Table 2.
Summary of survival characteristics of novel attenuated mutants
| Chr | BME | GENE FUNCTION | Gene | S v. Ra | In vitro Survival Ratio | M∅ Survival Ratioc | Mouse Survival Ratioc,d |
| Intracellular trafficking, secretion, and vesicular transport | |||||||
| I | 1894 | Gramicidin S biosynthesis | grsT | S | -0.4 | 1.5 | 0.9 |
| II | 0029–30 | VirB5-B6 | virB5/6 | S | |||
| II | 0034 | Type IV secretion (channel protein) | virB10 | S | 2.5* | ||
| II | 0035 | Type IV secretion (ATPase) | virB11 | S | 2.3*** | ||
| Nucleotide transport and metabolism | |||||||
| I | 0233 | Phosphoribosylaminoimidazolecarboxamide formyltransfease | purH | S | 0.4 | 0.9 | |
| I | 0295 | Phosphoribosylaminoimidazole carboxylase ATPase subunit | purK | S | 0.3 | 0.9 | |
| I | 1123 | Phosphoribosylformylglycinamidine (FGAM) synthase | purS | S | 0.9 | 2.9 | |
| I | 1124 | Phosphoribosylformylglycinamidine (FGAM) synthase | purQ | S | 1.9 | 2.2 | |
| I | 1204 | Exopolyphosphatase (Ppx/GppA) | ppx | S | 0.4 | 1.2 | |
| Signal transduction mechanisms | |||||||
| I | 0066 | Response regulator (two component) | ompR | S | 0.6 | 1.3 | 1.9* |
| I | 1327 | Glutamate-ammonia-ligase adenylyltransferase (signal transduction) | glnE | S | -0.1 | 1.2 | 1.4 |
| I | 1807 | Universal stress protein | uspA | S | 0.4 | 0.1 | |
| Transcription | |||||||
| I | 0304/5 | Hypothetical thioesterase-transcriptional regulator | deoR | S | -0.5 | 1.9 | |
| I | 0371 | Regulatory factor (rpoE) | virF | S | -0.1 | 1.1 | 0.3 |
| I | 0508 | Transcription elongation factor | greA | S | 1.0 | 0.7 | 0.6 |
| I | 0513 | Transcription factor | lysR | S | 0.9 | ||
| I | 0513/4 | Transcription factor-hypothetical protein | lysR/hyp | S | 0.2 | 0.7 | |
| I | 0731/2 | Cold shock protein-hypothetical cytosolic protein | csp/hyp | S | 0.2 | 1.1 | 0.9 |
| I | 0808 | Transcriptional regulator (soxR) | merR | S | 1.0 | 0.8 | |
| I | 1178 | Transcriptional regulator (soxR) | merR | S | 0.0 | ||
| I | 1297/8 | RNA polymerase omega subunit-hypothetical protein | rpoZ/hyp | S | 0.2 | 1.3 | |
| I | 1364 | Transcriptional regulatory protein | mucR | S | 0.3 | 2.0*** | 3.0** |
| I | 1647/8 | Hyp-RegM (ATP-binding) | hyp/regM | S | 1.0 | -0.2 | |
| II | 1116/7 | Transcriptional activator LuxR-TetR families | luxR/tetR | S | 0.1 | 1.6** | 1.1 |
| Cell wall/membrane/envelope biogenesis | |||||||
| I | 0498/9 | Cold shock protein/soluble lytic transglycosylase | cspA/slt | R | 0.0 | 1.7*** | 2.9 |
| I | 1302 | Soluble lytic murein transglycosylase | mltE | R | 0.6 | 0.7 | 0.4 |
| I | 1415 | O-antigen export system permease protein | rfbD | R | 0.1 | 1.3 | |
| I | 1416 | O-antigen export system ATP-binding protein | rfbE | R | 0.3 | 1.9** | |
| I | 1427 | UDP-N-acetylglucosamine 4,6-dehydratase | capD | R | -0.3 | 1.4 | |
| II | 0380 | Acriflavin resistance protein A (membrane biogenesis) | acrA(hlyD) | S | 3.4 | ||
| II | 0472 | Membrane fusion protein (membrane biogenesis) | mtrC(hlyD) | S | 0.9 | 0.3 | |
| Energy production and conversion | |||||||
| I | 0972 | Glutathione reductase | gor | S | 0.3 | 0.5 | |
| I | 1749 | FAD-dependent glycerol phosphate dehydrogenase | glpD | S | 0.2 | 0.7 | |
| Amino acid transport and metabolism | |||||||
| II | 0285 | Dipeptide transport system (inner membrane permease) | dppB | S | 0.4 | 0.7 | |
| Replication, recombination and repair | |||||||
| I | 0334 | AAA ATPase/DNA helicase/DNA damage checkpoint | ruvB | S | 1.3 | 0.9 | |
| I | 1307 | Integrase/recombinase (phage) | xerC (int) | S | 0.1 | 1.1 | |
| I | 2023 | ATP-dependent nuclease subunit A | uvrD/rep | S | 0.9 | ||
| II | 0260 | GTP-binding protein | lepA | S | 0.9 | 0.1 | |
| I | 1296 | GTP-binding protein | lepA | S | 1.0 | ||
| Translation, ribosome structure and biogenesis | |||||||
| I | 0983 | Ribosomal large subunit pseudouridine synthase C | rluA | S | 0.5 | 1.1 | 1.0 |
| I | 1057 | Ribonuclease E/Zn metalloprotease | cafA | S | 0.5 | 1.1 | 1.6 |
| I | 1775–6 | Rnase PH-HrcA (heat shock protein repressor) | rph/hrcA | S | -0.2 | 1.0 | 0.6 |
| Function unknown | |||||||
| I | 0186 | Hypothetical cytosolic protein | dut | S | 0.6 | 0.4 | |
| I | 0193 | Hypothetical protein | hyp | S | 0.4 | 2.4 | 1.5 |
| I | 0490 | Hypothetical cytosolic protein | hycp | S | 0.6 | 0.6 | |
| I | 0540 | Hypothetical protein | hyp | S | 0.0 | 0.6 | |
| I | 0603 | Hypothetical protein | hyp | S | -0.1 | 1.1 | |
| I | 0732 | Hypothetical cytosolic protein | hycp | S | 1.4 | ||
| II | 0045–6 | Hypothetical protein-Hypothetical protein | hyp/hyp | S | |||
| Posttranslational modification, protein turnover and chaperones | |||||||
| I | 0816 | ATP-dependent clp protease ATP-binding subunit | clpA | S | 1.0 | 0.8 | 1.5 |
| II | 0932 | Glutaredoxin | nrdH | S | 1.1 | -0.3 | |
| Defense mechanisms | |||||||
| I | 0926 | Multidrug resistance locus | emrA | 2.2 | |||
| General function prediction | |||||||
| I | 1143 | Metal-dependent hydrolase (β-lactamase) | mbl | S | -0.1 | 0.9 | |
| I | 1282 | Glycine cleavage T protein (aminomethyltransferase) | gcvT | S | 1.2 | 0.4 | |
| I | 1487 | Colicin V production protein | colV | S | 1.2 | 0.5 | 0.8 |
| I | 1499 | Pirin (cupin) | pirA | S | -0.1 | 1.3 | 1.7 |
| I | 1867 | Florfenicol resistance protein | nifB/elp3 | S | 0.4 | 0.5 | |
| Carbohydrate transport and metabolism | |||||||
| I | 2031 | Phosphocarrier protein HPr | ptsH | S | -0.3 | 0.9 | 0.4 |
| II | 1095 | L-fuculose phosphate aldolase | sbgE | S | 0.7 | 2.2* | |
| Lipid transport and metabolism | |||||||
| Coenzyme transport and metabolism | |||||||
| I | 0657 | Outer membrane receptor (TonB-dependent transport) | btuB | S | 0.2 | 0.6 | |
a S v. R denotes smooth versus rough colony morphology
b In vitro growth: 1:1 mixtures of 16 M and select mutants used to inoculate culture to evaluate growth defects of the mutant. The output ratios are corrected for variance from the input ratios (approximately 1.0) and presented as a log10 value, also refrerred to as Δlog10. Macrophage survival is presented as a log10 of the ratio of recovery 16 M to mutant which may also be presented as Δlog10.
c Statistical analysis of macrophage and mouse splenic clearance revealed significant differences among means using 1-way ANOVA, Dunn's multiple comparisons using a non-attenuated mutant revealed statistically valid differences with p-values of 0.05 (*), 0.01 (**) and 0.001 (***).
d Mouse splenic clearance: 1:1 mixtures of 16 M and select mutants were used to inoculate mice i.p. to evaluate degree of in vivo attenuation of survival at one week.
/ Indicates intergenic insertion between two genes.