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

The genetic and molecular basis of flagellar motility has been investigated for several decades, with innovative research strategies propelling advances at a steady pace. Furthermore, as the phenomenon is examined in diverse bacteria, new taxon-specific regulatory and structural features are being elucidated. Motility is also a straightforward bacterial phenotype that can allow undergraduate researchers to explore the palette of molecular genetic tools available to microbiologists. This study, driven primarily by undergraduate researchers, evaluated hundreds of flagellar motility mutants in the Gram-negative plant-associated bacterium Agrobacterium fabrum. The nearly saturating screen implicates a total of 37 genes in flagellar biosynthesis, including genes of previously unknown function

Synteny of four gene cluster required for motility in <i>Sinorhizobium meliloti</i> and <i>Agrobacterium fabrum</i>.

ATU0585 was renamed flgN and ATU8132 was renamed motF.</p

Plasmid sequences.

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Characterization of the Δ<i>visNR</i> deletion strain.

(A) Complementation test in which ΔvisNR strains harbor plasmids indicated in S1 Fig. Shown are the averages of swim ring diameters in millimeters (mm) in four replicates per strain and standard deviation from the mean. Statistical analysis is shown in S9 Fig. (B) The left image shows a plate with ΔvisNR colonies, and the right image shows a plate with parent strain BB01. Both strains were grown under the same conditions and images are shown at the same scale.</p

Cell-cell hyper-adherence test.

BB01 was modified to constitutively express lacZ from a synthetic transposon (lacZ+; blue colonies). This lacZ+ strain and ΔvisNR were grown as individual cultures in 5 ml of LB+Sm overnight at 30°C. Equal portions of these overnight cultures were mixed together, diluted, cultured for several hours, and plated on LB+Sm+X-Gal to test for cell hyper-adherence, which would have shown as sectored colonies. (TIF)</p

Primers used in this study.

(DOCX)</p

A transposon screen for motility mutants in <i>A</i>. <i>fabrum</i> aided by pre-enrichment.

(A) Transposon delivery plasmid used to carry out the mutagenesis. TE labels show the location of the repeated transposon elements. (B) Triparental mating scheme involving helper plasmid pRK600 and A. fabrum recipient strain UBAPF2 (SmR). (C) Enrichment strategy enabling high-efficiency screening for motility mutations. (TIF)</p