The authors have declared that no competing interests exist.
Conceived and designed the experiments: YM SRF CCH. Performed the experiments: YM SRF JSK PH MJQ WJF. Analyzed the data: YM JSK JS JFS CCH. Wrote the paper: YM SRF JSK AMA JS CCH.
Current address: Department of Microbiology, University of Minnesota, Minneapolis, Minnesota, United States of America
The Na+ translocating NADH:quinone oxidoreductase (Na+-NQR) is a unique respiratory enzyme catalyzing the electron transfer from NADH to quinone coupled with the translocation of sodium ions across the membrane. Typically,
Na+-translocating NADH:quinone oxidoreductases (Na+-NQR) are found in the respiratory chains of a number of marine and pathogenic bacteria (reviewed in
Besides Na+-NQR, two more types of respiratory NADH-dehydrogenases have been reported, the proton-pumping NADH dehydrogenases (NDH-1 or Complex I) and NDH-2
Lack of the major NADH dehydrogenase activity usually significantly affect bacterial physiology.
In this study, we aimed to investigate the effects of a Δ
Phenotype microarrays were performed using PM3 to PM10 MicroPlate (Biolog) at Biolog's PM Services group. All plates used pyruvate as sole carbon sources.
Cells of
Mass spectrometry based metabolomic profiling was performed as previously described
To account for analytical and sample preparation variation, samples were normalized to total ion count. Central energy metabolites (metabolites of the tricarboxylic acid cycle, pentose phosphate pathway, and glycolysis and amino acids) were targeted post-data acquisition and in addition, untargeted statistical analysis (Student's t-test p-value plotted against fold-change) revealed large (>10-fold) changes in cyclic AMP and cyclic GMP, and subsequently, related metabolites (purines and pyrimidines) were targeted post-data acquisition.
Growth measurement assays were performed as previously described
Acetate excretions from bacterial cells were measured as previously described
Motility of
We had previously performed a Phenotype Microarray (Biolog) analysis to address how a Δ
We next performed DNA microarray analyses to investigate how the lack of Na+-NQR broadly affects gene expression pattern in
VC number | Functions | Fold change (early) | Fold change (mid) |
VC0280 | lysine/cadaverine antiporter, cadB | 17.836 up | 8.313 up |
VC0281 | lysine decarboxylase, cadA | 26.554 up | 4.988 up |
VC0479 | hypothetical protein | 2.075 up | 1.516 up |
VC0615 | endoglucanase-related protein | 1.709 up | 1.568 up |
VC0620 | peptide ABC transporter, periplasmic peptide-binding protein | 1.686 up | 2.131 up |
VC0786 | D-amino acid dehydrogenase small subunit | 2.512 up | 1.881 up |
VC1203 | urocanate hydratase | 3.598 up | 2.000 up |
VC1204 | formimidoylglutamase | 3.152 up | 2.211 up |
VC1205 | imidazolonepropionase | 2.674 up | 2.062 up |
VC1480 | hypothetical protein | 1.677 up | 1.811 up |
VC1481 | hypothetical protein | 1.654 up | 1.640 up |
VC1627 | pH-dependent sodium/proton antiporter, nhaA | 2.199 up | 2.089 up |
VC1689 | hypothetical protein | 2.292 up | 1.634 up |
VC1752 | hypothetical protein | 1.611 up | 1.592 up |
VC1827 | mannose-6-phosphate isomerase | 6.706 up | 1.662 up |
VC1828 | hypothetical protein | 2.703 up | 1.629 up |
VC2216 | hypothetical protein | 2.216 up | 1.527 up |
VC2361 | autonomous glycyl radical cofactor GrcA | 2.197 up | 1.920 up |
VC2556 | hypothetical protein | 1.572 up | 1.519 up |
VC2699 | anaerobic C4-dicarboxylate transporter | 3.431 up | 1.509 up |
VCA0029 | transcriptional regulator, putative | 3.633 up | 2.241 up |
VCA0562 | hypothetical protein | 1.674 up | 1.585 up |
VCA0702 | iron-containing alcohol dehydrogenase | 1.770 up | 1.841 up |
VCA0732 | hypothetical protein | 2.365 up | 1.746 up |
VCA0744 | glycerol kinase | 1.653 up | 2.770 up |
VCA0773 | methyl-accepting chemotaxis protein | 2.453 up | 1.937 up |
VCA0811 | N-acetylglucosamine-binding protein A | 7.632 up | 2.174 up |
VCA0827 | pterin-4-alpha-carbinolamine dehydratase | 1.542 up | 2.797 up |
VCA0948 | hypothetical protein | 2.456 up | 1.554 up |
VCA1045 | PTS system, mannitol-specific IIABC component | 1.732 up | 2.479 up |
VCA1046 | mannitol-1-phosphate 5-dehydrogenase | 1.997 up | 1.712 up |
VC number | Functions | Fold change (early) | Fold change (mid) |
VC0022 | hypothetical protein | 1.657 down | 1.644 down |
VC0061 | thiamine biosynthesis protein ThiC | 1.916 down | 1.739 down |
VC0062 | thiamine-phosphate pyrophosphorylase | 1.935 down | 1.724 down |
VC0063 | thiF protein | 1.730 down | 1.864 down |
VC0302 | putative 3-phenylpropionic acid transporter | 2.403 down | 1.821 down |
VC0730 | copper homeostasis protein | 1.675 down | 1.647 down |
VC0734 | malate synthase | 2.689 down | 1.789 down |
VC0751 | co-chaperone HscB | 1.651 down | 1.828 down |
VC0754 | hypothetical protein | 1.899 down | 1.755 down |
VC0766 | exodeoxyribonuclease VII large subunit | 1.792 down | 2.076 down |
VC0769 | chitinase, putative | 2.752 down | 1.532 down |
VC0916 | phosphotyrosine protein phosphatase | 3.258 down | 1.622 down |
VC0917 | UDP-N-acetylglucosamine 2-epimerase | 2.858 down | 2.191 down |
VC1070 | phosphatase, putative | 1.752 down | 1.591 down |
VC1124 | hypothetical protein | 1.561 down | 1.519 down |
VC1267 | hypothetical protein | 1.761 down | 1.515 down |
VC1312 | alanine racemase | 1.625 down | 1.767 down |
VC1454 | RstA1 protein | 3.940 down | 1.617 down |
VC1461 | colonization factor | 2.246 down | 2.188 down |
VC1777 | sialic acid-specific TRAP transporter, SiaP | 2.354 down | 1.992 down |
VC1778 | sialic acid-specific TRAP transporter, SiaQ | 3.571 down | 1.923 down |
VC1779 | sialic acid-specific TRAP transporter, SiaM | 3.150 down | 2.187 down |
VC1782 | N-acetylmannosamine kinase | 7.239 down | 1.986 down |
VC1783 | N-acetylglucosamine-6-phosphate deacetylase | 5.481 down | 1.767 down |
VC1784 | neuraminidase | 2.475 down | 2.646 down |
VC1927 | C4-dicarboxylate transport protein | 1.745 down | 1.763 down |
VC1928 | C4-dicarboxylate transport protein DctQ, putative | 1.970 down | 1.947 down |
VC1929 | C4-dicarboxylate-binding periplasmic protein | 2.449 down | 2.796 down |
VC2037 | Na+/H+ antiporter, nhaC-1 | 1.680 down | 1.599 down |
VC2127 | flagellar basal body-associated protein FliL | 1.885 down | 1.602 down |
VC2128 | flagellar hook-length control protein FliK, putative | 4.759 down | 1.826 down |
VC2130 | flagellum-specific ATP synthase | 2.060 down | 1.915 down |
VC2131 | flagellar assembly protein H | 1.807 down | 1.805 down |
VC2132 | flagellar motor switch protein G | 1.519 down | 1.663 down |
VC2133 | flagellar MS-ring protein | 1.654 down | 1.547 down |
VC2136 | sensory box sensor histidine kinase | 1.682 down | 1.533 down |
VC2140 | flagellar capping protein | 1.824 down | 1.562 down |
VC2141 | flagellar protein FlaG | 1.914 down | 1.611 down |
VC2187 | flagellin | 1.617 down | 1.554 down |
VC2190 | flagellar hook-associated protein FlgL | 5.302 down | 1.728 down |
VC2192 | peptidoglycan hydrolase | 5.239 down | 1.591 down |
VC2195 | flagellar basal body rod protein FlgG | 4.720 down | 1.538 down |
VC2197 | flagellar hook protein FlgE | 2.561 down | 1.514 down |
VC2705 | sodium/solute symporter, putative | 4.691 down | 1.963 down |
VCA0176 | methyl-accepting chemotaxis protein | 2.111 down | 1.667 down |
VCA0186 | hypothetical protein | 3.613 down | 1.670 down |
VCA0204 | ATP-dependent RNA helicase RhlE | 1.708 down | 1.677 down |
VCA0699 | glucose-1-phosphate adenylyltransferase | 1.700 down | 1.620 down |
VCA0700 | chitodextrinase | 4.589 down | 1.619 down |
VCA0835 | hypothetical protein | 1.876 down | 1.611 down |
VCA0836 | hexapeptide repeat-containing acetyltransferase | 1.722 down | 1.679 down |
VCA0847 | inner membrane protein YjeH | 2.362 down | 1.638 down |
VCA0848 | GGDEF family protein | 2.354 down | 1.502 down |
VCA0862 | long-chain fatty acid transport protein | 4.995 down | 1.830 down |
VCA0864 | methyl-accepting chemotaxis protein | 1.582 down | 1.694 down |
The
Several transporter genes including tripartite ATP-independent periplasmic (TRAP) transporters,
We also found that two methyl-accepting chemotaxis proteins (MCPs), encoded by VCA0176 and VCA0864, were down-regulated in the
The Class II, III and IV flagellar genes were systematically down regulated in the
When lactate was added, both parent and mutant strain exhibited diminished swarming, and the improved motility of the parent strain was no longer apparent (p = 1,
Swarming assays were performed in LB medium supplemented with 100 mM NaCl and buffered to pH6.5 either with or without the addition of 33 mM D, L-lactate. Mean values and standard error from 16 experiments are presented. P values were calculated using Student's t test.
To further understand how lack of Na+-NQR affects
Metabolite | Fold change (nqr/WT) | p-value (t-test) |
Adenine | 0.737 | 0.0246 |
Adenosine | 0.248 | 0.0299 |
AMP | 1.105 | 0.6629 |
Arginine | 0.964 | 0.4638 |
Asparagine | 0.936 | 0.1727 |
Cadaverine | 2.400 | < 0.0001 |
Cyclic AMP | 0.095 | 0.0066 |
Cyclic GMP | 0.094 | 0.0035 |
Deoxyribose | 0.769 | 0.1178 |
dGMP | 0.260 | 0.0025 |
Fructose-1,6-bisphosphate | 0.772 | 0.2946 |
Glucose | 0.795 | 0.1613 |
Glutamate | 0.875 | 0.0220 |
GMP | ∼ 0.10 | N/A |
Guanine | 0.772 | 0.0065 |
Guanosine | 0.758 | 0.0056 |
Histidine | 0.956 | 0.4699 |
Hypoxanthine | 1.411 | 0.0222 |
Inosine | 0.594 | 0.0003 |
Iso/citrate | 0.756 | 0.0027 |
Isoleucine | 0.927 | 0.1445 |
Lactate | 0.739 | 0.0024 |
Lysine | 0.596 | 0.0004 |
Malate | 1.809 | 0.0003 |
Methionine | 0.873 | 0.1405 |
Phenylalanine | 0.886 | 0.0961 |
Phosphoglycerate | 1.094 | 0.7458 |
Proline | 0.896 | 0.2215 |
Ribose phosphate | 0.821 | 0.1549 |
Serine | 0.899 | 0.1519 |
Succinate | 1.664 | 0.0934 |
Threonine | 0.898 | 0.0837 |
Tryptophan | 0.818 | 0.0599 |
Tyrosine | 0.866 | 0.1397 |
Uracil | 1.461 | 0.0010 |
Valine | 0.861 | 0.1073 |
We had previously suggested that TCA cycle activity is decreased in the
Red solid squares show metabolites that are increased in the Δ
We found that intracellular lysine levels were decreased, while cadaverine levels were increased in the
We had previously shown that the
The results of the phenotype microarrays showed that the
Lack of Na+-NQR broadly affects
(XLSX)
We thank Caprice Rosato of the CGRB for her excellent support with microarray experiment.