Edited by: George Grant, University of Aberdeen, United Kingdom
Reviewed by: Paul Edward Carlson, United States Food and Drug Administration, United States; Isabelle Martin-Verstraete, Paris Diderot University, France; Jiri Dresler, Military Medical Agency of Czech Armed Forces, Czechia
†These authors have contributed equally to this work
This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
The response to iron limitation of several bacteria is regulated by the ferric uptake regulator (Fur). The Fur-regulated transcriptional, translational and metabolic networks of the Gram-positive, pathogen
The Fur regulons of
Here we describe a combined RNA sequencing-based transcriptomic, proteomic, metabolomic and electron microscopy approach to characterize multiple functional and metabolic changes induced by the Fur-mediated low iron response. Multiple cellular processes aside of iron transport including mainly energy metabolism, but also flagella formation and motility, cell wall architecture and antibiotic/CAMP resistance were controlled by iron and partly by Fur in
The vector for
Integration of the transcriptome (RNA-Seq), proteome, metabolome and bioinformatics-based Fnr –binding site analyses for the analysis of
Gene | Wild type: low vs. | Fur box | |||||
---|---|---|---|---|---|---|---|
Locus_tag | name | high iron (log2 FC) | at high iron (log2 FC) | ||||
T | P | T | P | ||||
CD630_16470 CDIF630erm_01824 | Iron family ABC transporter permease | 4.79 | 0.18 | 5.90 | – | + | |
CD630_16480 CDIF630erm_01825 | Iron family ABC transporter permease | 3.43 | ON | 4.74 | ON | ||
CD630_16490 CDIF630erm_01826 | Iron family ABC transporter ATP-binding protein | 5.16 | 0.62 | 5.19 | ON | ||
CD630_16500 CDIF630erm_01827 | Iron family ABC transporter substrate-binding protein | 4.98 | 7.46 | 4.65 | 8.55 | + | |
CD630_28740 CDIF630erm_03142 | MATE family drug/sodium antiporter | 2.42 | – | 4.12 | – | ||
CD630_28750 CDIF630erm_03143 | Ferrichrome-specific ABC transporter ATP-binding protein | 1.49 | – | 1.86 | ON | ||
CD630_28760 CDIF630erm_03144 | Ferrichrome-specific ABC transporter permease | 1.00 | ON | 3.85 | ON | ||
CD630_28770 CDIF630erm_03145 | Ferrichrome-specific ABC transporter permease | 1.15 | ON | 3.27 | ON | + | |
CD630_28780 CDIF630erm_03146 | Ferrichrome-specific ABC transporter, substrate-binding | 0.78 | 2.43 | 2.73 | 4.47 | + | |
CD630_29890 CDIF630erm_03273 | Sulfonate family ABC transporter substrate-binding protein | 2.60 | 2.29 | 4.39 | 3.73 | ||
CD630_29900 CDIF630erm_03274 | Sulfonate family ABC transporter ATP-binding protein | 2.91 | 3.09 | 4.50 | 4.81 | ||
CD630_29910 CDIF630erm_03275 | Sulfonate family ABC transporter permease | 3.53 | – | 6.10 | – | ||
CD630_29920 CDIF630erm_03276 | Uncharacterized protein, iron hydrogenase-like | 2.93 | 1.07 | 4.38 | 5.83 | ||
CD630_10870 CDIF630erm_01231 | Zinc transporter ZupT | 1.85 | ON | 6.19 | ON | + | |
CD630_05910 CDIF630erm_00704 | ATPase | 2.41 | 6.77 | 2.80 | 7.54 | ||
CD630_05920 CDIF630erm_00705 | Hypothetical protein | 2.04 | 5.21 | 1.93 | 5.64 | + | |
CD630_29970 CDIF630erm_03281 | Iron family ABC transporter ATP-binding protein | 1.02 | 0.19 | 1.08 | 1.16 | ||
CD630_29980 CDIF630erm_03282 | Iron family ABC transporter permease | 0.42 | 0.06 | 1.95 | – | ||
CD630_29990 CDIF630erm_03283 | Iron family ABC transporter substrate-binding protein | 0.17 | -0.83 | 0.65 | 0.48 | ||
CD630_14770 CDIF630erm_01641 | Ferrous iron transport protein FeoA | 1.51 | ON | -1.18 | ON | + | |
CD630_14780 CDIF630erm_01642 | Ferrous iron transport protein FeoA1 | 1.02 | 5.70 | -1.13 | 3.69 | ||
CD630_14790 CDIF630erm_01643 | Ferrous iron transport protein FeoB1 | 0.77 | 7.19 | -0.16 | 4.78 | + | |
CD630_14800 CDIF630erm_01644 | Hypothetical protein | 0.40 | – | -0.01 | – | ||
CD630_15170 CDIF630erm_01684 | Ferrous iron transport protein FeoB | -0.63 | – | -0.92 | – | ||
CD630_15180 CDIF630erm_01685 | Ferrous iron transport protein FeoA | – | - | -0.14 | – | ||
CD630_17451 CDIF630erm_01939 | Ferrous iron transport protein | -2.34 | 0.36 | -2.08 | 0.98 | + | |
CD630_21680 CDIF630erm_02400 | Hydroxylamine reductase | -0.81 | 0.29 | -2.21 | OFF | ||
CD630_21690 CDIF630erm_02401 | Iron-sulfur binding protein | -1.86 | – | -3.06 | – | ||
CD630_03240 CDIF630erm_00452 | Cobalamin transport protein CbiM | -2.19 | ON | -2.55 | – | ||
CD630_03250 CDIF630erm_00453 | Cobalt ABC transporter substrate-binding protein CbiN | -1.81 | – | -2.81 | – | ||
CD630_03260 CDIF630erm_00454 | Cobalt ABC transporter permease CbiQ | -2.05 | ON | -1.13 | ON | ||
CD630_03270 CDIF630erm_00455 | Cobalt ABC transporter ATP-binding protein CbiO | -0.47 | -0.18 | -1.81 | -0.10 | ||
CD630_35040 CDIF630erm_03817 | Type IV prepilin peptidase | -0.48 | – | -0.33 | – | ||
CD630_35050 CDIF630erm_03818 | Twitching motility protein PilT | -0.91 | -0.52 | -1.71 | 0.12 | ||
CD630_35060 CDIF630erm_03819 | Hypothetical protein | -0.64 | – | -2.01 | – | ||
CD630_35070 CDIF630erm_03820 | Type IV pilin | -0.29 | – | -2.62 | – | ||
CD630_35080 CDIF630erm_03821 | Type IV pilin | -1.40 | – | -1.74 | – | ||
CD630_35090 CDIF630erm_03822 | Type IV pilus assembly protein | -1.01 | – | -2.43 | – | ||
CD630_35100 CDIF630erm_03823 | Membrane protein | -1.30 | 0.08 | -1.91 | -0.21 | ||
CD630_35110 CDIF630erm_03824 | Type IV pilus secretion protein | 0.79 | -1.43 | -2.74 | OFF | ||
CD630_35120 CDIF630erm_03825 | Type IV pilus transporter system | -0.93 | 0.40 | -2.50 | -0.02 | ||
CD630_35130 CDIF630erm_03826 | Pilin protein | -1.61 | 0.21 | -3.13 | -0.52 | 03828 | |
CD630_02520 CDIF630erm_00375 | Flagellar protein FliJ | 2.37 | ON | 2.25 | ON | ||
CD630_02530 CDIF630erm_00376 | Flagellar hook-length control protein FliK | 2.60 | 1.15 | 2.96 | 1.36 | ||
CD630_02540 CDIF630erm_00377 | Basal-body rod modification protein FlgD | 2.68 | – | 2.41 | – | ||
CD630_02550 CDIF630erm_00378 | Flagellar hook protein FlgE | 1.42 | 0.43 | 2.27 | -0.18 | ||
CD630_02551 CDIF630erm_00379 | Flagellar protein FlbD | 3.26 | 1.56 | 1.28 | 0.91 | ||
CD630_02560 CDIF630erm_00380 | Flagellar motor rotation protein MotA | 2.38 | -0.73 | 2.19 | -0.39 | ||
CD630_02570 CDIF630erm_00381 | Flagellar motor rotation protein MotB | 1.97 | -0.16 | 2.04 | -0.79 | ||
CD630_02580 CDIF630erm_00382 | Flagellar basal body-associated protein FliL | 1.84 | 0.53 | 2.60 | 0.24 | ||
CD630_02590 CDIF630erm_00383 | Flagellar protein FliZ | 1.05 | ON | 2.28 | ON | ||
CD630_02600 CDIF630erm_00384 | Flagellar biosynthesis protein FliP | 1.80 | OFF | 3.06 | -0.14 | ||
CD630_02610 CDIF630erm_00385 | Flagellar biosynthetic protein FliQ | 1.07 | – | 2.50 | – | ||
CD630_02620 CDIF630erm_00386 | Bifunctional flagellar biosynthesis protein FliR/FlhB | 2.38 | – | 3.59 | – | ||
CD630_02630 CDIF630erm_00387 | Flagellar biosynthesis protein FlhA | 3.84 | 0.18 | 4.44 | 0.10 | ||
CD630_02640 CDIF630erm_00388 | Flagellar biosynthesis regulator FlhF | 3.61 | 1.04 | 3.84 | 1.30 | ||
CD630_02650 CDIF630erm_00389 | Flagellar biosynthesis protein FlhG | 1.99 | 0.28 | 3.57 | 0.21 | ||
CD630_02660 CDIF630erm_00390 | Flagellar operon RNA polymerase sigma-28 factor | 3.54 | -0.01 | 4.15 | OFF | ||
CD630_02670 CDIF630erm_00391 | Flagellar protein | 2.80 | 0.65 | 3.76 | OFF | ||
CD630_02671 | Flagellar protein | 3.57 | – | 4.06 | ON | ||
CD630_02680 CDIF630erm_00392 | Flagellar basal body rod protein FlgG | 3.44 | 0.74 | 2.92 | 2.00 | ||
CD630_02690 CDIF630erm_00393 | Flagellar basal body rod protein FlgG | 3.25 | 0.78 | 3.47 | OFF | ||
CD630_02700 CDIF630erm_00394 | Flagellar motor switch protein FliM | 2.98 | 0.18 | 3.41 | -0.12 | ||
CD630_02710 CDIF630erm_00395 | Flagellar motor switch phosphatase FliN | 3.33 | 0.43 | 2.97 | 0.04 | ||
CD630_02260 CDIF630erm_00348 | Lytic transglycosylase | -3.23 | 0.78 | -3.18 | 1.96 | + | |
CD630_02270 CDIF630erm_00349 | Hypothetical protein | -1.99 | 0.13 | -3.99 | -0.01 | ||
CD630_02280 CDIF630erm_00350 | Flagellar motor switch protein FliN | -2.25 | 0.25 | -2.84 | 0.16 | ||
CD630_02290 CDIF630erm_00351 | Negative regulator of flagellin synthesis | -2.08 | 1.19 | -3.40 | OFF | ||
CD630_02300 CDIF630erm_00352 | Flagellar biosynthesis protein | -2.41 | 0.92 | -3.12 | 0.59 | ||
CD630_02310 CDIF630erm_00353 | Flagellar hook-associated protein FlgK | -2.08 | 1.58 | -3.51 | 0.08 | ||
CD630_02320 CDIF630erm_00354 | Flagellar hook-associated protein FlgL | -2.21 | 0.22 | -3.88 | -0.46 | ||
CD630_02330 CDIF630erm_00355 | Flagellar assembly factor FliW | -2.66 | -0.81 | -3.16 | 0.15 | ||
CD630_02340 CDIF630erm_00356 | Carbon storage regulator CsrA | -1.87 | 0.09 | -2.57 | -0.46 | ||
CD630_02350 CDIF630erm_00357 | Flagellar protein FliS1 | -1.33 | 1.91 | -3.04 | 1.89 | ||
CD630_02360 CDIF630erm_00358 | Flagellar protein FliS2 | -1.03 | 0.15 | -2.30 | 0.20 | ||
CD630_02370 CDIF630erm_00359 | Flagellar hook-associated protein FliD | -0.94 | 1.65 | -2.45 | 0.30 | ||
CD630_02380 CDIF630erm_00360 | Hypothetical protein | -0.69 | 1.30 | -3.06 | -0.56 | ||
CD630_02390 CDIF630erm_00361 | Flagellin C | -0.09 | 0.14 | -1.57 | -0.93 | ||
CD630_22140 CDIF630erm_02447 | HTH-type transcriptional regulator | 1.85 | OFF | 2.46 | 0.72 | ||
CD630_22150 CDIF630erm_02448 | HTH-type transcriptional regulator | 2.17 | – | 2.68 | – | 02449 | |
CD630_19970 CDIF630erm_02215 | Proline iminopeptidae | 2.04 | 0.26 | 4.07 | 1.63 | ||
CD630_19980 CDIF630erm_02216 | TetR family transcriptional regulator | 1.85 | ON | 3.73 | ON | ||
CD630_08880 CDIF630erm_01008 | Arginine decarboxylase | 3.92 | ON | 4.48 | ON | + | |
CD630_08890 CDIF630erm_01009 | 2.57 | ON | 2.95 | ON | |||
CD630_08900 CDIF630erm_01010 | Polyamine aminopropyl transferase | 4.53 | 0.38 | 3.78 | 0.59 | ||
CD630_08910 CDIF630erm_01011 | Agmatinase | 5.41 | -0.31 | 4.95 | 0.53 | ||
CD630_10230 CDIF630erm_01159 | Transcriptional regulator | 3.64 | -0.32 | 2.21 | 0.66 | ||
CD630_10240 CDIF630erm_01160 | Spermidine/putrescine ABC transporter ATP-binding protein | 3.43 | -0.06 | 3.05 | 0.43 | ||
CD630_10250 CDIF630erm_01161 | Spermidine/putrescine ABC transporter permease | 3.24 | -0.66 | 3.22 | -0.66 | ||
CD630_10260 CDIF630erm_01162 | Spermidine/putrescine ABC transporter permease | 2.98 | – | 3.23 | – | ||
CD630_10270 CDIF630erm_01163 | Spermidine/putrescine ABC transporter substrate-binding protein | 2.57 | -0.58 | 2.94 | 1.78 | ||
CD630_28510 CDIF630erm_03118 | 1.51 | – | 2.10 | – | |||
CD630_28520 CDIF630erm_03119 | 2.84 | OFF | 2.79 | OFF | |||
CD630_28530 CDIF630erm_03120 | 1.37 | -0.80 | 1.68 | -1.19 | |||
CD630_28540 CDIF630erm_03122 | 1.49 | 0.13 | 1.37 | 0.01 | |||
CD630_16260 CDIF630erm_01803 | -1.00 | – | 2.91 | – | |||
CD630_16270 CDIF630erm_01804 | -0.89 | – | 3.13 | – | |||
CD630_16280 CDIF630erm_01805 | Alanine racemase 1 | 0.64 | – | 3.47 | – | ||
CD630_03160 CDIF630erm_00443 | ABC transporter permease | 1.10 | – | 2.42 | – | ||
CD630_03170 CDIF630erm_00444 | ABC transporter permease | 0.67 | – | 2.61 | – | ||
CD630_03180 CDIF630erm_00445 | Bacitracin/multidrug family ABC transporter ATP-binding protein | 1.51 | -0.19 | 2.85 | 0.45 | ||
CD630_08200 CDIF630erm_00938 | Two-component response regulator | 0.72 | ON | 1.58 | ON | ||
CD630_08210 CDIF630erm_00939 | Two-component sensor histidine kinase | 1.39 | ON | 2.76 | ON | ||
CD630_08220 CDIF630erm_00940 | Multidrug family ABC transporter ATP-binding protein | 1.04 | – | 2.35 | – | ||
CD630_08230 CDIF630erm_00941 | Multidrug family ABC transporter permease | -0.38 | – | 1.62 | – | ||
CD630_08240 CDIF630erm_00943 | Multidrug family ABC transporter permease | 0.78 | – | 3.41 | – | ||
CD630_20030 CDIF630erm_02221 | MATE family drug/sodium antiporter | -3.56 | – | -1.20 | – | ||
CD630_20040 CDIF630erm_02222 | MarR family transcriptional regulator | -5.25 | -0.75 | -2.98 | 3.12 | ||
CD630_15570 CDIF630erm_01726 | Peptidyl-prolyl isomerase | 1.07 | -0.58 | 3.69 | 0.16 | + | |
CD630_15580 CDIF630erm_01727 | ECF RNA polymerase sigma factor CsfV | 2.29 | 3.42 | – | |||
CD630_15590 CDIF630erm_01728 | Anti ECF RNA polymerase sigma factor RsiV | 1.55 | – | 2.73 | – | ||
CD630_26510 CDIF630erm_02905 | UDP- |
1.30 | 0.63 | 1.54 | 1.34 | ||
CD630_26520 CDIF630erm_02906 | Cell division/stage V sporulation protein | 0.91 | – | 2.04 | – | ||
CD630_26530 CDIF630erm_02907 | UDP- |
0.77 | 0.11 | 1.45 | 0.42 | ||
CD630_26540 CDIF630erm_02908 | Phospho- |
0.82 | – | 2.37 | – | ||
CD630_26550 CDIF630erm_02909 | UDP- |
2.59 | -0.26 | 2.74 | 0.21 | ||
CD630_27780 CDIF630erm_03041 | Glycosyl transferase family protein | 0.98 | -0.10 | 1.26 | 0.10 | ||
CD630_27790 CDIF630erm_03042 | Mannose-1-phosphate guanylyltransferase | 1.33 | 0.81 | 2.47 | 0.33 | ||
CD630_27800 CDIF630erm_03043 | Phosphoglucomutase | 2.75 | 0.22 | 3.20 | 0.06 | ||
CD630_27810 CDIF630erm_03044 | Transmembrane virulence factor | 3.67 | -0.93 | 4.13 | 0.78 | ||
CD630_02840 CDIF630erm_00408 | PTS system mannose/fructose/sorbose transporter subunit IIA | – | -0.04 | – | 0.88 | ||
CD630_02850 CDIF630erm_00409 | PTS system mannose/fructose/sorbose transporter subunit IIB | 1.86 | 0.14 | 2.61 | 0.62 | ||
CD630_02860 CDIF630erm_00410 | PTS system mannose/fructose/sorbose transporter subunit IIA | 2.27 | -0.52 | 2.43 | 0.64 | ||
CD630_02870 CDIF630erm_00411 | PTS system mannose/fructose/sorbose transporter subunit IIB | 1.90 | 0.20 | 2.70 | 1.00 | ||
CD630_02880 CDIF630erm_00412 | PTS system mannose/fructose/sorbose transporter subunit IIC | 1.51 | -0.14 | 2.68 | 0.06 | ||
CD630_02890 CDIF630erm_00413 | PTS system mannose/fructose/sorbose transporter subunit IID | 1.90 | -0.08 | 1.68 | 0.46 | ||
CD630_32070 CDIF630erm_03501 | Multi antimicrobial extrusion protein | 1.30 | – | 2.93 | – | ||
CD630_32080 CDIF630erm_03502 | MarR family transcriptional regulator | 0.99 | ON | 2.77 | ON | ||
CD630_35140 CDIF630erm_03828 | Ribose-phosphate pyrophosphokinase | 1.89 | -0.10 | 2.31 | 0.30 | ||
CD630_35150 CDIF630erm_03829 | Bifunctional |
1.51 | -0.27 | 2.04 | 0.40 | + | |
CD630_10090 CDIF630erm_01145 | GntR family transcriptional regulator | -3.62 | – | -2.36 | – | ||
CD630_10100 CDIF630erm_01146 | -2.69 | -2.60 | -2.02 | -0.22 | |||
CD630_10110 CDIF630erm_01147 | Glucosamine-6-phosphate deaminase | -2.74 | 1.69 | -1.74 | 0.19 | ||
CD630_32370 CDIF630erm_03533 | Proline racemase | 3.03 | -1.48 | 4.04 | -1.14 | ||
CD630_32380 CDIF630erm_03534 | Proline reductase PrdE-like protein | 4.31 | – | 4.34 | – | ||
CD630_32390 CDIF630erm_03535 | Proline reductase PrdE | 4.14 | -2.05 | 4.48 | -2.14 | ||
CD630_32400 CDIF630erm_03536 | Proline reductase PrdD | 4.18 | -1.83 | 4.88 | -1.00 | ||
CD630_32410 CDIF630erm_03537 | Proline reductase | 1.74 | -0.53 | 2.69 | -0.80 | ||
CD630_32430 CDIF630erm_03539 | Hypothetical protein | 2.90 | -0.79 | 2.07 | -0.39 | ||
CD630_32440 CDIF630erm_03540 | 2.98 | -0.67 | 3.05 | -0.83 | |||
CD630_32450 CDIF630erm_03541 | Sigma-54 dependent transcriptional regulator | 0.61 | 0.02 | -1.02 | -0.80 | ||
CD630_32460 CDIF630erm_03542 | Surface protein | -1.35 | – | -2.85 | – | ||
CD630_32470 CDIF630erm_03544 | Electron transfer protein | 4.62 | -0.96 | 4.78 | -0.90 | ||
CD630_11370 CDIF630erm_01284 | Electron transport complex protein RnfC | 2.64 | -0.73 | 2.91 | -0.49 | ||
CD630_11380 CDIF630erm_01285 | Electron transport complex protein RnfD | 2.01 | -1.19 | 3.58 | -0.62 | ||
CD630_11390 CDIF630erm_01286 | Electron transport complex protein RnfG | 2.67 | 0.47 | 3.31 | 0.41 | ||
CD630_11400 CDIF630erm_01287 | Electron transport complex protein RnfE | 1.17 | OFF | 3.47 | OFF | ||
CD630_11410 CDIF630erm_01288 | Electron transport complex protein RnfA | 1.88 | – | 3.55 | – | ||
CD630_11420 CDIF630erm_01289 | Electron transport complex protein RnfB | 1.61 | -1.38 | 2.47 | -0.91 | ||
CD630_11700 CDIF630erm_01318 | Lactate racemase | 1.41 | – | 2.86 | – | ||
CD630_11710 CDIF630erm_01319 | Lactate dehydrogenase, electron transfer flavoprotein beta subunit | 0.67 | OFF | 2.43 | OFF | ||
CD630_11720 CDIF630erm_01320 | Lactate dehydrogenase, electron transfer flavoprotein alpha subunit | -0.66 | – | 0.63 | – | ||
CD630_11730 CDIF630erm_01321 | lactate dehydrogenase (electron bifurcating), catalytic subunit | -0.30 | -0.57 | 0.73 | OFF | ||
CD630_03940 CDIF630erm_00522 | (R)-2-hydroxyisocaproate dehydrogenase | -8.37 | OFF | -8.66 | OFF | ||
CD630_03950 CDIF630erm_00523 | Isocaprenoyl-CoA:2-hydroxyisocaproate CoA-transferase | -8.33 | OFF | -11.10 | OFF | 00519 | |
CD630_03960 CDIF630erm_00524 | 2-hydroxyisocaproyl-CoA dehydratase activator | -0.79 | OFF | -11 | OFF | ||
CD630_03970 CDIF630erm_00525 | Oxygen-sensitive 2-hydroxyisocaproyl-CoA dehydratase subunit B | -7.47 | OFF | -10.52 | OFF | ||
CD630_03980 CDIF630erm_00526 | Oxygen-sensitive 2-hydroxyisocaproyl-CoA dehydratase subunit C | -7.81 | -4.86 | -10.94 | OFF | ||
CD630_03990 CDIF630erm_00527 | Acyl-CoA dehydrogenase | -7.43 | -4.27 | -10.17 | -4.76 | ||
CD630_04000 CDIF630erm_00528 | Electron transfer flavoprotein subunit beta | -7.28 | -3.90 | -10.48 | -4.50 | ||
CD630_04010 CDIF630erm_00529 | Electron transfer flavoprotein subunit alpha | -7.50 | -3.90 | -10.39 | -4.98 | ||
CD630_10540 CDIF630erm_01194 | Butyryl-CoA dehydrogenase | -5.89 | OFF | -8.00 | OFF | ||
CD630_10550 CDIF630erm_01195 | Electron transfer flavoprotein subunit beta | -7.47 | OFF | -7.70 | OFF | ||
CD630_10560 CDIF630erm_01196 | Electron transfer flavoprotein subunit alpha | -6.75 | OFF | -7.75 | OFF | ||
CD630_10570 CDIF630erm_01197 | 3-hydroxybutyryl-CoA dehydratase | -6.37 | – | -7.48 | OFF | ||
CD630_10580 CDIF630erm_01198 | 3-hydroxybutyryl-CoA dehydrogenase | -5.21 | OFF | -5.97 | OFF | ||
CD630_10590 CDIF630erm_01199 | Acetyl-CoA acetyltransferase | -6.22 | OFF | -6.43 | OFF | ||
CD630_29660 CDIF630erm_03250 | Bifunctional acetaldehyde-CoA/alcohol dehydrogenase | -8.19 | -4.52 | -9.86 | -5.91 | ||
CD630_23380 CDIF630erm_02577 | 4-hydroxybutyrate dehydrogenase | -1.00 | -0.87 | -1.23 | -1.06 | 02575 | |
CD630_23390 CDIF630erm_02578 | 4-hydroxybutyrate CoA-transferase | -0.87 | OFF | -0.50 | OFF | ||
CD630_23400 CDIF630erm_02579 | Hypothetical protein | -2.16 | – | -1.55 | – | ||
CD630_23410 CDIF630erm_02580 | Gamma-aminobutyrate metabolism dehydratase/isomerase | -1.41 | -1.74 | -1.23 | -1.16 | ||
CD630_23420 CDIF630erm_02581 | Succinate-semialdehyde dehydrogenase | -2.47 | – | -3.77 | – | ||
CD630_23430 CDIF630erm_02582 | Succinyl-CoA:coenzyme A transferase | -3.35 | – | -3.58 | – | ||
CD630_23440 CDIF630erm_02583 | Membrane protein | -4.79 | – | -5.28 | – | ||
CD630_23480 CDIF630erm_02587 | Glycine reductase complex component D | – | OFF | – | OFF | ||
CD630_23490 CDIF630erm_02588 | Glycine reductase complex component C | -3.70 | – | -3.70 | – | ||
CD630_23510 CDIF630erm_02589 | Glycine reductase complex component B | – | OFF | – | OFF | ||
CD630_23520 CDIF630erm_02592 | Glycine reductase complex component A | – | - | – | - | ||
CD630_23540 CDIF630erm_02594 | Glycine reductase complex component E | -5.02 | -2.84 | -5.22 | OFF | ||
CD630_23550 CDIF630erm_02595 | Thioredoxin 2 | -3.66 | – | -3.66 | – | + | |
CD630_23560 CDIF630erm_02596 | Thioredoxin reductase | – | - | – | OFF | ||
CD630_23570 CDIF630erm_02597 | – | OFF | – | ||||
CD630_08530 CDIF630erm_00972 | Oligopeptide family ABC transporter permease | -2.81 | OFF | -2.26 | OFF | ||
CD630_08540 CDIF630erm_00973 | Oligopeptide family ABC transporter permease | -2.67 | – | -2.28 | – | ||
CD630_08550 CDIF630erm_00974 | Oligopeptide family ABC transporter substrate-binding protein | -2.48 | -0.37 | -3.22 | -1.02 | ||
CD630_08560 CDIF630erm_00975 | ABC transporter ATP-binding protein | -2.38 | – | -3.65 | – | ||
CD630_15360 CDIF630erm_01704 | NADH-dependent reduced ferredoxin:NADP oxidoreductase | -2.56 | -1.18 | -2.43 | -0.71 | ||
CD630_15370 CDIF630erm_01705 | NADH-dependent reduced ferredoxin:NADP oxidoreductase | -2.69 | -1.77 | -3.01 | -1.53 | ||
CD630_08820 CDIF630erm_01002 | Glucose-1-phosphate adenylyltransferase | -1.89 | – | -3.38 | – | ||
CD630_08830 CDIF630erm_01003 | Glycogen biosynthesis protein | -1.70 | -2.42 | -4.22 | OFF | ||
CD630_08840 CDIF630erm_01004 | Glycogen synthase | -2.45 | 1.98 | -3.24 | -0.37 | ||
CD630_08850 CDIF630erm_01005 | Glycogen phosphorylase | -1.62 | 0.49 | -3.38 | 0.14 | ||
CD630_08860 CDIF630erm_01006 | Amylopullulanase | -0.12 | -0.29 | -2.68 | 0.05 | ||
CD630_23180 CDIF630erm_02556 | Phosphohexomutase | 2.01 | 0.58 | 1.56 | 0.21 | ||
CD630_23190 CDIF630erm_02557 | Ribulose-phosphate 3-epimerase | 1.24 | ON | 1.19 | ON | ||
CD630_23200 CDIF630erm_02558 | Ribose-5-phosphate isomerase B | 3.19 | 0.00 | 2.23 | -0.02 | ||
CD630_23210 CDIF630erm_02559 | Transketolase | 1.75 | 0.44 | 2.01 | 0.22 | ||
CD630_23220 CDIF630erm_02560 | Transketolase | 1.48 | 0.23 | 2.63 | 0.29 | ||
CD630_11200 CDIF630erm_01264 | Formate acetyltransferase | -0.51 | -0.12 | 2.03 | 1.70 | ||
CD630_11210 CDIF630erm_01265 | Pyruvate formate-lyase activating enzyme | -0.32 | – | 2.21 | – | ||
CD630_11220 CDIF630erm_01266 | Transcriptional regulator | -1.51 | -0.63 | 1.48 | 0.05 | ||
CD630_32820 CDIF630erm_03582 | Pyruvate formate-lyase | -1.38 | -2.38 | -2.88 | -2.94 | ||
CD630_32830 CDIF630erm_03583 | Pyruvate formate-lyase | -2.11 | – | -3.53 | – | ||
CD630_33130 CDIF630erm_03614 | Oxidoreductase Fe-S subunit | -2.44 | – | -3.60 | – | ||
CD630_33140 CDIF630erm_03615 | Iron hydrogenase | -2.59 | OFF | -3.08 | -1.30 | ||
CD630_33150 CDIF630erm_03616 | Oxidoreductase Fe-S subunit | -3.54 | -1.13 | -3.00 | -0.86 | + | |
CD630_33151 CDIF630erm_03617 | Hypothetical protein | -1.53 | – | -3.00 | – | ||
CD630_33160 CDIF630erm_03618 | Formate dehydrogenase accessory protein FdhD | -2.22 | – | -3.03 | – | ||
CD630_33170 CDIF630erm_03619 | Formate dehydrogenase-H | -2.88 | -1.63 | -3.37 | -1.72 | ||
CD630_01740 CDIF630erm_00296 | Carbon monoxide dehydrogenase | -3.57 | -1.46 | -4.70 | -2.05 | ||
CD630_01750 CDIF630erm_00297 | Oxidoreductase Fe-S subunit | -2.43 | – | -4.33 | – | ||
CD630_01760 CDIF630erm_00298 | Oxidoreductase NAD/FAD binding subunit | -2.12 | OFF | -3.96 | OFF | ||
CD630_12780 CDIF630erm_01431 | Rrf2 family transcriptional regulator | 4.43 | 3.30 | -0.27 | 1.44 | ||
CD630_12790 CDIF630erm_01432 | Cysteine desulfurase | 0.55 | 2.69 | -3.25 | 0.03 | ||
CD630_12800 CDIF630erm_01433 | NifU family iron-sulfur cluster assembly protein | 0.83 | 3.83 | -2.97 | 0.31 | ||
CD630_34670 CDIF630erm_03778 | ATP synthase subunit epsilon | 3.83 | 0.56 | 1.37 | 1.04 | ||
CD630_34680 CDIF630erm_03779 | ATP synthase subunit beta | 2.25 | 0.60 | 2.04 | 0.94 | ||
CD630_34690 CDIF630erm_03780 | ATP synthase subunit gamma | 3.91 | 0.51 | 2.83 | 0.55 | ||
CD630_34700 CDIF630erm_03781 | ATPase subunit alpha | 3.72 | 0.61 | 2.44 | 1.00 | ||
CD630_34710 CDIF630erm_03782 | ATP synthase subunit delta | 3.65 | 0.49 | 2.67 | 0.72 | ||
CD630_34720 CDIF630erm_03783 | ATP synthase subunit B | 3.33 | 0.16 | 2.31 | 0.47 | ||
CD630_34730 CDIF630erm_03784 | ATP synthase subunit C | 2.81 | ON | 1.58 | ON | ||
CD630_34740 CDIF630erm_03785 | ATP synthase subunit A | 1.59 | – | 1.77 | 0.94 | ||
CD630_29540 CDIF630erm_03237 | V-type ATP synthase subunit D | -1.47 | – | -2.46 | – | ||
CD630_29550 CDIF630erm_03238 | V-type ATP synthase subunit B | -3.00 | -0.98 | -2.58 | -1.34 | ||
CD630_29560 CDIF630erm_03239 | V-type ATP synthase subunit A | -2.35 | -0.73 | -2.54 | -0.95 | + | |
CD630_29561 CDIF630erm_03240 | V-type ATP synthase subunit F | -2.40 | – | -1.52 | – | ||
CD630_29570 CDIF630erm_03241 | V-type ATP synthase subunit C | -2.32 | – | -2.56 | – | ||
CD630_29580 CDIF630erm_03242 | V-type ATP synthase subunit E | -1.47 | OFF | -2.92 | OFF | ||
CD630_29590 CDIF630erm_03243 | V-type ATP synthase subunit K | -1.47 | -2.10 | -2.48 | -1.20 | ||
CD630_29600 CDIF630erm_03244 | V-type ATP synthase subunit I | -2.73 | OFF | -3.24 | OFF | ||
CD630_29610 CDIF630erm_03245 | Hypothetical protein | -1.78 | 0.00 | -2.92 | -0.80 | ||
CD630_19990 CDIF630erm_02217 | Flavodoxin | 4.54 | ON | 4.54 | ON | ||
CD630_16970 CDIF630erm_01882 | 6.7-dimethyl-8-ribityllumazine synthase | 2.82 | 0.31 | 3.86 | 0.68 | + | |
CD630_16980 CDIF630erm_01883 | Riboflavin biosynthesis bifunctional 3.4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase | 2.10 | 0.40 | 2.62 | 0.99 | ||
CD630_16990 CDIF630erm_01884 | Riboflavin synthase subunit alpha | 1.82 | 0.26 | 1.55 | -0.07 | ||
CD630_17000 CDIF630erm_01885 | Riboflavin biosynthesis bifunctional diaminohydroxyphosphoribosylamino pyrimidine deaminase/5-amino-6-(5-phosphoribosylamino)uracil reductase | 1.39 | 0.35 | 1.81 | 1.02 | ||
CD630_23310 CDIF630erm_02570 | Mannitol-1-phosphate 5-dehydrogenase | -6.28 | -2.60 | -5.79 | -1.91 | ||
CD630_23320 CDIF630erm_02571 | PTS system mannitol-specific transporter subunit IIA | -5.98 | OFF | -5.53 | -1.54 | ||
CD630_23330 CDIF630erm_02572 | PTS operon transcription antiterminator | -5.78 | OFF | -6.17 | OFF | ||
CD630_23340 CDIF630erm_02573 | PTS system mannitol-specific transporter subunit IICB | -6.29 | OFF | -5.92 | -3.14 | ||
CD630_11770 CDIF630erm_01326 | Fatty acid biosynthesis transcriptional regulator | 2.53 | 0.17 | 3.16 | 0.11 | ||
CD630_11780 CDIF630erm_01327 | Phosphate acyltransferase | 1.93 | -1.29 | 3.25 | -0.22 | ||
CD630_11790 CDIF630erm_01328 | 3-oxoacyl-ACP synthase | 2.12 | 0.09 | 4.00 | 0.92 | ||
CD630_11800 CDIF630erm_01329 | Enoyl-(acyl-carrier-protein) reductase II | 2.86 | -0.16 | 3.61 | 0.50 | ||
CD630_11810 CDIF630erm_01330 | Malonyl CoA-acyl carrier protein transacylase | 3.03 | -0.18 | 3.87 | 0.41 | ||
CD630_11820 CDIF630erm_01331 | 3-oxoacyl-ACP reductase | 3.65 | -0.06 | 3.94 | 0.23 | ||
CD630_11830 CDIF630erm_01332 | Acyl carrier protein | 2.95 | – | 4.28 | – | ||
CD630_11840 CDIF630erm_01333 | 3-oxoacyl-ACP synthase | 2.98 | 0.07 | 4.71 | 0.69 | 01334 | |
CD630_29750 CDIF630erm_03259 | CRISPR-associated endoribonuclease Cas2 | -0.74 | – | -2.13 | – | ||
CD630_29760 CDIF630erm_03260 | CRISPR-associated endonuclease Cas1 | -0.87 | – | -1.67 | – | ||
CD630_29770 CDIF630erm_03261 | CRISPR-associated Cas4 family protein | -0.41 | – | -2.72 | – | ||
CD630_29780 CDIF630erm_03262 | CRISPR-associated Cas3 family helicase | -0.26 | – | -2.20 | – | ||
CD630_29790 CDIF630erm_03263 | CRISPR-associated Cas5 family protein | -1.30 | – | -1.66 | – | ||
CD630_29800 CDIF630erm_03264 | CRISPR-associated autoregulatorDevR family protein | 0.20 | -0.55 | -1.97 | -1.28 | ||
CD630_29810 CDIF630erm_03265 | CRISPR-associated protein | -1.27 | -1.20 | -1.78 | OFF | ||
CD630_29820 CDIF630erm_03266 | CRISPR-associated Cas6 family protein | -0.60 | – | -2.92 | – | ||
CD630_13650 CDIF630erm_01522 | XkdN-like protein | – | - | -2.30 | – | + | |
CD630_13660 CDIF630erm_01524 | Tail protein | -1.33 | ON | -3.28 | – | ||
CD630_13680 CDIF630erm_01526 | Cell wall XkdQ-like hydrolase | 0.02 | – | -3.93 | – | ||
CD630_13700 CDIF630erm_01528 | XkdS-like protein | – | - | -1.07 | – | ||
CD630_13710 CDIF630erm_01529 | Baseplate assembly protein | -1.33 | – | -2.06 | – | ||
CD630_13720 CDIF630erm_01530 | XkdT-like protein | -1.27 | – | -2.90 | – | ||
CD630_13740 CDIF630erm_01532 | Beta-lactamase-inhibitor protein II | 0.64 | ON | -1.58 | – | ||
CD630_02180 CDIF630erm_00340 | 5-carboxyaminoimidazole ribonucleotide mutase | 0.09 | -0.24 | 2.12 | 0.80 | ||
CD630_02190 CDIF630erm_00341 | Phosphoribosylaminoimidazolesuccino carboxamide synthase | -0.22 | -0.21 | 2.59 | 0.41 | ||
CD630_02200 CDIF630erm_00342 | Amidophosphoribosyltransferase | 1.65 | -0.69 | 2.96 | 0.09 | ||
CD630_02210 CDIF630erm_00343 | Phosphoribosylformylglycinamidinecyclo-ligase | 0.60 | -0.76 | 2.94 | -0.69 | ||
CD630_02220 CDIF630erm_00344 | Phosphoribosylglycinamideformyl transferase | 2.66 | -0.41 | 3.49 | -0.04 | ||
CD630_02230 CDIF630erm_00345 | Bifunctional phosphoribosylaminoimidazole carboxamideformyltransferase/IMP cyclohydrolase | 2.49 | -0.08 | 3.37 | 0.24 | ||
CD630_02240 CDIF630erm_00346 | Phosphoribosylamine–glycine ligase | 2.19 | -0.28 | 3.07 | 0.32 | ||
CD630_02250 CDIF630erm_00347 | Phosphoribosylformylglycinamidine synthase | 1.24 | -0.06 | 2.23 | 0.19 | 00348 | |
CD630_01840 CDIF630erm_00305 | Aspartate carbamoyltransferase | 0.19 | 0.47 | 1.01 | 0.60 | ||
CD630_01850 CDIF630erm_00306 | Dihydroorotate dehydrogenase electron transfer subunit | 2.94 | 0.82 | 2.82 | 0.74 | ||
CD630_01860 CDIF630erm_00307 | Dihydroorotate dehydrogenase 1B | 1.96 | 0.21 | 2.72 | 0.16 | ||
CD630_01870 CDIF630erm_00308 | Orotatephosphoribosyltransferase | 3.92 | 0.19 | 3.38 | 0.54 | ||
CD630_25940 CDIF630erm_02848 | Uracil-specific ABC transporter permease | 0.96 | 0.17 | 3.83 | -0.46 | ||
CD630_25950 CDIF630erm_02849 | Bifunctional pyrimidine operon regulatory protein/uracil phosphoribosyltransferase | – | 0.05 | 2.17 | 0.62 | + | |
CD630_25960 CDIF630erm_02850 | Pseudouridylate synthase | 1.85 | – | 2.05 | – | ||
CD630_25970 CDIF630erm_02851 | Lipoprotein signal peptidase | 2.47 | – | 3.02 | – | ||
CD630_27690 CDIF630erm_03032 | Polysaccharide biosynthesis protein | 1.95 | 0.29 | 2.73 | -0.39 | ||
CD630_27700 CDIF630erm_03033 | Group 1 glycosyl transferase | 1.22 | -0.32 | 2.55 | 0.20 | ||
CD630_27710 CDIF630erm_03034 | UDP-glucose 6-dehydrogenase | 2.13 | -0.05 | 2.59 | 0.41 | ||
CD630_27720 CDIF630erm_03035 | Family 2 glycosyl transferase | 2.23 | -0.23 | 3.25 | 0.35 | ||
CD630_27730 CDIF630erm_03036 | Family 2 glycosyl transferase | 2.45 | -0.53 | 2.59 | 0.06 | ||
CD630_27740 CDIF630erm_03037 | Family 2 glycosyl transferase | 1.24 | -0.52 | 2.30 | 0.64 | ||
CD630_27750 CDIF630erm_03038 | Glycerophosphotransferase | 2.64 | -0.61 | 2.73 | -0.07 | ||
CD630_27760 CDIF630erm_03039 | Family 2 glycosyl transferase | 0.78 | -0.06 | 1.99 | 0.17 | ||
Bacteria were grown as outlined for the RNA-seq experiments. Cell pellets were suspended in 700 μl of ice-cold urea-containing buffer (7 M urea, 2 M thiourea, 50 mM dithiothreitol (DTT), 4% (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM Tris-HCl). Cell lysis was performed by sonication (probe MS73, Sonoplus, Bandelin, Berlin, Germany) in six cycles of 60s (amplitude 60%, 0.1 s pulse every 0.5 s) on ice. Cell debris was removed by centrifugation at 6,000
The eluted peptides were subjected to LC-MS/MS analyses performed on a Proxeon nLC 1000 coupled online to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Waltham, MA, United States). In-house self-packed columns [i.d. 100 μm, o.d. 360 μm, length 150 mm; packed with 1.7 μm Aeris XB-C18 reversed-phase material (Phenomenex, Aschaffenburg, Germany)] were loaded and washed with 10 μl of buffer A [0.1% (v/v) acetic acid] at a maximum pressure of 750 bar. For coupled LC-MS/MS analysis, elution of peptides took place with a non-linear 80 min gradient from 1 to 99% buffer B [0.1% (v/v) acetic acid in acetonitrile] at a constant flow rate of 300 nl/min. Eluting peptides were recorded in the mass spectrometer at a resolution of
Cells were grown to the mid-exponential growth phase and harvested anaerobically as indicated above for the transcriptome and proteome experiments. The supernatant was removed and the cells were immediately quenched by suspension in pre-cooled isotonic sodium chloride-methanol [50% (v/v), -32°C]. Cells were pelleted at -20°C, 8,000
Coenzyme A (CoA)-esters were isolated by cell breakage using a Precellys 24 homogenizer (Peqlab, Erlangen, Germany) at -10°C. The procedure included three cycles of homogenization (6,800 rpm, 30 s with equivalent breaks). The lysate was transferred to 10 ml of ice-cold ammonium acetate (25 mM, pH 6) and centrifuged (5 min at 10,000
Motif search was performed with the standalone version of MEME (
We performed genome-wide motif search in the promoters [-250,0] of
The overarching aim of this study was identification and characterization of the Fur regulon at the transcriptional, translational, metabolomics and the phenotypic level. For this purpose a
We explicitly circumvented the utilization of the chelator 2,2′-dipyridyl (DPP) to achieve low/no iron conditions. Cernat and Scott failed after DPP treatment of
Growth of wild type and
Next, different iron sources were analyzed for the ability to restore iron limited growth of the wild type and the
We aimed at a multi-level, holistic view on iron-regulation in
The RNA-Seq approach identified 3,156 individual transcripts. First, we compared the 4 different transcriptomes (wild type low/high iron,
Analyzing iron limitation in
Combined GC/MS- and LC/MS-based metabolome approaches were employed for the analyses of intracellular metabolites including CoA-esters and for the elucidation of the metabolic composition of the growth medium and corresponding volatiles. Overall, we identified 113 intracellular metabolites including 23 coenzyme A-esters. Extracellularly, 45 metabolites were identified. Using a fold change cutoff of 1.5 and at an adjusted
Finally, a bioinformatics approach for the definition of the Fur regulon was taken. Fur binding sites upstream of Fur-regulated genes in
As expected various iron and other metal uptake systems encoded by
Overview of the overall adaptation strategies of
With standard Western diet the iron concentrations in the gut is about 100 mg Fe/g wet weight feces (
Certain systems found still enhanced at both the RNA and protein level (
The precursor of many catechol siderophores is spermidine (
Comparative inspection of the wild type and
Scanning/transmission electron microscopic images and motility assays of
The basic principles of energy generation of
At low iron conditions the ferredoxin-dependent pathways of phenylalanine, leucine (
Moreover, the synthesis of the flavodoxin (FldX, CDIF630erm_02217) and of enzymes of riboflavin biosynthesis (
Looking at the identified potential Fur binding sites, most of the observed changes are not directly regulated by Fur. Two open readings frames upstream of the
Obviously, the low iron stress was counteracted via increased resistance to external attacks. Firstly, the transcription of the
We challenged the wild type and the
Growth of wild type and
Additionally, two potential ABC transporter systems of the bacitracin/multidrug family (CDIF630erm_00443 – 00445,CDIF630erm_00938 – 00943) and one multi antimicrobial extrusion protein with a downstream MarR family transcriptional regulator gene (CDIF630erm_03501 – 03502) were also found approximately 2- to 3-fold induced at the transcriptional level (Table
Dihydroorotate dehydrogenase (PyrDK), aspartate carbamoyltransferase (PyrB), and orotate phosphoribosyltransferase (PyrE), all enzyme of pyrimidine biosynthesis (
The highly specialized energy metabolism of
In the closely related
AN, MaB, and PD were responsible for the RNA-Seq experiments. CL, KR, SS, SM, DB, and AO performed and interpreted the proteomic approaches. MN-S performed and interpreted the metabolome experiments, bioinformatics came from DE and MiB. MR, MaB and AMM did the electron microscopy studies. DJ, MN-S, MJ, JMBdA and AMM were responsible for data integration and manuscript writing.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at:
Clostron-based knock out of the
Growth of wild type and
Complementation of the
Comparison of the growth behavior of
Principle component analysis (PCA) of the RNA-Seq based transcriptome samples from this study in biological triplicates. Cultures were grown in CDM medium under high (760 μg/l) and low (11 μg/l) iron conditions, harvested at mid-log phase (see Figure
Comparative transcriptome (RNA-Seq) analysis of
Comparative transcriptome (RNA-Seq) analysis of
Comparative transcriptome (RNA-Seq) analysis of the
Comparative proteome analysis of
Genome annotation conversion table for the
Comparative metabolome and exo-metabolome analysis of
Bioinformatics-based investigation of the Fur-binding sites in the
Iron-binding proteins in