M503 was isolated and could efficiently degrade tetracycline, doxycycline, and chlorotetracyline. The characteristics of tetracycline degradation were investigated under a broad range of cultural conditions. Response surface methodology (RSM) predicted that the highest degradation rate of tetracycline could be obtained under the following conditions: 39.69 °C, pH of 8.79, and inoculum dose of 4.0% (v/v, ~3.5 × 106 cells/mL in the medium). In accordance with the five identified degradation products of tetracycline, two putative degradation pathways, which included the shedding of methyl and amino groups, were proposed. Moreover, the well diffusion method showed that the strain of M503 decreases the antibacterial potency of tetracycline, doxycycline, and chlorotetracycline. These findings proposed a putative mechanism of tetracycline degradation by a fungus strain and contributed to the estimation of the fate of tetracycline in the aquatic environment.
                    Cover page: Biodegradation of Tetracycline Antibiotics by the Yeast Strain Cutaneotrichosporon dermatis M503
                    • Article
                    • Peer Reviewed
                    UC Davis Previously Published Works (2020)
                    Malachite green is a common environmental pollutant that poses a great threat to non-target organisms, including humans. This study reports the characterization of a bacterial strain, Pseudomonas veronii JW3-6, which was isolated from a malachite green enrichment culture. This strain degraded malachite green efficiently in a wide range of temperature and pH levels. Under optimal degradation conditions (32.4 °C, pH 7.1, and inoculum amount of 2.5 × 107 cfu/mL), P. veronii JW3-6 could degrade 93.5% of 50 mg/L malachite green within seven days. Five intermediate products from the degradation of malachite green were identified: leucomalachite green, 4-(dimethylamino) benzophenone, 4-dimethylaminophenol, benzaldehyde, and hydroquinone. We propose a possible degradation pathway based on these findings. The present study is the first to report the degradation of malachite green by P. veronii and the identification of hydroquinone as a metabolite in the degradation pathway.
                      Cover page: Pathway and kinetics of malachite green biodegradation by Pseudomonas veronii
                      Top