skip to main content


Title: Nitroreductases of bacterial origin in Giardia lamblia : Potential role in detoxification of xenobiotics
Abstract

The anaerobic parasiteGiardia lamblia, causative agent of persistent diarrhea, contains a family of nitroreductase genes most likely acquired by lateral transfer from anaerobic bacteria or archaebacteria. Two of these nitroreductases, containing a ferredoxin domain at their N‐terminus, NR1, and NR2, have been characterized previously. Here, we present the characterization of a third member of this family, NR3. In functional assays, recombinant NR1 and NR3 reduced quinones like menadione and the antibiotic tetracycline, and—to much lesser extents—the nitro compound dinitrotoluene. Conversely, recombinant NR2 had no activity on tetracycline.Escherichia coliexpressing NR3 were less susceptible to tetracycline, but more susceptible to the nitro compound metronidazole under semi‐aerobic growth conditions.G. lambliaoverexpressing NR1 and NR3, but not lines overexpressing NR2, are more susceptible to the nitro drug nitazoxanide. These findings suggest that NR3 is an active quinone reductase with a mode of action similar to NR1, but different from NR2. The biological function of this family of enzymes may reside in the use of xenobiotics as final electron acceptors. Thereby, these enzymes may provide at least two evolutionary advantages namely a higher potential to recycle NAD(P) as electron acceptors for the (fermentative) energy and intermediary metabolism, and the possibility to inactivate toxic xenobiotics produced by microorganisms living in concurrence inside the intestinal habitat.

 
more » « less
NSF-PAR ID:
10459553
Author(s) / Creator(s):
 ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
MicrobiologyOpen
Volume:
8
Issue:
10
ISSN:
2045-8827
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Glutaredoxins (GRXs) are small oxidoreductase enzymes that can reduce disulfide bonds in target proteins. The class III GRX gene family is unique to land plants, andArabidopsis thalianahas 21 class III GRXs, which remain largely uncharacterized. About 80% ofA. thalianaclass III GRXs are transcriptionally regulated by nitrate, and several recent studies have suggested roles for these GRXs in nitrogen signaling. Our objective was to functionally characterize two nitrate‐induced GRX genes,AtGRXS5andAtGRXS8, defining their roles in signaling and development in theA. thalianaroot. We demonstrated thatAtGRXS5andAtGRXS8are primarily expressed in root and shoot vasculature (phloem), and that the corresponding GRX proteins display nucleo‐cytosolic subcellular localization. Ectopic expression ofAtGRXS8in transgenic plants caused major alterations in root system architecture: Normal primary root development, but a near absence of lateral roots. RNA sequencing demonstrated that the roots ofAtGRXS8‐overexpressing plants show strongly reduced transcript abundance for many primary nitrate response genes, including the major high‐affinity nitrate transporters. Correspondingly, high‐affinity nitrate uptake and the transport of nitrate from roots to shoots are compromised inAtGRXS8‐overexpressing plants. Finally, we demonstrated that the AtGRXS8 protein can physically interact with the TGA1 and TGA4 transcription factors, which are central regulators of early transcriptional responses to nitrate inA. thalianaroots. Overall, these results suggest thatAtGRXS8acts to quench both transcriptional and developmental aspects of primary nitrate response, potentially by interfering with the activity of the TGA1 and TGA4 transcription factors.

     
    more » « less
  2. Abstract

    Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungusCaecomyces churrovisand the methanogenMethanobacterium bryantii(not native to the rumen) were formed, demonstrating that microbes from different environments can be paired based on metabolic ties. Transcriptional and metabolic changes induced by methanogen co-culture were evaluated inC. churrovisacross a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome ofC. churroviswas obtained and annotated, which is the first sequenced genome of a non-rhizoid-forming anaerobic fungus.C. churrovispossess an abundance of CAZymes and carbohydrate binding modules and, in agreement with previous studies of early-diverging fungal lineages, N6-methyldeoxyadenine (6mA) was associated with transcriptionally active genes. Co-culture with the methanogen increased overall transcription of CAZymes, carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose and caused upregulation of genes coding associated enzymatic machinery including carbohydrate binding modules in family 18 and dockerin domains across multiple growth substrates relative toC. churrovismonoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2-fold change values for upregulation of genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of theC. churrovisstrain with a methanogen may enhance pyruvate formate lyase (PFL) function for growth on xylan and fructose and production of bottleneck enzymes in sugar utilization pathways, further supporting the hypothesis that co-culture with a methanogen may enhance certain fungal metabolic functions. Upregulation of CBM18 may play a role in fungal–methanogen physical associations and fungal cell wall development and remodeling.

     
    more » « less
  3. Sun, Qiu (Ed.)
    Nicotinamide adenine dinucleotide (NAD) is a central molecule in cellular metabolism that has been implicated in human health, the aging process, and an array of human diseases. NAD is well known as an electron storage molecule, cycling between NAD and the reduced NADH. In addition, NAD is cleaved into nicotinamide and Adenine diphosphate ribose by NAD-consuming enzymes such as sirtuins, PARPs and CD38. There are numerous pathways for the biosynthesis of NAD to maintain a baseline concentration and thus avoid cellular death. The NAD salvage pathway, a two-step process to regenerate NAD after cleavage, is the predominant pathway for humans. Nicotinamide PhosphribosylTransferase (NAMPT) is the rate-limiting enzyme within the salvage path. Exposure to pharmacological modulators of NAMPT has been reported to either deplete or increase NAD levels. This study used a curated set of virtual compounds coupled with biochemical assays to identify novel activators of NAMPT. Autodock Vina generated a ranking of the National Cancer Institute’s Diversity Set III molecular library. The library contains a set of organic molecules with diverse functional groups and carbon skeletons that can be used to identify lead compounds. The target NAMPT surface encompassed a novel binding location that included the NAMPT dimerization plane, the openings to the two active site channels, and a portion of the known binding location for NAMPT substrate and product. Ranked molecules were evaluated in a biochemical assay using purified recombinant NAMPT enzyme. Two novel carbon skeletons were confirmed to stimulate NAMPT activity. Compound 20 (NSC9037) is a polyphenolic xanthene derivative in the fluorescein family, while compound 2 (NSC19803) is the polyphenolic myricitrin nature product. Micromolar quantities of compound 20 or compound 2 can double NAMPT’s product formation. In addition, natural products that contain high concentrations of polyphenolic flavonoids, similar to myricitrin, also stimulate NAMPT activity. Confirmation of a novel binding site for these compounds will further our understanding of the cellular mechanism leading to NAD homeostasis and better human health outcomes. 
    more » « less
  4. Abstract

    Hydrophobins are small highly surface‐active fungal proteins with potential as biosurfactants in a wide array of applications. However, practical implementation of hydrophobins at large scale has been hindered by low recombinant yields. In this study, the effects of increasing hydrophobin gene copy number and overexpressing endoplasmic reticulum resident chaperone proteins Kar2p, Pdi1p, and Ero1p were explored as a means to enhance recombinant yields of the class II hydrophobin HFBI in the eukaryotic expression hostPichia pastoris. One‐, 2‐, and 3‐copy‐HFBI strains were attained using an in vitro multimer ligation approach, with strains displaying copy number stability following subsequent transformations as measured by quantitative polymerase chain reaction. Increasing HFBI copy number alone had no effect on increasing HFBI secretion, but increasing copy number in concert with chaperone overexpression synergistically increased HFBI secretion. Overexpression ofPDI1orERO1caused insignificant changes in HFBI secretion in 1‐ and 2‐copy strains, but a statistically significant HFBI secretion increase in 3‐copy strain.KAR2overexpression consistently resulted in enhanced HFBI secretion in all copy number strains, with 3‐copy‐HFBI secreting 22±1.6 fold more than the 1‐copy‐HFBI/no chaperone strain. The highest increase was seen in 3‐copy‐HFBI/Ero1p overexpressing strain with 30±4.0 fold increase in HFBI secretion over 1‐copy‐HFBI/no chaperone strain. This corresponded to an expression level of approximately 330 mg/L HFBI in the 5 ml small‐scale format used in this study.

     
    more » « less
  5. Abstract

    Electron-deficient π-conjugated functional dyes lie at the heart of organic optoelectronics. Adding nitro groups to aromatic compounds usually quenches their fluorescence via inter-system crossing (ISC) or internal conversion (IC). While strong electronic coupling of the nitro groups with the dyes ensures the benefits from these electron-withdrawing substituents, it also leads to fluorescence quenching. Here, we demonstrate how such electronic coupling affects the photophysics of acceptor–donor–acceptor fluorescent dyes, with nitrophenyl acceptors and a pyrrolo[3,2-b]pyrrole donor. The position of the nitro groups and the donor-acceptor distance strongly affect the fluorescence properties of thebis-nitrotetraphenylpyrrolopyrroles. Concurrently, increasing solvent polarity quenches the emission that recovers upon solidifying the media. Intramolecular charge transfer (CT) and molecular dynamics, therefore, govern the fluorescence of these nitro-aromatics. While balanced donor-acceptor coupling ensures fast radiative deactivation and slow ISC essential for large fluorescence quantum yields, vibronic borrowing accounts for medium dependent IC via back CT. These mechanistic paradigms set important design principles for molecular photonics and electronics.

     
    more » « less