Actinobacillus utilizes a binding protein–dependent ABC transporter to acquire the active form of vitamin B6

Pan, Chuxi; Zimmer, A.; Shah, Megha; Huynh, Minh Sang; Lai, Christine Chieh-Lin; Sit, Brandon; Hooda, Yogesh; Curran, David M.; Moraes, Trevor F.

doi:10.1016/j.jbc.2021.101046

Cited by 8 publications

(4 citation statements)

References 52 publications

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“…Two ATP molecules bind and are hydrolyzed at the interface of the NBD dimer, driving conformational changes of the dimeric transmembrane domains (TMDs) that eventually lead to translocation of the substrate molecule. ABC Transporters play important roles in bacterial physiology and virulence 2 by transporting substrates including, but not limited to, vitamins 3–5 , amino acids 6 , oligopeptides 7 , sugars 8 , and metal ions 9 . Import of iron chelation complexes by type II ABC importers makes important contributions to both bacterial fitness and pathogenesis 2 .…”

Section: Introductionmentioning

confidence: 99%

Mechanistic implications of the interaction of the soluble substrate-binding protein with a type II ABC importer

Hunt

Kim

Leng

et al. 2022

Preprint

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ATP-Binding Cassette (ABC) Transporters employ homologous ATPase domains to drive transmembrane transport of diverse substrates ranging from small molecules to large polymers. Bacterial ABC importers require an extramembranous substrate binding protein (SBP) to deliver the transport substrate to the extracellular side of the transporter complex. Previous studies suggest significant differences in the transport mechanisms of type I vs. type II bacterial ABC importers, which contain unrelated transmembrane domains. We herein use ensemble fluorescence resonance energy transfer (FRET) experiments to characterize the kinetics of SBP interaction in the E. coli BtuCD-F complex, a canonical type II ABC importer that transports vitamin B12 . We demonstrate that, in the absence of B12 , BtuF (the SBP) forms a 'locked' (kinetically hyper-stable) complex with nanodisc-reconstituted BtuCD that can only be dissociated by ATP hydrolysis, which represents a futile reaction cycle. Notably, no type I importer has been observed to form an equivalent locked complex. We also show that either ATP or vitamin B12 binding substantially slows formation of the locked BtuCD-F complex, which will limit the occurrence of futile hydrolysis under physiological conditions. Mutagenesis experiments demonstrate that efficient locking requires concerted interaction of BtuCD with residues on both sides of the B12 binding pocket in BtuF. Combined with the kinetic inhibition of locking by ATP binding, these observations imply that the transition state for the locking reaction involves a global alteration in the conformation of BtuCD that extends from its BtuF binding site in the periplasm to its ATP-binding sites on the opposite side of the membrane in the cytoplasm. These observations suggest that locking, which seals the extracellular B12 entry site of the transporter, may help push B12 through the transporter and directly contribute to the transport mechanism in type II ABC importers.

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Section: Introductionmentioning

confidence: 99%

Mechanistic implications of the interaction of the soluble substrate-binding protein with a type II ABC importer

Hunt

Kim

Leng

et al. 2022

Preprint

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“…Thus, these compounds or precursors thereof must be taken up via known (porphyrins) or yet-to-be-identified transporters (pyridoxal-phosphate) or could be dispensable during standard growth (ubiquinones and molybdopterin). A hemin transporter is known in L. monocytogenes ( Xiao et al 2011 ), and an ABC transporter for pyridoxal-phosphate was recently discovered in the γ-proteobacterium Actinobacillus pleuropneumoniae ( Pan et al 2021 ). A homolog of this transporter is not present in L. monocytogenes , but one of the many other ABC transporters could execute this function.…”

Section: Discussionmentioning

confidence: 99%

Listeria monocytogenes genes supporting growth under standard laboratory cultivation conditions and during macrophage infection

et al. 2022

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The Gram-positive bacterium Listeria monocytogenes occurs widespread in the environment and infects humans when ingested along with contaminated food. Such infections are particularly dangerous for risk group patients, for whom they represent a life-threatening disease. To invent novel strategies to control contamination and disease, it is important to identify those cellular processes that maintain pathogen growth inside and outside the host. Here, we have applied transposon insertion sequencing (Tn-Seq) to L. monocytogenes for the identification of such processes on a genome-wide scale. Our approach identified 394 open reading frames that are required for growth under standard laboratory conditions and 42 further genes, which become necessary during intracellular growth in macrophages. Most of these genes encode components of the translation machinery and act in chromosome-related processes, cell division, and biosynthesis of the cellular envelope. Several cofactor biosynthesis pathways and 29 genes with unknown functions are also required for growth, suggesting novel options for the development of antilisterial drugs. Among the genes specifically required during intracellular growth are known virulence factors, genes compensating intracellular auxotrophies, and several cell division genes. Our experiments also highlight the importance of PASTA kinase signaling for general viability and of glycine metabolism and chromosome segregation for efficient intracellular growth of L. monocytogenes.

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“…Thus, these compounds or precursors thereof must be taken up via known (porphyrines) or yet to be identified transporters (pyridoxal-phosphate) or could be dispensable during standard growth (ubiquinones and molybdopterin). A haemin transporter is known in L. monocytogenes (109) and an ABC transporter for pyridoxal-phosphate was recently discovered in the γ-proteobacterium Actinobacillus pleuropneumoniae (110). A homolog of this transporter is not present in L. monocytogenes , but one of the many other ABC transporters could execute this function.…”

Section: Discussionmentioning

confidence: 99%

Listeria monocytogenes gene essentiality under laboratory conditions and during macrophage infection

Fischer

Engelgeh

Rothe

et al. 2022

Preprint

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The Gram-positive bacterium Listeria monocytogenes occurs widespread in the environment and infects humans when ingested along with contaminated food. Such infections are particularly dangerous for risk group patients, for whom they represent a life-threatening disease. To invent novel strategies to control contamination and disease, it is important to identify those cellular processes that maintain pathogen growth in- and outside the host. We here have applied transposon insertion sequencing (Tn-Seq) to L. monocytogenes for the identification of such processes on a genome-wide scale. Our approach classified 394 open reading frames as essential for growth under standard laboratory conditions and identified 42 further genes, which become additionally essential during intracellular growth in macrophages. Most essential genes encode components of the translation machinery, act in chromosome-related processes, cell division and biosynthesis of the cellular envelope. Several cofactor biosynthesis pathways and 29 genes with unknown functions were also essential, opening novel options for the development of anti- listerial drugs. Among the genes specifically required during intracellular growth were known virulence factors, genes compensating intracellular auxotrophies and several cell division genes. Our experiments also highlight the importance of PASTA kinase signalling, glycine metabolism and chromosome segregation for efficient intracellular growth of L. monocytogenes.

show abstract

Actinobacillus utilizes a binding protein–dependent ABC transporter to acquire the active form of vitamin B6

Cited by 8 publications

References 52 publications

Mechanistic implications of the interaction of the soluble substrate-binding protein with a type II ABC importer

Mechanistic implications of the interaction of the soluble substrate-binding protein with a type II ABC importer

Listeria monocytogenes genes supporting growth under standard laboratory cultivation conditions and during macrophage infection

Listeria monocytogenes gene essentiality under laboratory conditions and during macrophage infection

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