Automated genomic context analysis and experimental validation platform for discovery of prokaryote transcriptional regulator functions

BMC Genomics

Pat J Unkefer²  Clifford J Unkefer²  Melinda S Wren²  Kristy L Nowak-Lovato²  Fangping Mu¹  Ricardo Martí-Arbona³ 

[1]Theoretical Division, Los Alamos National Laboratory, PO Box 1663, Los Alamos, NM 87545, USA

[2]Bioscience Division, Los Alamos National Laboratory, PO Box 1663, Los Alamos, NM 87545, USA

[3]Los Alamos National Laboratory, P.O. Box 1663, MS E529, Los Alamos, New Mexico 87545, USA

关键词: Protein function discovery;    Burkholderia xenovorans LB400;    rtPCR;    FAC-MS;    EMSA;    Anisotropy;    Transcriptional regulator;    methylglyoxal;    Bxe_B3018;    Comparative genomics;   

Others  :  1122716
DOI  :  10.1186/1471-2164-15-1142

 received in 2014-05-19, accepted in 2014-12-12,  发布年份 2014

【 摘 要 】

Background

The clustering of genes in a pathway and the co-location of functionally related genes is widely recognized in prokaryotes. We used these characteristics to predict the metabolic involvement for a Transcriptional Regulator (TR) of unknown function, identified and confirmed its biological activity.

Results

A software tool that identifies the genes encoded within a defined genomic neighborhood for the subject TR and its homologs was developed. The output lists of genes in the genetic neighborhoods, their annotated functions, the reactants/products, and identifies the metabolic pathway in which the encoded-proteins function. When a set of TRs of known function was analyzed, we observed that their homologs frequently had conserved genomic neighborhoods that co-located the metabolically related genes regulated by the subject TR. We postulate that TR effectors are metabolites in the identified pathways; indeed the known effectors were present. We analyzed Bxe_B3018 from Burkholderia xenovorans, a TR of unknown function and predicted that this TR was related to the glycine, threonine and serine degradation. We tested the binding of metabolites in these pathways and for those that bound, their ability to modulate TR binding to its specific DNA operator sequence. Using rtPCR, we confirmed that methylglyoxal was an effector of Bxe_3018.

Conclusion

These studies provide the proof of concept and validation of a systematic approach to the discovery of the biological activity for proteins of unknown function, in this case a TR. Bxe_B3018 is a methylglyoxal responsive TR that controls the expression of an operon composed of a putative efflux system.

【 授权许可】

   
2014 Martí-Arbona et al.; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Santos MA, Turinsky AL, Ong S, Tsai J, Berger MF, Badis G, Talukder S, Gehrke AR, Bulyk ML, Hughes TR, Wodak SJ: Objective sequence-based subfamily classifications of mouse homeodomains reflect their in vitro DNA-binding preferences. Nucleic Acids Res 2010, 38:7927-7942.
• [2]Rocha EP: The organization of the bacterial genome. Annu Rev Genet 2008, 42:211-233.
• [3]Staehelin LA, Golecki JR, Drews G: Supramolecular organization of chlorosomes (chlorobium vesicles) and of their membrane attachment sites in Chlorobium limicola. Biochim Biophys Acta 1980, 589:30-45.
• [4]Wada T, Shirouzu M, Terada T, Kamewari Y, Park SY, Tame JR, Kuramitsu S, Yokoyama S: Crystal structure of the conserved hypothetical protein TT1380 from Thermus thermophilus HB8. Proteins 2004, 55:778-780.
• [5]Handa N, Terada T, Doi-Katayama Y, Hirota H, Tame JR, Park SY, Kuramitsu S, Shirouzu M, Yokoyama S: Crystal structure of a novel polyisoprenoid-binding protein from Thermus thermophilus HB8. Protein Sci 2005, 14:1004-1010.
• [6]Rohmer M, Knani M, Simonin P, Sutter B, Sahm H: Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate. Biochem J 1993, 295(Pt 2):517-524.
• [7]Schriemer DC, Hindsgaul O: Deconvolution approaches in screening compound mixtures. Comb Chem High Throughput Screen 1998, 1:155-170.
• [8]Yu X, Lin J, Masuda T, Esumi N, Zack DJ, Qian J: Genome-wide prediction and characterization of interactions between transcription factors in Saccharomyces cerevisiae. Nucleic acids research 2006, 34:917-927.
• [9]Kim J, Cunningham R, James B, Wyder S, Gibson JD, Niehuis O, Zdobnov EM, Robertson HM, Robinson GE, Werren JH, Sinha S: Functional characterization of transcription factor motifs using cross-species comparison across large evolutionary distances. PLoS Comput Biol 2010, 6:e1000652.
• [10]Berger MF, Badis G, Gehrke AR, Talukder S, Philippakis AA, Pena-Castillo L, Alleyne TM, Mnaimneh S, Botvinnik OB, Chan ET, Khalid F, Zhang W, Newburger D, Jaeger SA, Morris QD, Bulyk ML, Hughes TR: Variation in homeodomain DNA binding revealed by high-resolution analysis of sequence preferences. Cell 2008, 133:1266-1276.
• [11]Philippakis AA, Qureshi AM, Berger MF, Bulyk ML: Design of compact, universal DNA microarrays for protein binding microarray experiments. J Comput Biol 2008, 15:655-665.
• [12]Yurkov V, Stackebrandt E, Holmes A, Fuerst JA, Hugenholtz P, Golecki J, Gad’on N, Gorlenko VM, Kompantseva EI, Drews G: Phylogenetic positions of novel aerobic, bacteriochlorophyll a-containing bacteria and description of Roseococcus thiosulfatophilus gen. nov., sp. nov., Erythromicrobium ramosum gen. nov., sp. nov., and Erythrobacter litoralis sp. nov. Int J Syst Bacteriol 1994, 44:427-434.
• [13]Zhu C, Byers KJ, McCord RP, Shi Z, Berger MF, Newburger DE, Saulrieta K, Smith Z, Shah MV, Radhakrishnan M, Philippakis AA, Hu Y, De Masi F, Pacek M, Rolfs A, Murthy T, LaBaer J, Bulyk ML: High-resolution DNA-binding specificity analysis of yeast transcription factors. Genome Res 2009, 19:556-566.
• [14]Choi Y, Qin Y, Berger MF, Ballow DJ, Bulyk ML, Rajkovic A: Microarray analyses of newborn mouse ovaries lacking Nobox. Biol Reprod 2007, 77:312-319.
• [15]Berger MF, Bulyk ML: Universal protein-binding microarrays for the comprehensive characterization of the DNA-binding specificities of transcription factors. Nat Protoc 2009, 4:393-411.
• [16]Alleyne TM, Pena-Castillo L, Badis G, Talukder S, Berger MF, Gehrke AR, Philippakis AA, Bulyk ML, Morris QD, Hughes TR: Predicting the binding preference of transcription factors to individual DNA k-mers. Bioinformatics 2009, 25:1012-1018.
• [17]Pompeani AJ, Irgon JJ, Berger MF, Bulyk ML, Wingreen NS, Bassler BL: The Vibrio harveyi master quorum-sensing regulator, LuxR, a TetR-type protein is both an activator and a repressor: DNA recognition and binding specificity at target promoters. Mol Microbiol 2008, 70:76-88.
• [18]Schena M, Shalon D, Davis RW, Brown PO: Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 1995, 270:467-470.
• [19]Marti-Arbona R, Teshima M, Anderson PS, Nowak-Lovato KL, Hong-Geller E, Unkefer CJ, Unkefer PJ: Identification of new ligands for the methionine biosynthesis transcriptional regulator (MetJ) by FAC-MS. J Mol Microbiol Biotechnol 2012, 22:205-214.
• [20]Maity TS, Close DW, Valdez YE, Nowak-Lovato K, Marti-Arbona R, Nguyen TT, Unkefer PJ, Hong-Geller E, Bradbury AR, Dunbar J: Discovery of DNA operators for TetR and MarR family transcription factors from Burkholderia xenovorans. Microbiology 2012, 158:571-582.
• [21]Kanehisa M, Goto S: KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 2000, 28:27-30.
• [22]Rkenes TP, Lamark T, Strom AR: DNA-binding properties of the BetI repressor protein of Escherichia coli: the inducer choline stimulates BetI-DNA complex formation. J Bacteriol 1996, 178:1663-1670.
• [23]Parsek MR, Shinabarger DL, Rothmel RK, Chakrabarty AM: Roles of CatR and cis, cis-muconate in activation of the catBC operon, which is involved in benzoate degradation in Pseudomonas putida. J Bacteriol 1992, 174:7798-7806.
• [24]Eaton RW: p-Cumate catabolic pathway in Pseudomonas putida Fl: cloning and characterization of DNA carrying the cmt operon. J Bacteriol 1996, 178:1351-1362.
• [25]Iwanicka-Nowicka R, Zielak A, Cook AM, Thomas MS, Hryniewicz MM: Regulation of sulfur assimilation pathways in Burkholderia cenocepacia: identification of transcription factors CysB and SsuR and their role in control of target genes. J Bacteriol 2007, 189:1675-1688.
• [26]Schweizer HP, Po C: Regulation of glycerol metabolism in Pseudomonas aeruginosa: characterization of the glpR repressor gene. J Bacteriol 1996, 178:5215-5221.
• [27]Galan B, Kolb A, Sanz JM, Garcia JL, Prieto MA: Molecular determinants of the hpa regulatory system of Escherichia coli: the HpaR repressor. Nucleic Acids Res 2003, 31:6598-6609.
• [28]Knoten CA, Hudson LL, Coleman JP, Farrow JM 3rd, Pesci EC: KynR, a Lrp/AsnC-type transcriptional regulator, directly controls the kynurenine pathway in Pseudomonas aeruginosa. J Bacteriol 2011, 193:6567-6575.
• [29]Hall RS, Marti-Arbona R, Hennelly SP, Maity TS, Dunbar J, Unkefer CJ, Unkefer PJ: In-vitro Characterization of an L-Kynurenine-Responsive Transcription Regulator of the Oxidative Tryptophan Degradation Pathway in Burkholderia xenovorans. J Mol Biology Res 2013, 3:55-67.
• [30]Bender RA: Regulation of the histidine utilization (hut) system in bacteria. Microbiol Mol Biol Rev 2012, 76:565-584.
• [31]Marti-Arbona R, Xu C, Steele S, Weeks A, Kuty GF, Seibert CM, Raushel FM: Annotating enzymes of unknown function: N-formimino-L-glutamate deiminase is a member of the amidohydrolase superfamily. Biochemistry 2006, 45:1997-2005.
• [32]Santiago B, Schubel U, Egelseer C, Meyer O: Sequence analysis, characterization and CO-specific transcription of the cox gene cluster on the megaplasmid pHCG3 of Oligotropha carboxidovorans. Gene 1999, 236:115-124.
• [33]Grunden AM, Shanmugam KT: Molybdate transport and regulation in bacteria. Arch Microbiol 1997, 168:345-354.
• [34]Serebrovsky AS: Analysis of allelomorphism. IV. Transgeneration of scute-6 and a case of “non-allelomorphic” limbs of an allelomorphic sequence in Drosophila Melanogaster. Roux Arch Dev Biol 1930, 122:88-104.
• [35]Jaeger SA, Chan ET, Berger MF, Stottmann R, Hughes TR, Bulyk ML: Conservation and regulatory associations of a wide affinity range of mouse transcription factor binding sites. Genomics 2010, 95:185-195.
• [36]Moreno-Hagelsieb G, Collado-Vides J: A powerful non-homology method for the prediction of operons in prokaryotes. Bioinformatics 2002, 18(Suppl 1):S329-S336.
• [37]Moreno-Hagelsieb G, Collado-Vides J: Operon conservation from the point of view of Escherichia coli, and inference of functional interdependence of gene products from genome context. In Silico Biol 2002, 2:87-95.
• [38]Nguyen TT, Marti-Arbona R, Hall RS, Maity TS, Valdez YE, Dunbar J, Unkefer CJ, Unkefer PJ: Identification and In-vitro Characterization of a Novel OhrR Transcriptional Regulator in Burkholderia xenovorans LB400. J Mol Biology Res 2013, 3:37-46.
• [39]Martí-Arbona R, Maity TS, Dunbar J, Unkefer CJ, Unkefer PJ: Discovery of a Choline-Responsive Transcriptional Regulator in Burkholderia xenovorans. J Mol Biology Res 2013, 3:91-96.
• [40]Lo TWC, Westwood ME, Mclellan AC, Selwood T, Thornalley PJ: Binding and Modification of Proteins by Methylglyoxal under Physiological Conditions - a Kinetic and Mechanistic Study with N-Alpha-Acetylarginine, N-Alpha-Acetylcysteine, and N-Alpha-Acetyllysine, and Bovine Serum-Albumin. J Biol Chem 1994, 269:32299-32305.
• [41]Vattanaviboon P, Sriprang R, Mongkolsuk S: Catalase has a novel protective role against electrophile killing of Xanthomonas. Microbiol 2001, 147:491-498.
• [42]De Groote VN, Fauvart M, Kint CI, Verstraeten N, Jans A, Cornelis P, Michiels J: Pseudomonas aeruginosa fosfomycin resistance mechanisms affect non-inherited fluoroquinolone tolerance. J Med Microbiol 2011, 60:329-336.
• [43]Hansen JL, Moore PB, Steitz TA: Structures of five antibiotics bound at the peptidyl transferase center of the large ribosomal subunit. J Mol Biol 2003, 330:1061-1075.
• [44]Schlunzen F, Pyetan E, Fucini P, Yonath A, Harms JM: Inhibition of peptide bond formation by pleuromutilins: the structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin. Mol Microbiol 2004, 54:1287-1294.
• [45]El-Halfawy OM, Valvano MA: Chemical communication of antibiotic resistance by a highly resistant subpopulation of bacterial cells. PloS One 2013, 8:e68874.
• [46]Sisinni L, Cendron L, Favaro G, Zanotti G: Helicobacter pylori acidic stress response factor HP1286 is a YceI homolog with new binding specificity. FEBS J 2010, 277:1896-1905.
• [47]Ispolnov K, Gomes RA, Silva MS, Freire AP: Extracellular methylglyoxal toxicity in Saccharomyces cerevisiae: role of glucose and phosphate ions. J Appl Microbiol 2008, 104:1092-1102.
• [48]Ferguson GP, Totemeyer S, MacLean MJ, Booth IR: Methylglyoxal production in bacteria: suicide or survival? Arch Microbiol 1998, 170:209-218.
• [49]Chandrangsu P, Dusi R, Hamilton CJ, Helmann JD: Methylglyoxal resistance in Bacillus subtilis: contributions of bacillithiol-dependent and independent pathways. Mol Microbiol 2014, 91:706-714.
• [50]Kanehisa M, Goto S, Sato Y, Kawashima M, Furumichi M, Tanabe M: Data, information, knowledge and principle: back to metabolism in KEGG. Nucleic Acids Res 2014, 42:D199-D205.
• [51]LiCata VJ, Wowor AJ: Applications of fluorescence anisotropy to the study of protein-DNA interactions. Methods Cell Biol 2008, 84:243-262.
• [52]Schmittgen TD, Livak KJ: Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008, 3:1101-1108.