Functional genetics of human gut commensal Bacteroides thetaiotaomicron reveals metabolic requirements for growth across environments

Liu, Hualan; Shiver, Anthony L.; Price, Morgan N.; Carlson, Hans K.; Trotter, Valentine; Chen, Yan; Escalante, Veronica; J, Ray; Hern, Kelsey E.; Kim, Young-Mo; Turnbaugh, Peter J.; Huang, Kerwyn Casey; Arkin, Adam P.; Deutschbauer, Adam M.

doi:10.1016/j.celrep.2021.108789

Cited by 117 publications

(165 citation statements)

References 123 publications

(154 reference statements)

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“…The rbpC mutants also had an increased growth on melibiose suggesting that rbpC also plays a role in utilization of RFOs. In the same study (62), colonization of germ-free mice fed a plant polysaccharide-rich diet with the B. thetaiotaomicron Tn-mutant pool led to increased fitness of rbpC mutants. BT1871 mutants showed decreased fitness over time, whereas Tn-insertions into several other PUL24 genes led to increased fitness in vivo.…”

Section: Discussionmentioning

confidence: 81%

“…We have yet to explore the role of RBPs in helping B. thetaiotaomicron colonize or be maintained in the host gut. In a recent study (62) screening transposon (Tn) mutants for a wide variety of in vitro and in vivo phenotypes, there were no reported insertions in rbpA or rbpB. Insertions in rbpC led to reduced fitness on glucose-containing media (62), possibly explaining our inability to generate ∆rbpC mutants in our standard glucose-rich media.…”

Section: Discussionmentioning

confidence: 91%

“…In a recent study (62) screening transposon (Tn) mutants for a wide variety of in vitro and in vivo phenotypes, there were no reported insertions in rbpA or rbpB. Insertions in rbpC led to reduced fitness on glucose-containing media (62), possibly explaining our inability to generate ∆rbpC mutants in our standard glucose-rich media. The rbpC mutants also had an increased growth on melibiose suggesting that rbpC also plays a role in utilization of RFOs.…”

Section: Discussionmentioning

confidence: 91%

“…Previous work showed that BT1871 has in vitro melibiase activity (61), and transposon insertions in BT1871 led to decreased fitness in melibiose in a carbohydrate utilization screen (62). To confirm that BT1871 was important for B. thetaiotaomicron utilization of RFOs including melibiose as a sole carbon source, we deleted BT1871 and cultured the ∆BT1871 and wild-type strains in minimal media with melibiose, raffinose, stachyose, and sucrose (Fig.…”

Section: Loss Of Rbpb Leads To Decreased Expression Of An Essential Melibiase In Pul24mentioning

confidence: 96%

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A Novel Family of RNA-Binding Proteins Regulate Polysaccharide Metabolism inBacteroides thetaiotaomicron

Adams

Azam

Costliow

et al. 2021

Preprint

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Human gut microbiome composition is constantly changing, and diet is a major driver of these changes. Gut microbial species that persist in mammalian hosts for long periods of time must possess mechanisms for sensing and adapting to nutrient shifts to avoid being outcompeted. Global regulatory mechanisms mediated by RNA-binding proteins (RBPs) that govern responses to nutrient shifts have been characterized in Proteobacteria and Firmicutes but remain undiscovered in the Bacteroidetes. Here we report the identification of RBPs that are broadly distributed across the Bacteroidetes, with many genomes encoding multiple copies. Genes encoding these RBPs are highly expressed in many Bacteroides species. A purified RBP, RbpB, from Bacteroides thetaiotaomicron binds to single-stranded RNA in vitro with an affinity similar to other characterized regulatory RBPs. B. thetaiotaomicron mutants lacking RBPs show dramatic shifts in expression of polysaccharide utilization and capsular polysaccharide loci, suggesting that these RBPs may act as global regulators of polysaccharide metabolism. A B. thetaiotaomicron ΔrbpB mutant shows a growth defect on dietary sugars belonging to the raffinose family of oligosaccharides (RFOs). The ΔrbpB mutant had reduced expression of BT1871, encoding a predicted RFO-degrading melibiase, compared to the wild-type strain. Mutation of BT1871 confirmed that the enzyme it encodes is essential for growth on melibiose and promotes growth on the RFOs raffinose and stachyose. Our data reveal that RbpB is required for optimal expression of BT1871 and other polysaccharide-related genes, suggesting that we have identified an important new family of global regulatory proteins in the Bacteroidetes.

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Section: Discussionmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 91%

Section: Discussionmentioning

confidence: 91%

Section: Loss Of Rbpb Leads To Decreased Expression Of An Essential Melibiase In Pul24mentioning

confidence: 96%

See 2 more Smart Citations

A Novel Family of RNA-Binding Proteins Regulate Polysaccharide Metabolism inBacteroides thetaiotaomicron

Adams

Azam

Costliow

et al. 2021

Preprint

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“…BarSeq PCR was performed using ~200ng of DNA for each sample. Barcodes were amplified using unique, indexed primers, as described in Liu et al, 2021 [35]. Samples were pooled and sequenced using Illumina HiSeq single-end reads.…”

Section: Methodsmentioning

confidence: 99%

Mapping the genetic landscape of biomineralization in Magnetospirillum magneticum AMB-1 with RB-Tnseq

McCausland

Wetmore

Arkin

et al. 2021

Preprint

Self Cite

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Magnetotactic bacteria (MTB) are a phylogenetically diverse group of bacteria remarkable for their ability to biomineralize magnetite (Fe3O4) or greigite (Fe3S4) in organelles called magnetosomes. The majority of genes required for magnetosome formation are encoded by a magnetosome gene island (MAI). Here, we conducted random barcoded transposon mutagenesis (RB-TnSeq) in Magnetospirillum magneticum AMB-1 to identify the global genetic requirements for magnetosome formation under different growth conditions. We generated a library of 184,710 unique strains in a wild-type background, generating ~34 mutant strains for each gene. RB-TnSeq also allowed us to determine the essential gene set of AMB-1 under standard laboratory growth conditions. To pinpoint novel genes that are important for magnetosome formation, we subjected the library to magnetic selection screens in varied growth conditions. We compared biomineralization in standard growth conditions to biomineralization in high iron and anaerobic conditions, respectively. Strains with transposon insertions in the MAI gene mamT had an exacerbated biomineralization defect under both high iron and anerobic conditions compared to standard conditions, adding to our knowledge of the role of MamT in magnetosome formation. Mutants in amb4151, a gene outside of the MAI, are more magnetic than wild-type cells under anaerobic conditions. All three of these phenotypes were validated by creating a markerless deletion strain of the gene and evaluating with TEM imaging. Overall, our results indicate that growth conditions affect which genes are required for biomineralization and that some MAI genes may have more nuanced functions than was previously understood.

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Repurposing Microbes for Therapeutic Applications in Humans

Kim

Choe

Kang

et al. 2022

Principles in Microbiome Engineering

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Functional genetics of human gut commensal Bacteroides thetaiotaomicron reveals metabolic requirements for growth across environments

Cited by 117 publications

References 123 publications

A Novel Family of RNA-Binding Proteins Regulate Polysaccharide Metabolism inBacteroides thetaiotaomicron

A Novel Family of RNA-Binding Proteins Regulate Polysaccharide Metabolism inBacteroides thetaiotaomicron

Mapping the genetic landscape of biomineralization in Magnetospirillum magneticum AMB-1 with RB-Tnseq

Repurposing Microbes for Therapeutic Applications in Humans

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