Through the dedicated efforts of myriad talented researchers, the following have been achieved in the Wood lab:
Cryptic Prophages and CRISPR-Cas
We discovered that the main physiological role of CRISPR-Cas may be to control resident prophages in the bacterial chromosome (Int. J. Molec. Sci 23, 16195 (2022)) as well as control non-phage-related genes in the chromosome; i.e., that CRISPR-Cas regulates the bacterial chromosome. The mechanism appears to be non-canonical and includes RNA inhibition rather than DNA cleavage.
Discovered that cryptic prophages are not genomic junk and are used by the cell to respond to stress. Nature Communications 1: 147 (2010).
Discovered that cryptic prophages control persister cell resuscitation based on sensing phosphate Environ. Microbiol. 23: 7245-7254 (2021).
Discovered that through YfdM of cryptic prophage CPS-53, T1-type, lytic phage SW1. SW1 is involved in kin recognition and controls formation of a demarcation line between swimming E. coli Cell Reports 27: 737–749 (2019).
Discovered that cryptic prophage proteins affect biofilm formation Nature ISME Journal 3: 1164-1179 (2009).
Reversing Methanogenesis
Reversed methanogenesis for the first time by producing methane reductase from unculturable Archaea from the Black Sea in the methanogen Methanosarcina acetivorans to create the first pure culture that grows anaerobically on methane; this allows the inexpensive carbon source methane to now be used for biotechnology and proves that cells grow on methane anaerobically by reversing methanogenesis. Microbial Cell Factories 15: 11 (2016).
Reversed methanogenesis for the first time such methane can be converted into electricity for the first time in a microbial fuel cell. Nature Communications 8: 15419 (2017). This process was also improved by adding electron carriers such that power at 216 mW/m2 was obtained at a current density 7.3 A/m2; this performance is one of the best achieved for any substrate in a microbial fuel cell. Biotechnology for Biofuels 11: 211 (2018).
By reversing methanogenesis in our archaeal methanogen host by producing ANME methane reductase from the Black Sea and by producing 3-hydroxybutyryl-CoA dehydrogenase (Hbd) from Clostridium acetobutylicum, we converted methane into optically pure L-lactate. Our process is 10-fold superior compared to the aerobic process in terms of the yield and represents the first metabolic engineering of a methanogen. Biotechnology & Bioengineering on-line (2016).
Toxin/Antitoxin Systems
Discovered the first type VII TA system in which the antitoxin (TomB) is an enzyme that inactivates toxin Hha by oxidizing its cysteine residues; the reaction by TomB is controlled by oxygen levels in biofilms. Nature Communications 7: 13634 (2016).
Characterized the first toxin/antitoxin system (HigB/HigA) of the opportunistic pathogen Pseudomonas aeruginosa and showed it helped regulate virulence since toxin HigB reduces production of the virulence factors pyochelin, pyocyanin, swarming, and biofilm formation Microbiology Open 5: 499–511 (2016).
Discovered the first solo toxin, OrtT of E. coli OrtT lacks an antitoxin, and we found its physiological role is to damage the membrane to reduce ATP during nutrient stress, thereby reducing metabolism so the cell can survive the nutrient stress Toxins 7: 299-321 (2015).
Discovered the physiological role of the MqsR/MqsA TA system is to protect the E. coli cell from bile acid stress (bile dissolves the membrane of cells); toxin MqsR degrades the mRNA of YgiS and thereby prevents influx of the bile acid Environ. Microbiol. on-line (2015).
Determined that toxin YafQ increases persister cell formation by reducing indole signaling; hence, indole signaling reduces persistence Environ. Micro. on-line (2014).
Discovered that toxin GhoT reduces cell metabolism in the presence of antibiotics to increase long-term cell fitness; hence, a physiological role was determined for this toxin Environmental Microbiology 16: 1741-1754 (2014).
Demonstrated that TA systems may be rapidly evolved by converting antitoxin GhoS to toxin ArT with two substitutions and that both a type III antitoxin (ToxI) and a type II antitoxin (MqsA) could be evolved to mask the membrane toxicity of the new toxin Scientific Reports 4: 4807 (2014).
Discovered the first toxin of a TA system (RalR) that cleaves DNA. Nucleic Acids Research on-line (2014).
Engineered the first toxin (MqsR) of a TA system (MqsR/MqsA) for greater toxicity to determine that cells that are less fit are more likely to become persister cells. Microb. Biotechnol 5: 509–522 (2012).
Discovered that TA systems directly control other TA systems through cleavage of their antitoxin mRNAs. Environmental Microbiology (2013).
Discovered that TA systems affect biofilm formation. Appl. Microbiol. Biotechnol. 64: 515-524 (2004); J. Bacteriol. 188: 305-316 (2006); PLoS ONE 3(6): e2394 (2008); J. Bacteriol. 191: 1258-1267 (2009).
Discovered that antitoxins (e.g., MqsA, DinJ) are global regulators. Nat. Chem. Biol. 7: 359-366 (2011); Environ. Microbiol. 14: 669–679 (2012).
Discovered that toxins (e.g., MqsR) are global regulators J. Bacteriol. 188: 305-316 (2006).
Discovered the first Type V TA system in which the protein antitoxin GhoS cleaves the mRNA of the toxin GhoT. Nat. Chem. Biol. 8: 855-861 (2012).
Discovered the general phenomenon that TA systems exclude phage via the Hok/sok TA system; this is one of the few systems that allows the cell to behave altruistically. J. Bacteriol. 178: 2044-2050, 1996.
Quantified the ability of the hok locus of the E. coli R1 plasmid to stabilize plasmids (200 generations) and introduced the idea of using multiple loci for increasing plasmid stability. Appl. Environ. Microbiol. 63: 1917-1924 (1997).
Discovered the Hha/TomB TA system and its novel mechanism of toxicity via control of rare tRNAs. PLoS ONE 3(6): e2394 (2008).
Discovered that TA systems are linked to cell motility and quorum sensing. J. Bacteriol. 188: 305-316 (2006).
Persistence
Discovered the molecular mechanism of persister cell formation: dimerization of ribosomes. Biochem. Biophys. Res. Commun. 523: 281-286 (2020).
Discovered the molecular mechanism of persister cell resuscitation: reduction of ppGpp & cAMP as a result of the recognition of nutrients via chemotaxis sensors (for amino acids) and PTS transport (for sugars), which leads to activation of dimerized ribosomes by HflX and chemotaxis. iScience. 23: 100792 (2020).
Discovered that the viable population of VBNCs are the same as persister cells. Environment. Microbiol. on-line (2018).
Discovered that the FDA-approved, anti-cancer drug cisplatin eradicates a broad range of persister cells (e.g., EHEC, S. aureus, and P. aeruginosa) by crosslinking their DNA as they sleep. Biotech. & Bioengr. on-line (2016).
Discovered that the FDA-approved, anti-cancer drug mitomycin C eradicates a broad range of persister cells (e.g., EHEC, S. aureus, and P. aeruginosa) by crosslinking their DNA as they sleep. This study was highlighted by Nature Medicine. Environ. Micro. on-line (2015).
Demonstrated clearly that persistence is the result of metabolic inactivity brought about by a precipitous drop in transcription, translation, or ATP production. Antimicrobial Agents and Chemotherapy 57: 1468–1473 (2013).
Discovered the first toxin that upon inactivation, reduces persistence (MqsR). Biochem. Biophys. Res. Commun. 391: 209-213 (2010).
Discovered that persistence increases as cell fitness decreases. Microb. Biotechnol. 5: 509–522 (2012).
Post-translational Modifications and Cell Physiology
Discovered the first tyrosine phosphatase that controls c-di-GMP levels and eDNA levels. PLoS Pathogens 5: e1000483 (2009); Environ. Microbiol. Reports 2: 449-455 (2010).
Discovered that the post-translational process of acetylation is for rapid stress response. Biochem. Biophys. Res. Comm.. 410: 846-851 (2011).
Biofilms
Created the first beneficial biofilm that limits its own thickness (through a novel quorum-sensing circuit) while it protects reverse osmosis membranes by secreting nitric oxide to prevent and to remove deleterious biofilms; the beneficial biofilm also degrades the reference pollutant epichlorohydrin. Proceedings of the National Academy of Sciences U.S.A. E2802–E2811 (2016).
Pioneered the use of directed evolution of proteins for biofilm control (used protein engineering to develop a toolset of regulatory proteins for controlling biofilm formation by engineering the biofilm regulators SdiA [Appl. Environ. Microbiol. 75: 1703-1716 (2009)]; Hha [Microb. Biotechnol. 3: 717-728 (2010)]; H-NS [Microb. Biotechnol. 3: 344-356 (2010)]; and BdcA [Environ. Microbiol. 13: 631-642 (2011)].
Developed the first genetically-engineered biofilm for any application and used it to secrete successfully antimicrobials that inhibit the growth of deleterious sulfate-reducing bacteria (SRB); used this biofilm to reduce the corrosion by inhibiting the growth of SRB. J. Ind. Microbiol. Biotechnol. 22: 167-175 (1999).
Developed the first QS circuit to control biofilm formation (by engineering a consortium by adding toluene o-monooxygenase to Pseudomonas aeruginosa in order to control indole concentrations so that E. coli biofilm formation could be controlled). BMC Microbiology 7: 42 (2007).
Developed the first synthetic QS circuit for biofilm dispersal using two proteins we engineered for dispersal (Hha and BdcA); this allowed consortial biofilm formation to be controlled for the first time. Nature Comm. 3: 613(2012).
Discovered BdcA is a c-di-GMP binding protein that controls biofilm dispersal and engineered this protein for greater dispersal. Environ. Microbiol. 13: 631-642 (2011).
Identified some of the genes responsible for biofilm formation in E. coli and B. subtilis via DNA microarrays.
Determined that stress increases biofilm formation and this effect is mediated by YcfR (renamed to BhsA) in E. coli. J. Bacteriol. 189: 3051-3062 (2007).
Showed biofilms can decrease corrosion on mild steel (by 40-fold), stainless steel, copper, brass, and aluminum.
Found the X-ray crystallography structure of the first biofilm protein, YmgB (renamed AriR) with collaborator Wolfgang Peti. J. Mol. Biol. 373: 11-26 (2007).
Found that biofilms that produce the peptide antimicrobial gramicidin S may be used to inhibit the corrosion-causing bacteria Leptothrix discophora SP-6 and Desulfosporosinus orientis, and that this biofilm is active in process water from the Three Mile Island nuclear facility.
Discovered that OmpA represses biofilm formation through CpxRA. Environ. Microbiol. 11: 2735-2746 (2009).
Pioneered the use of beneficial biofilms to reduce the corrosion of aluminum, mild steel, brass, and copper by secreting compounds such as gramicidin-S, polyaspartate, gamma-polyglutamate, indolicin, and bactenecin (19 publications in this field).
Cell Signaling
Discovered that quorum sensing control of the stress response promotes resistance to quorum quenching and prevents social cheating. Nature ISMEJ on-line (2014).
Discovered cells are chemotactic toward AI-2. Appl. Microbiol. Biotechnol. 78: 811-819 (2006).
Discovered that uracil is a cell signal. Microb. Biotechnol. 2: 62-74 (2009).
Discovered why E. coli has two quorum sensing systems: one for the human host (37oC, AI-2) and another for lower temperatures (indole). Nature ISME Journal 2: 1007-1023 (2008).
Discovered cell signals are promiscuous in that indole impacts biofilms of cells that cannot synthesize it. BMC Microbiology 7: 42 (2007).
Discovered that E. coli signal indole inhibits P. aeruginosa pathogenicity and QS phenotypes. Microb. Biotechnol. 2: 75-90 (2009).
Discovered that indole is a quorum-sensing signal and that it reduces the biofilm formation of pathogenic E. coli. Appl. Environ. Microbiol. 73: 4100-4109 (2007).
Discovered the protein that exports AI-2 from E. coli (TqsA). J. Bacteriol. 188: 587-598 (2006).
Discovered that the cell signal AI-2 increases biofilms in E. coli (first report of AI-2 controlling a non-luminescence phenotype) and found the uncharacterized protein MqsR mediates this effect. J. Bacteriol. 188: 305-316 (2006).
Discovered that bacterial quorum sensing signals are involved in insect attraction. Nature ISME Journal 6: 1356–1366 (2012).
Discovered that the bacterial signal indole increases epithelial-cell tight-junctions. Proc. Natl. Acad. Sci. U.S.A. 107: 228-233 (2010).
Discovered that YliH and YceP regulate biofilm formation via indole signaling. Appl. Environ. Microbiol. 72: 2449-2459 (2006).
Discovered that indole decreases biofilm formation. BMC Microbiology 7: 42 (2007).
Quorum Quenching
Discovered that QS is used primarily to allow the cells to respond to stress by controlling private goods like catalase, that QS and stress promote resistance to quorum quenching compounds, and that QS and stress prevent social cheating. Nature ISMEJ on-line (2014).
Discovered that cells evolve resistance to quorum-quenching compounds. Nature ISME Journal 6: 493 (2012).
Showed that furanone from the seaweed Delisea pulchra inhibits both known autoinducers for bacterial cell communication. Environ. Microbiol. 3: 731-736 (2001).
Discovered the QS inhibitors indole [BMC Microbiology 7: 42 (2007)]; hydroxylated indoles [Appl. Environ. Microbiol. 73: 4100-4109 (2007)]; 5-fluorouracil [Microb. Biotechnol. 2: 62-74 (2009); Appl. Microbiol. Biotechnol. 82: 525-533 (2009)]; ursolic acid [Appl. Environ. Microbiol. 71: 4022-4034 (2005)]; and adenosine [Microbiol. Biotechnol. 5: 560-572 (2012)]. These discoveries have led to development of the first anti-biofilm compound based on cell signaling, 5-fluoruracil, being adopted for use as a coating for catheters by Angiotech Pharmaceuticals.
Discovered that furanone inactivate quorum sensing by binding LuxR in V. harveyi [Environ. Microbiol. 9: 2486-2495 (2007)].
Bioremediation
Discovered the first bacterium, Pseudomonas stutzeri OX1, capable of degrading aerobically one of the world’s most serious pollutants, tetrachloroethylene [Nature Biotechnology 18: 775-778 (2000)].
Determined for the first time what genes are expressed in the rhizosphere for both a plant and a bacterial pathogen and discovered seven new virulence factors [Microb. Biotechnol. 1: 17-29 (2008)].
Evolved the aromatic monooxygenase toluene o-monooxygenase (TOM) from Burkholderia cepacia G4 for the synthesis of 1-naphthol from naphthalene and for the degradation of chlorinated ethenes which at the time was the largest protein complex to be subject to DNA shuffling, then used saturation mutagenesis to improve chloroform degradation [J. Bacteriol. 184: 344-349 (2002)].
Created the first rhizoremediation system using a genetically-engineered bacterium and plant root for the degradation of trichloroethylene (coined the word “rhizoremediation”) [Appl. Environ. Microbiol. 64: 112-118 (1998)].
Developed one of the first two-stage systems for the destruction of pollutants: treat first with a general physical technique (energetic electrons from pulsed-electric discharge) and then complete the degradation with bacteria (used this technique to degrade perchloroethylene and chlorinated phenols in a fixed-film fluidized-bed bioreactor).
Created the first tree-colonizing bacteria for bioremediation (competitive TCE-degrading bacteria for use with poplar trees [Appl. Environ. Microbiol. 66: 4673-4678 (2000)].
Pieced together a novel metabolic pathway involving 8 genes for enhanced degradation of trichloroethylene and cis-DCE by cloning a DNA-shuffled toluene o-monooxygenase (that initiates attack on the chlorinated aliphatic), a novel glutathione S-transferase (to remove the toxic epoxide formed by the monooxygenase), and gamma-glutamylcysteine synthetase (that provides to cofactor glutathione for the glutathione S-transferase) [Environ. Microbiol. 6: 491-500 (2004)].
Evolved the epoxide hydrolase from Agrobacterium radiobacter AD1 to enhance aerobic mineralization of cis-1,2-dichloroethylene in cells expressing an evolved toluene ortho-monooxygenase [J. Biol. Chem. 279: 46810-46817 (2004)].
Achieved expression in a recombinant host for the first time of soluble methane monooxygenase from Methylosinus trichosporium OB3b and used it to degrade TCE and chloroform without inducers and without competitive inhibition [Appl. Environ. Microbiol. 60: 2473-2482 (1994)].
Quantified TCE degradation in the first fixed-film bioreactor that uses genetically-engineered bacteria to degrade TCE for an appreciable period (developed a mathematical model for the biofilm reactor).
Evolved dioxygenases for bioremediation including evolving the first two enzymes in the pathway for 2,4-dinitrotoluene (2,4-DNT) degradation, evolving the large subunit of 2,4-DNT dioxygenase for an increase in the rates of degradation of 2,3-DNT, 2,4-DNT, 2,5-DNT, 2,6-DNT, 2-NT, and 4-NT, and evolving the first enzyme capable of degrading 2,3-DNT and 2,5-DNT; the evolution of 4-methyl-5-nitrocatechol monooxygenase (the second enzyme in the degradation of 2,4-DNT) led to a broadening of the enzyme substrate range to include 4-nitrophenol and 3-methyl-4-nitrophenol.
Discovered Pseudomonas stutzeri OX1 is chemotactic toward chlorinated compounds (e.g., TCE, PCE, cis-DCE, trans-DCE, 1,1-DCE, vinyl chloride) so this one strain moves toward PCE, PCE induces ToMO formation, then ToMO degrades PCE.
Discovered toluene/o-xylene monooxygenase from Pseudomonas stutzeri can degrade chlorinated aliphatics as well as mixtures of chlorinated aliphatics (including tetrachloroethylene). Appl. Microbiol. Biotechnol. 56: 265-269 (2001).
Bioenergy
Created one of the best strains for producing hydrogen from bacteria through formate [Microb. Biotechnol. 1: 30-39 (2008)] and glucose [Appl. Microbiol. Biotechnol. 77: 879-890 (2007)].
Used strain evolution to make hydrogen from glycerol [Biochem. Biophys. Res. Comm. 391: 1033-1038 (2010)].
Evolved the first hydrogenase for hydrogen production [Appl. Microbiol. Biotechnol. 79: 77-86 (2008)].
Green Chemistry
Engineered the first enzyme for complete control of regio-specific hydroxylation (TpMO for toluene oxidation) by combining rational engineering with random engineering to create the first meta-hydroxylating enzyme for toluene [J. Biol. Chem. 280: 506-514 (2005)].
Identified the key amino acids responsible indole oxidation and color formation (e.g., indigo, indirubin, isoindigo, isatin) via toluene o-monooxygenase of B. cepacia G4 [Appl. Microbiol Biotechnol. 66: 422-429 (2005)].
Discovered aromatic monooxygenases such as T3MO of R. pickettii and T4MO of P. mendocina KR1 catalyze three successive hydroxylations to convert benzene to trihydroxybenzene, opening the possibility of using these strains to form important dihydroxy and trihydroxy compounds (Appl. Environ. Microbiol. 70: 3814-3820 (2004)).
Evolved monooxygenases for green chemistry, including those for the production of 8 industrial compounds that could not previously be made by bacteria or enzymes (nitrohydroquinone, 4-methylresorcinol, 1-hydroxyfluorene, 3-hydroxyfluorene, 4-hydroxyfluorene, 2-naphthol, 2,6-dihydroxynaphthalene, and 3,6-dihydroxyfluorene).
Evolved (using DNA shuffling and saturation mutagenesis) the family of aromatic monooxygenases (ToMO of P. stutzeri OX1, TOM of B. cepacia G4, T3MO of R. pickettii, and T4MO of P. mendocina KR1) to produce industrially-significant compounds such as methyl dihydroxy aromatics (e.g., 4-methylresorcinol, methylhydroquinone, pyrogallol), methoxy dihydroxy aromatics (3-methoxycatechol, methoxyhydroquinone), and nitro dihydroxy aromatics (4-nitrocatechol) to demonstrate that protein engineering of monooxygenases may be used to control the regiospecific oxidation of nitroaromatics, methylaromatics, and methoxyaromatics.
Discovered six residues that influence catalysis for monooxygenases.
Miscellany
Determined via DNA microarrays that furanone, the anti-biofilm compound from the seaweed Delisea pulchra that does not affect the growth of Gram-negative strains, inhibits AI-2 quorum sensing in Gram-negative strains.
Elucidated the genetic basis of the inhibition of Gram-positive strains by furanone from the seaweed Delisea pulchra; this compound may have importance as a novel antimicrobial.
Found that furanone from the seaweed Delisea pulchra both stimulates and inhibits siderophore formation in pseudomonads and that it may be used to decrease corrosion.
Expressing bacterial dioxygenases for the bioremediation of nitroaromatics and green chemical synthesis (using the combinatorial method of directed evolution, with Prof. Barth Smets).
Enhanced expression of lignin peroxidase from Streptomyces viridosporus by formulating a novel corn-starch-based medium.
Discerned that the translational machinery of the E. coli cell limits recombinant protein production (for high gene dosage and strong promoters) and used specialized ribosomes to enhance productivity.