A large-scale genetic screen identifies genes essential for motility in Agrobacterium fabrum

Calvopina-Chavez, Diana G; Howarth, Robyn E; Memmott, Audrey K; Gonzalez, Oscar H Pech; Hafen, Caleb B; Jensen, Kyson T; Benedict, Alex B.; Altman, Jessica D.; Burnside, Brittany S; Childs, Justin S; Dallon, Samuel W; DeMarco, Alexa C; Flindt, Kirsten C; Grover, Sarah A; Heninger, Elizabeth; Iverson, Christina S; Johnson, A.K.; Lopez, Jack B; Meinzer, McKay A; Moulder, Brook A; Moulton, Rebecca; Russell, Hyrum S; Scott, Tiana M.; Shiobara, Yuka; Taylor, Mason D.; Tippets, Kathryn E; Vainerere, Kayla M; Wallwitz, Isabella C Von; Wagley, Madison; Wiley, Megumi S; Young, Naomi; Griffitts, Joel S.

doi:10.1371/journal.pone.0279936

Cited by 3 publications

(4 citation statements)

References 38 publications

(69 reference statements)

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“…Bacterial template DNA for arbitrary-PCR was prepared by cell lysis as described above. Arbitrary-PCR was carried out as described by Calvopina-Chavez et al ( 47 ), except primers 2,133 and 2,135 were used for the first-round PCR, and primers 2,134 and 2,137 were used for the second-round PCR. DNA products were purified as described above and Sanger sequenced using primer 2,134.…”

Section: Methodsmentioning

confidence: 99%

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Three genes controlling streptomycin susceptibility in Agrobacterium fabrum

Howarth,

Pattillo,

Griffitts

et al. 2023

J Bacteriol

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Streptomycin (Sm) is a commonly used antibiotic for its efficacy against diverse bacteria. The plant pathogen Agrobacterium fabrum is a model for studying pathogenesis and interkingdom gene transfer. Streptomycin-resistant variants of A. fabrum are commonly employed in genetic analyses, yet mechanisms of resistance and susceptibility to streptomycin in this organism have not previously been investigated. We observe that resistance to a high concentration of streptomycin arises at high frequency in A. fabrum , and we attribute this trait to the presence of a chromosomal gene ( strB ) encoding a putative aminoglycoside phosphotransferase. We show how strB , along with rpsL (encoding ribosomal protein S12) and rsmG (encoding a 16S rRNA methyltransferase), modulates streptomycin sensitivity in A. fabrum . IMPORTANCE The plant pathogen Agrobacterium fabrum is a widely used model bacterium for studying biofilms, bacterial motility, pathogenesis, and gene transfer from bacteria to plants. Streptomycin (Sm) is an aminoglycoside antibiotic known for its broad efficacy against gram-negative bacteria. A. fabrum exhibits endogenous resistance to somewhat high levels of streptomycin, but the mechanism underlying this resistance has not been elucidated. Here, we demonstrate that this resistance is caused by a chromosomally encoded streptomycin-inactivating enzyme, StrB, that has not been previously characterized in A. fabrum . Furthermore, we show how the genes rsmG , rpsL , and strB jointly modulate streptomycin susceptibility in A. fabrum .

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Section: Methodsmentioning

confidence: 99%

“…Allelic exchange plasmid pJG1108 ( 47 ), containing the gus and sacB genes, was used for strB deletion in UBAPF2. Primers oDC103 and oDC104 were designed to amplify the strB left homology region, and oDC105 and oDC106 were designed to amplify the right homology region.…”

Section: Methodsmentioning

confidence: 99%

Three genes controlling streptomycin susceptibility in Agrobacterium fabrum

Howarth,

Pattillo,

Griffitts

et al. 2023

J Bacteriol

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“…Bacterial template DNA for arbitrary-PCR was prepared by cell lysis as described above. Arbitrary-PCR was carried out as described by Calvopina-Chavez et al (34), except primers 2133 and 2135 were used for the first-round PCR, and primers 2134 and 2137 were used for the secondround PCR. DNA products were purified as described above and Sanger sequenced using primer 2134.…”

Section: Transposon Mutagenesis and Determination Of Insertion Sitesmentioning

confidence: 99%

“…Allelic exchange plasmid pJG1108 (34), containing the gus and sacB genes, was used for strB deletion in UBAPF2. Primers oDC103 and oDC104 were designed to amplify the strB left homology region, and oDC105 and oDC106 were deigned to amplify the right homology region.…”

Section: Construction Of the δStrb Strain D272mentioning

confidence: 99%

Three genes controlling streptomycin susceptibility inAgrobacterium fabrum

Howarth

Pattillo

Griffitts

et al. 2023

Preprint

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Streptomycin is a commonly used antibiotic for its efficacy against diverse bacteria. The plant pathogenAgrobacterium fabrumis a model for studying pathogenesis and interkingdom gene transfer. Streptomycin-resistant variants ofA. fabrumare commonly employed in genetic analyses, yet mechanisms of resistance and susceptibility to streptomycin in this organism have not previously been investigated. We observe that resistance to a high concentration of streptomycin arises at high frequency inA. fabrumand we link this trait to the presence of a chromosomal gene (strB) encoding a putative aminoglycoside phosphotransferase. We show howstrB, along withrpsL(encoding ribosomal protein S12) andrsmG(encoding a 16S rRNA methyltransferase) modulate streptomycin sensitivity inA. fabrum.

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