Table 1.
Characterization of transposon mutants with greatly reduced (<30% of normal) soft-agar motility
| Function | ORF | Gene | Description | % wild-type motility1 | FlrA activation2 | # independent mutants | Predicted operon structure3 |
|---|---|---|---|---|---|---|---|
| Regulation | VF_0387 | rpoN | RNA polymerase sigma-54 factor | 0 | NS4 | 7 | kdsDC-0390-lptAB-rpoN-hpf-ptsN-yhbJ-03835 |
| VF_1856 | flrA | Sigma-54-dependent regulator | 0 | ND | 2 | flrA | |
| VF_1854 | flrC | Two-component response regulator | 0 | 8.1 | 4 | flrBC | |
| VF_1855 | flrB | Two-component sensor kinase | 17 | 16 | 1 | flrBC | |
| VF_1834 | fliA | RNA polymerase sigma-28 factor | 0 | NS | 2 | flhAFG-fliA-cheYZAB-1829-1828-cheW-1825 | |
| VF_1835 | flhG | Flagellar synthesis regulator | 0 | NS | 1 | flhAFG-fliA-cheYZAB-1829-1828-cheW-1825 | |
| VF_1836 | flhF | Flagellar regulator | 23 | 2.6 | 4 | flhAFG-fliA-cheYZAB-1829-1828-cheW-1825 | |
| Structure/Secretion | VF_1837 | flhA | Flagellar biosynthesis protein | 0 | NS | 5 | flhAFG-fliA-cheYZAB-1829-1828-cheW-1825 |
| VF_1840 | fliR | Flagellar biosynthesis protein | 0 | 2.9 | 1 | fliL1MNOPQR | |
| VF_1844 | fliN | Flagellar motor switch component | 0 | 6.4 | 1 | fliL1MNOPQR | |
| VF_1845 | fliM | Flagellar motor switch protein | 0 | 6.8 | 1 | fliL1MNOPQR | |
| VF_1846 | fliL1 | Flagellar basal body–associated protein | 0 | 8.1 | 1 | fliL1MNOPQR | |
| VF_1847 | fliK | Flagellar hook length control protein | 0 | 9.2 | 2 | fliK | |
| VF_1849 | fliI | Flagellum-specific ATP synthase | 0 | NS | 2 | fliHIJ | |
| VF_1850 | fliH | Flagellar assembly protein | 0 | NS | 3 | fliHIJ | |
| VF_1851 | fliG | Flagellar motor switch protein | 0 | 6.8 | 5 | fliEFG | |
| VF_1852 | fliF | Flagellar M ring protein | 0 | 6.6 | 2 | fliEFG | |
| VF_1860 | fliD | Flagellar hook-associated protein 2 | 19 | 7.4 | 3 | flaG-fliD -flaI-fliS | |
| VF_1868 | flgK | Flagellar hook-associated protein 1 | 0 | 41 | 4 | flgKL | |
| VF_1870 | flgI | Flagellar P ring protein | 0 | 3.5 | 5 | flgFGHI | |
| VF_1871 | flgH | Flagellar L ring protein | 0 | 6.3 | 1 | flgFGHI | |
| VF_1872 | flgG | Flagellar distal rod protein | 0 | 12 | 2 | flgFGHI | |
| VF_1873 | flgF | Flagellar proximal rod protein | 0 | 9.6 | 2 | flgFGHI | |
| VF_1874 | flgE | Flagellar hook protein | 0 | 5.7 | 1 | flgE | |
| igVF_1874 | (intergenic region) | 0 | ND | 1 | |||
| VF_1875 | flgD | Flagellar hook capping protein | 0 | 7.5 | 2 | flgBCD | |
| VF_1882 | flgN | Flagellar chaperone | 0 | 2.5 | 1 | flgMN | |
| Motor | VF_0714 | motA1 | Flagellar motor protein | 0 | 6.3 | 1 | motA1B1 |
| VF_0715 | motB1 | Flagellar motor protein | 0 | 3.4 | 1 | motA1B1 | |
| VF_0926 | motY | Flagellar motor protein | 0 | 2.6 | 2 | motY | |
| VF_2317 | motX | Flagellar motor protein | 0 | 11 | 2 | motX | |
| Chemotaxis | VF_1826 | cheW | Chemotaxis coupling protein | 10 | 2.6 | 1 | flhAFG-fliA-cheYZAB-1829-1828-cheW-1825 |
| VF_1830 | cheB | Chemotaxis methyl esterase | 24 | 2.1 | 2 | flhAFG-fliA-cheYZAB-1829-1828-cheW-1825 | |
| VF_1831 | cheA | Chemotaxis histidine autokinase | 7 | 2.0 | 6 | flhAFG-fliA-cheYZAB-1829-1828-cheW-1825 | |
| VF_1832 | cheZ | CheY phosphatase | 8 | 2.2 | 1 | flhAFG-fliA-cheYZAB-1829-1828-cheW-1825 | |
| VF_1833 | cheY | Chemotaxis response regulator | 6 | 2.2 | 1 | flhAFG-fliA-cheYZAB-1829-1828-cheW-1825 | |
| Unexpected | igVF_0135 | (intergenic region) | 21 | ND | 1 | ||
| VF_0534 | mutS5 | Methyl-directed mismatch repair protein | 0 | NS | 1 | mutS | |
| VF_1491 | Hypothetical protein | 9 | NS | 3 | 1491 | ||
| VF_1883 | flgP | Flagellar motility-associated protein | 0 | 8.7 | 4 | flgOP | |
| VF_1884 | flgO | Flagellar motility-associated protein | 0 | 10 | 2 | flgOP | |
| VF_1885 | flgT | Flagellar motility-associated protein | 0 | NS | 1 | flgT | |
| VF_2326 | amiB | N-acetylmuramoyl-l-alanine amidase II | 0 | NS | 1 | yjeE-amiB-mutL-miaA | |
| VF_A0430 | mukF | Calcium-binding protein involved in chromosome partioning | 23 | NS | 1 | smtA-mukFEB | |
| VF_A0432 | mukB | Fused chromosome partitioning protein | 22 | NS | 2 | smtA-mukFEB |
ORF, open reading frame; ND, not determined.
As scored by the normalized soft-agar motility assay described in Experimental Procedures.
FlrA activation is defined as the fold change of gene expression in wild type as compared to ΔflrA, as determined by microarray analysis.
Predicted operon structure based on DOOR (Database of prOkaryotic OpeRons) analysis of the Vibrio fischeri genome. Bold font indicates gene of interest in the predicted operon.
NS, not significant at fold change ≥2 and P ≤ 0.01.
Four-digit numbers indicate locus tags and should be read as preceded by “VF_”.
When mutS is expressed in trans, the complemented strain does not regain the ability to swim through soft agar (data not shown), indicating that mutS expression does not directly mediate flagellar motility in this strain. MutS is a protein involved in DNA mismatch repair, and the loss of this function results in strains with higher mutation rates. We hypothesize that the mutagenic nature of the mutS strain enabled a secondary mutation that is responsible for the amotile phenotype, and we will not follow up on this mutant in this study.