skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


  • NSF Public Access
  • Search Results
  • Gene products and processes contributing to lanthanide homeostasis and methanol metabolism in Methylorubrum extorquens AM1
Title: Gene products and processes contributing to lanthanide homeostasis and methanol metabolism in Methylorubrum extorquens AM1
Abstract Lanthanide elements have been recently recognized as “new life metals” yet much remains unknown regarding lanthanide acquisition and homeostasis. InMethylorubrum extorquensAM1, the periplasmic lanthanide-dependent methanol dehydrogenase XoxF1 produces formaldehyde, which is lethal if allowed to accumulate.This property enabled a transposon mutagenesis study and growth studies to confirm novel gene products required for XoxF1 function. The identified genes encode an MxaD homolog,an ABC-type transporter, an aminopeptidase, a putative homospermidine synthase, and two genes of unknown function annotated asorf6andorf7. Lanthanide transport and trafficking genes were also identified. Growth and lanthanide uptake were measured using strains lacking individual lanthanide transport cluster genes, and transmission electron microscopy was used to visualize lanthanide localization. We corroborated previous reports that a TonB-ABC transport system is required for lanthanide incorporation to the cytoplasm. However, cells were able to acclimate over time and bypass the requirement for the TonB outer membrane transporter to allow expression ofxoxF1and growth. Transcriptional reporter fusions show that excess lanthanides repress the gene encoding the TonB-receptor. Using growth studies along with energy dispersive X-ray spectroscopy and transmission electron microscopy, we demonstrate that lanthanides are stored as cytoplasmic inclusions that resemble polyphosphate granules.  more » « less
Award ID(s):
1750003
PAR ID:
10176830
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ; ; ; ;
Publisher / Repository:
Nature Publishing Group
Date Published:
Journal Name:
Scientific Reports
Volume:
10
Issue:
1
ISSN:
2045-2322
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; (, Journal of Virology)
    Parrish, Colin R (Ed.)
    ABSTRACT Bracoviruses (BVs) are endogenized nudiviruses in parasitoid wasps of the microgastroid complex (order Hymenoptera: Family Braconidae). BVs produce replication-defective virions that adult female wasps use to transfer DNAs encoding virulence genes to parasitized hosts. Some BV genes are shared with nudiviruses and baculoviruses with studies of the latter providing insights on function, whereas other genes are only known from nudiviruses or other BVs which provide no functional insights. A proteomic analysis ofMicroplitis demolitorbracovirus (MdBV) virions recently identified 16 genes encoding nucleocapsid components. In this study, we further characterized most of these genes. Some nucleocapsid genes exhibited early or intermediate expression profiles, while others exhibited late expression profiles. RNA interference (RNAi) assays together with transmission electron microscopy indicatedvp39,HzNVorf9-like2,HzNVorf93-like,HzNVorf106-like,HzNVorf118-like,and 27bare required to produce capsids with a normal barrel-shaped morphology. RNAi knockdown ofvlf-1a,vlf-1b-1,vlf-1b-2,int-1,andp6.9-1did not alter the formation of barrel-shaped capsids but each reduced processing of amplified proviral segments and DNA packaging as evidenced by the formation of electron translucent capsids. All of the genes required for normal capsid assembly were also required for proviral segment processing and DNA packaging. Collectively, our results deorphanize several BV genes with previously unknown roles in virion morphogenesis. IMPORTANCEUnderstanding how bracoviruses (BVs) function in wasps is of broad interest in the study of virus evolution. This study characterizes most of theMicroplitis demolitorbracovirus (MdBV) genes whose products are nucleocapsid components. Results indicate several genes unknown outside of nudiviruses and BVs are essential for normal capsid assembly. Results also indicate most MdBV tyrosine recombinase family members and the DNA binding proteinp6.9-1are required for DNA processing and packaging into nucleocapsids. 
    more » « less
  2. ; ; ; ; ; (, Molecular Genetics and Genomics)
    Abstract Natural transformation, the process whereby a cell acquires DNA directly from the environment, is an important driver of evolution in microbial populations, yet the mechanism of DNA uptake is only characterized in bacteria. To expand our understanding of natural transformation in archaea, we undertook a genetic approach to identify a catalog of genes necessary for transformation inMethanococcus maripaludis. Using an optimized method to generate random transposon mutants, we screened 6144 mutant strains for defects in natural transformation and identified 25 transformation-associated candidate genes. Among these are genes encoding components of the type IV-like pilus, transcription/translation associated genes, genes encoding putative membrane bound transport proteins, and genes of unknown function. Interestingly, similar genes were identified regardless of whether replicating or integrating plasmids were provided as a substrate for transformation. Using allelic replacement mutagenesis, we confirmed that several genes identified in these screens are essential for transformation. Finally, we identified a homolog of a membrane bound substrate transporter inMethanoculleus thermophilusand verified its importance for transformation using allelic replacement mutagenesis, suggesting a conserved mechanism for DNA transfer in multiple archaea. These data represent an initial characterization of the genes important for transformation which will inform efforts to understand gene flow in natural populations. Additionally, knowledge of the genes necessary for natural transformation may assist in identifying signatures of transformation machinery in archaeal genomes and aid the establishment of new model genetic systems for studying archaea. 
    more » « less
  3. ; ; ; (, Scientific Reports)
    Abstract Lanthanide (Ln) elements are utilized as cofactors for catalysis by XoxF-type methanol dehydrogenases (MDHs). A primary assumption is that XoxF enzymes produce formate from methanol oxidation, which could impact organisms that require formaldehyde for assimilation. We report genetic and phenotypic evidence showing that XoxF1 (MexAM1_1740) fromMethylobacterium extorquensAM1 produces formaldehyde, and not formate, during growth with methanol. Enzyme purified with lanthanum or neodymium oxidizes formaldehyde. However, formaldehyde oxidation via 2,6-dichlorophenol-indophenol (DCPIP) reduction is not detected in cell-free extracts from wild-type strain methanol- and lanthanum-grown cultures. Formaldehyde activating enzyme (Fae) is required for Ln methylotrophic growth, demonstrating that XoxF1-mediated production of formaldehyde is essential. Addition of exogenous lanthanum increases growth rate with methanol by 9–12% but does not correlate with changes to methanol consumption or formaldehyde accumulation. Transcriptomics analysis of lanthanum methanol growth shows upregulation ofxox1and downregulation ofmxagenes, consistent with the Ln-switch, no differential expression of formaldehyde conversion genes, downregulation of pyrroloquinoline quinone (PQQ) biosynthesis genes, and upregulation offdh4formate dehydrogenase (FDH) genes. Additionally, the Ln-dependent ethanol dehydrogenase ExaF reduces methanol sensitivity in thefaemutant strain when lanthanides are present, providing evidence for the capacity of an auxiliary role for ExaF during Ln-dependent methylotrophy. 
    more » « less
  4. ; ; ; ; (, Journal of Virology)
    van_Oers, Monique M (Ed.)
    ABSTRACT Venturia canescensis a parasitoid wasp that harbors a domesticated endogenous virus (DEV) and parasitizes host insects likeEphestia kuehniella. TheV. canescensDEV evolved from an alphanudivirus and produces virus-like particles (VLPs) in females that protect wasp eggs from a host immune defense called encapsulation. In contrast, very few DEV genes required for VLP formation and function have been identified. In this study, we characterized fiveV. canescensDEV genes of unknown function that all nudiviruses encode. Three of these genes are single copy (OrNVorf18-like,OrNVorf61-like, andOrNVorf76-like), whileOrNVorf41-likehas expanded into a six-member family andOrNVorf47-likehas expanded into a three-member family. Sequence analysis indicated all of these genes retain essential motifs present in nudivirus homologs, while transmission electron microscopy (TEM) studies characterized the timing of VLP formation during the wasp pupal stage. RNA interference (RNAi) assays identifiedOrNVorf18-like,OrNVorf61-like,OrNVorf41-like-1,andOrNVorf41-like-2as genes that are required for normal VLP formation. Knockdown ofOrNVorf47-likefamily members did not affect VLP formation but did disable binding of VLPs toV. canescenseggs and protection against encapsulation. Disabled formation of VLPs in response to RNAi knockdown ofOrNVorf18-like,OrNVorf61-like,OrNVorf41-like-1,andOrNVorf41-like-2also resulted in wasp eggs being encapsulated. In contrast, knockdown ofOrNVorf76-likehad no effect on VLP assembly, egg binding, or encapsulation. Altogether, reported results significantly advance our understanding ofV. canescensVLP (VcVLP) formation and function. IMPORTANCEUnderstanding howV. canescenscoopted an alphanudivirus to produce VcVLPs is of interest to the study of virus evolution. Our results show that three nudivirus core genes have essential functions in VcVLP formation, while one is essential for the novel function of binding to wasp eggs and protection from encapsulation, which is the most important immune defense of insects against parasitoids. 
    more » « less
  5. ; ; (, Frontiers in Plant Science)
    The model legumeMedicago truncatulaestablishes a symbiosis with soil bacteria (rhizobia) that carry out symbiotic nitrogen fixation (SNF) in plant root nodules. SNF requires the exchange of nutrients between the plant and rhizobia in the nodule that occurs across a plant-derived symbiosome membrane. One iron transporter, belonging to the Vacuolar iron Transporter-Like (VTL) family, MtVTL8, has been identified as essential for bacteria survival and therefore SNF. In this work we investigated the spatial expression ofMtVTL8in nodules and addressed whether it could be functionally interchangeable with a similar nodule-expressed iron transporter, MtVTL4. Using a structural model for MtVTL8 and the previously hypothesized mechanism for iron transport in a phylogenetically-related Vacuolar Iron Transporter (VIT), EgVIT1 with known crystal structure, we identified critical amino acids and obtained their mutants. Mutants were testedin plantafor complementation of an SNF defective line and in an iron sensitive mutant yeast strain. An extended phylogenetic assessment of VTLs and VITs showed that amino acids critical for function are conserved differently in VTLs vs. VITs. Our studies showed that some amino acids are essential for iron transport leading us to suggest a model for MtVTL8 function, one that is different for other iron transporters (VITs) studied so far. This study extends the understanding of iron transport mechanisms in VTLs as well as those used in SNF. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email