NITRATE BIOSENSOR

Abstract

A nitrate sensing biosensor and bacteria and applications relating to the use of same are described. The nitrate biosensor comprises a two component sensor system (TCS) comprising: a nitrate-sensing sensor kinase (SK) gene comprising a ligand binding domain operably coupled to a kinase domain, and, a cognate response regulator (RR) gene comprising a receiver domain operably coupled to an DNA binding domain (DBD), as well as an output promoter that binds said DBD that is operably coupled to a heterologous reporter gene.

Claims

1) A genetically engineered bacteria, said bacteria overexpressing: a) a two component sensor system (TCS) comprising: i) a nitrate-sensing sensor kinase (SK) gene comprising a ligand binding domain operably coupled to a kinase domain, and, ii) a cognate response regulator (RR) gene comprising a receiver domain operably coupled to an DNA binding domain (DBD), b) an output promoter that binds said DBD that is operably coupled to a heterologous reporter gene.

2) (canceled)

3) (canceled)

4) (canceled)

5) The bacteria of claim 1, which is gut-adapted for use in humans.

6) The bacteria of claim 5, wherein said SK gene or said RR gene or both genes are encoded on an expression vector, an inducible expression vector, and/or a constitutive expression vector.

7) (canceled)

8) (canceled)

9) The bacteria of claim 1, wherein said SK gene or said RR gene or both genes integrated into a genome of said bacteria.

10) The bacteria of claim 1, wherein said SK gene and said RR gene are encoded in a single operon.

11) The bacteria of claim 1, wherein said reporter gene is encoded on a plasmid.

12) The bacteria of claim 1, wherein said reporter gene is integrated into a genome of said bacteria.

13) The bacteria of claim 1, comprising SEQ ID NO. 1 and an amino terminal portion of SEQ ID NO. 3 operably fused to a carboxy terminal portion of SEQ ID NO 5 containing a DNA binding site.

14) The bacteria of claim 1, wherein said reporter gene encodes a fluorescent protein.

15) The bacteria of claim 1, wherein said reporter gene encodes green fluorescent protein, red fluorescent protein, far red fluorescent protein, blue fluorescent protein, orange fluorescent protein, yellow fluorescent protein, mCHERRY, mORANGE, mCITRINE, VENUS, YPET, EMERALD, or CERULEAN.

16) (canceled)

17) (canceled)

18) (canceled)

19) (canceled)

20) (canceled)

21) (canceled)

22) A method of measuring nitrate levels in a patient, comprising: a) combining a gut sample with a nitrate reporter bacteria comprising: i) a nitrate-sensing sensor kinase (SK) gene encoding an SK protein comprising a ligand binding domain that binds nitrate and activates a kinase domain, ii) a cognate RR gene encoding an RR protein comprising a receiver domain operably coupled to an DNA binding domain (DBD), wherein said cognate RR protein is activated by said activated kinase domain phosphorylating said receiver domain, and iii) a reporter gene comprising a DNA binding site that binds said DBD of said cognate activated RR protein operably coupled to an open reading frame encoding a reporter protein; b) measuring expression of said reporter gene; and, c) correlating a measured level of reporter gene expression with a level of nitrate using a standard curve.

23) The method of claim 22, wherein said bacteria is a gut-adapted bacteria and said combining step is by administering said bacteria to said patient.

24) The method of claim 22, wherein said combining step a) is by collecting a gut or stool sample from said patient and combining said gut or stool sample with said bacteria.

25) (canceled)

26) (canceled)

27) (canceled)

28) A fusion protein comprising the amino terminus of NarL operably fused to the DNA binding site domain of YdfI.

29) The fusion protein of claim 28, comprising an amino portion of SEQ ID NO 3 fused to a carboxy portion of SEQ ID NO. 5.

30) The fusion protein of claim 28, comprising SEQ ID NO 6-9.

31) A bacteria comprising an expression vector encoding the fusion protein of claim 28.

Description

BRIEF DESCRIPTION OF FIGURES

[0126] FIG. 1. Two component sensor systems.

[0127] FIG. 2. Overview of the human colonic microflora. Bacterial genera were classified as health positive, health negative, or health neutral. Bacterial enumeration was by selective media. Ps=Pseudomonas.

[0128] FIG. 3. Dose response of the described NarX and NarL/YdfI nitrate sensor in B. subtilis to NaNO.sub.3.

[0129] FIG. 4. Demonstration of the use of the B. subtilis nitrate sensor to detect both nitrate and fertilizer in a soil sample. Dose response curves of soil with increasing concentrations of NaNO.sub.3 and fertilizer are shown.

[0130] FIG. 5. Dose response of the described NarX and NarL/YdfI nitrate sensor in E. coli to NaNO.sub.3. An additional inactivated NarL-YdfI D59E mutant (lacking the needed aspartate residue for activation) is shown as a negative control to demonstrate specificity of the response to the described pathway.

[0131] FIG. 6. Preliminary data demonstrating the use of the E. coli nitrate sensor being used in vivo to determine the presence of the dextran sodium sulfate (DSS) inflammation disease model in mice.

DETAILED DESCRIPTION

[0132] In our proof of concept work, we used a NarX SK and a rewired NarL RR with a DBD domain from YdfI and a GFP reporter. These sequence are publically available, and are discussed in more detail below.

[0133] NarX: Acts as a sensor kinase (SK) for nitrate/nitrite and transduces signal of nitrate availability to the NarL protein and of both nitrate/nitrite to the NarP protein. NarX probably activates NarL and NarP by phosphorylation in the presence of nitrate. NarX also plays a negative role in controlling NarL activity, probably through dephosphorylation in the absence of nitrate.

TABLE-US-00003 narXfromE.coli[1](SEQIDNO.1): MLKRCLSPLTLVNQVALIVLLSTAIGLAGMAVSGWLVQGV QGSAHAINKAGSLRMQSYRLLAAVPLSEKDKPLIKEMEQT AFSAELTRAAERDGQLAQLQGLQDYWRNELIPALMRAQNR ETVSADVSQFVAGLDQLVSGFDRTTEMRIETVVLVHRVMA VFMALLLVFTIIWLRARLLQPWRQLLAMASAVSHRDFTQR ANISGRNEMAMLGTALNNMSAELAESYAVLEQRVQEKTAG LEHKNQILSFLWQANRRLHSRAPLCERLSPVLNGLQNLTL LRDIELRVYDTDDEENHQEFTCQPDMTCDDKGCQLCPRGV LPVGDRGTTLKWRLADSHTQYGILLATLPQGRHLSHDQQQ LVDTLVEQLTATLALDRHQERQQQLIVMEERATIARELHD SIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNELNA SWAQLRELLTTFRLQLTEPGLRPALEASCEEYSAKFGFPV KLDYQLPPRLVPSHQAIHLLQIAREALSNALKHSQASEVV VTVAQNDNQVKLTVQDNGCGVPENAIRSNHYGMIIMRDRA QSLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHE NarQ:anothernitratedetectorfromShewenella oneidensis.SEQIDNO.2: MKRGSLTSKILGLMLVLILLSSSLAIFAIINLSYSLGDAK AINASGSLRMQSYRLMFYANSGSEAAQEKITEFENTLHSE ALHPSKSWLSPKKIAAQYQLVIDKWLVMKYYIEQENSRDY AASLKDFVDTIDLLVLEMEHHAAFKLRLLAASQIFGLGLM LSIAFLAVRFTKRKVVVPLQQLMESANTISKGNFEIEMPE TEYIELTALTDALQKTARELATLYGNLESQVAEKTLALTR ANNELAFLYDTLLTLNAKKLDYKALKAALNQLKDYESIDY LRLIIQYPEQELEMIEANGGWPESADNSTRFPLQFEQANL GYLELISAQDINTPLFKNFAIMLTRSIVIHNATEQRQQLA LMEERGVIARELHDSLGQVLSFLKIQISLLRKNLDHSCRS PAVEVQLTEINEGVSTAYVQLRELLSTFRLTIKEPNLKNA MEAMLEQLRANTDIKIHLDYKLSPQWLEAKQHIHILQITR EATLNAIKHANASHINIRCYKDDRGMVNISVSDNGVGIGH IKERDQHFGIGIMHERASKLDGEVVFSSNDTHTNSTATTE QRHQENPDSPLESHNTSNLSQGTIVTLIFPSQQEPTHG

[0134] Other nitrate SK homologs that can be used include WP 042949651 from Salmonella (86% amino acid identity to NarX); WP_042949651 from Citrobacter (86%); WP_045142747.1 from Enterobacter (94%); and WP_059179795.1 from Lelliottia (81%). As can be seen, the degree of homology is quite high, indicating a high likelihood of having the same functionality.

[0135] Additional proteins that can substitute herein can be identified by homology search, and functionality can be confirmed as described herein. These are available by BLAST search of the above sequences at GenBank. Additionally, UniProt and other such databases have links to a large number of variants in the same and different species.

[0136] NarL: This response regulator (RR) protein activates the expression of the nitrate reductase (narGHJI) and formate dehydrogenase-N(fdnGHI) operons and represses the transcription of the fumarate reductase (frdABCD) operon in response to a nitrate/nitrite induction signal transmitted by either the NarX or NarQ proteins. The DNA binding element is 173-192 (underlined).

TABLE-US-00004 NarLfromE.coli.(SEQIDNO.3): MSNQEPATILLIDDHPMLRTGVKQLISMAPDITVVGEASN GEQGIELAESLDPDLILLDLNMPGMNGLETLDKLREKSLS GRIVVFSVSNHEEDVVTALKRGADGYLLKDMEPEDLLKAL HQAAAGEMVLSEALTPVLAASLRANRATTERDVNQLTPRE RDILKLIAQGLPNKMIARRLDITESTVKVHVKHMLKKMKL KSRVEAAVWVHQERIF NarP:anotherShewenellafrigidmarinaRRbelieved torespondtoNarXand/orNarQ.SEQIDNO.4: MGKPYSVLVVDDHPLLRRGICQLITSDGDFSLFGETGTGL EALTAVAEDEPDIILLDLNMKGMSGLDTLNAMRQEGVTAR IVILTVSDAKQDVVRLLRAGADGYLLKDTEPDLLLEQLKK AMLGHRVISDEVEAYLYELKNTIDDNSWIENLTPRELQIL QELAEGKSNRMIAEDLHISEGTVKVHVKNLLRKANAKSRT EMAVRYLNN

[0137] Additional nitrate RR homologs that can be used herein include WP_000070489.1 from Shigella (99%); WP_045443652.1 from Citrobacter (98%); WP_061496301.1 from Enterobacter (97%); WP_003856701.1 from Proteobacter (96%); WP_032641051.1 from Enterobacter (96%); WP_001064598.1 from Salmonella (96%); WP 020803248.1 from Kleibsella (94%); WP 032611305.1 from Leclercia (96%); and WP_035895589.1 from Kluyvera (95%).

[0138] YdfI: An RR member of the two-component regulatory system YdfH/YdfI. Regulates the transcription of ydfJ by binding to its promoter region. The DNA binding subsequence is aa 166-186 (underlined).

TABLE-US-00005 YdfIfromBacillussubtilis.(SEQIDNO.5): MNKVLIVDDHLVVREGLKLLIETNDQYTIIGEAENGKVAV RLADELEPDIILMDLYMPEMSGLEAIKQIKEKHDTPIIIL TTYNEDHLMIEGIELGAKGYLLKDTSSETLFHTMDAAIRG NVLLQPDILKRLQEIQFERMKKQRNETQLTEKEVIVLKAI AKGLKSKAIAFDLGVSERTVKSRLTSIYNKLGANSRTEAV TIAMQKGILTIDN ExemplaryNarL-YdfIfusionprotein(SEQIDNO.6), theNarLsplitataa170(Ydflunderlined): MSNQEPATILLIDDHPMLRTGVKQLISMAPDITVVGEASN GEQGIELAESLDPDLILLDLNMPGMNGLETLDKLREKSLS GRIVVFSVSNHEEDVVTALKRGADGYLLKDMEPEDLLKAL HQAAAGEMVLSEALTPVLAASLRANRATTERDVNQLTPRE RDILKLIAQGAKGLKSKAIAFDLGVSERTVKSRLTSIYNK LGANSRTEAVTIAMQKGILTIDN

[0139] As of yet, there are no examples of this technique succeeding with a DBD from a non-TCS, but it is possible (albeit unlikely) if the domain structure were such as to be activatable by an active REC domain. However, there are a large number (>10,000 TCS) of proteins available from which to choose, so this limitation is very modest. A homologous DBD from the native RR is predicted to give the best chance of success (>30%, >35%, >40%, or higher), but we have used non-homologous domains too.

[0140] Obviously, the heterologous DBD domain that is rewired to the RR should be functional in the bacterial species in which the nitrate sensor will be hosted. In making the change from disparate species, it may be necessary to select a DBD domain from the host species or a closely related species to ensure operability. In this way, we were able to move a heterologous TCS system from a gram negative (E. coli) to a gram positive (B. subtillus) species.

[0141] The exact fusion point of the two domains can vary somewhat, provided that the DNA binding subsequence (underlined) of NarL (or a homolog) is replaced with that of YdfI or another suitable DBD from a heterologous RR. By switching the DBD domains, we are able to transport the nitrate sensor system of E. coli into the probiotic strain of B. Subtilus.

[0142] Other potential DBDs that can be used herein include LiaR (UniProt 032197) at the linker region in the 20 amino acids surrounding the K120 residue and UhpA (P0AGA6) at the linker region in the 20 amino acids surrounding the T123 residue.

[0143] We have also constructed three other chimera RR proteins herein:

TABLE-US-00006 NarL131-Ydfl(SEQ.IDNO.7)whichissplitat the131.sup.staminoacidofNarL: MSNQEPATILLIDDHPMLRTGVKQLISMAPDITVVGEASN GEQGIELAESLDPDLILLDLNMPGMNGLETLDKLREKSLS GRIVVFSVSNHEEDVVTALKRGADGYLLKDMEPEDLLKAL HQAAAGEMVLSPDILKRLQEIQFERMKKQRNETQLTEKEV IVLKAIAKGLKSKAIAFDLGVSERTVKSRLTSIYNKLGAN SRTEAVTIAMQKGILTIDN

[0144] The other two chimeric proteins (SEQ. ID NO. 8 and 9) are split at the nearby amino acids NarL142 and NarL154 and have a similar but slightly decreased functionality.

TABLE-US-00007 NarL142-Ydfl(SEQ.IDNO.8): MSNQEPATILLIDDHPMLRTGVKQLISMAPDITVVGEASN GEQGIELAESLDPDLILLDLNMPGMNGLETLDKLREKSLS GRIVVFSVSNHEEDVVTALKRGADGYLLKDMEPEDLLKAL HQAAAGEMVLSEALTPVLAASLQFERMKKQRNETQLTEKE VIVLKAIAKGLKSKAIAFDLGVSERTVKSRLTSIYNKLGA NSRTEAVTIAMQKGILTIDN NarL154-Ydfl(SEQ.IDNO.9): MSNQEPATILLIDDHPMLRTGVKQLISMAPDITVVGEASN GEQGIELAESLDPDLILLDLNMPGMNGLETLDKLREKSLS GRIVVFSVSNHEEDVVTALKRGADGYLLKDMEPEDLLKAL HQAAAGEMVLSEALTPVLAASLRANRATTERDVNQLTEKE VIVLKAIAKGLKSKAIAFDLGVSERTVKSRLTSIYNKLGA NSRTEAVTIAMQKGILTIDN

[0145] For proof of concept experiments to characterize the nitrate sensor in B. subtilis, the sensor kinase NarX was expressed under the IPTG inducible Phyper_spank promoter in the AmyE locus and the NarL-YdfI gene was expressed from the xylose inducible PxylA promoter at the LacA locus.

[0146] Growth/assay protocol for in vitro B. subtilis experiments: [0147] Overnight pre-culture (13 hours) in CSE 0.5% glycerol [0148] Dilute overnight culture in CSE 0.5% glycerol with optimal IPTG and Xylose induction levels [0149] Grow 90-150 minutes [0150] Dilute to OD600=0.001 in CSE 0.5% glycerol with optimal IPTG and Xylose induction levels [0151] Grow shaking at 37 C. until cultures reach OD600=0.1 . . . 0.3 [0152] Put on ice, measure fluorescence by flow cytometry

[0153] Growth/assay protocol for measuring nitrate in soil: [0154] Overnight pre-culture (13 hours) in CSE 0.5% glycerol [0155] Dilute overnight culture in 1:100 CSE 0.5% glycerol with optimal IPTG and Xylose induction levels [0156] Grow for until OD600=0.1 [0157] Add appropriate amounts of either sodium nitrate or commercial fertilizer to the soil [0158] Add 250 L of culture to 0.1 g of soil and mix [0159] Grow standing at 37 C. for 2 hours [0160] Resuspend in 10 volume PBS [0161] Filter dirt through a Whatman filter [0162] Put on ice, measure fluorescence by flow cytometry

[0163] For proof of concept experiments to characterize the use of the engineered nitrate sensor in E. coli, the sensor kinase NarX was expressed under the constitutive promoter J23114 and translated with the ribosome binding site (RBS) apFAB655 on a p15a plasmid backbone. The engineered NarL-YdfI response regulator was expressed under the constitutive promoter Bba_J23115 and translated with the RBS BCD24 on a ColE1 plasmid backbone. Transcription of the various genes can terminated by the B0015, T1, or T0 terminators.

[0164] Growth/assay protocol for in vitro E. coli experiments: [0165] Overnight pre-culture (13 hours) in LB+Cm/Spec. [0166] Dilute to OD.sub.600=0.02 in M9+0.4% glycerol. [0167] Grow 3 hours to OD.sub.600=0.3. [0168] Dilute to OD.sub.600=0.0001 in M9+0.4% glycerol. [0169] Add nitrate. [0170] Grow shaking at 37 C. 6 hours to OD.sub.600=0.3. [0171] Put on ice, measure OD, measure fluorescence by flow cytometry (FL1=800, FL3=850).

[0172] Growth Assay protocol for detection of nitrate in inflammation mouse models: [0173] Treat mice DSS for 5 days to simulate inflammatory bowel disease [0174] Administer genetically engineered nitrate sensing bacteria prepared according the in vitro protocol above [0175] At 6 hours collect mouse fecal and organ samples [0176] Process samples via resuspension in PBS and subsequent filtration through a 10 M filter [0177] Put on ice and measure fluorescence by flow cytometry (FL1=800, FL3=850).

[0178] The above described vectors, promoters, terminators and other components of the system are exemplary only, and other components could be used. However, the above assays provided proof of concept and confirmed that the above system is indeed a nitrate two-component nitrate sensor system.

[0179] Although four SK/RR gene pairs were exemplified herein, and at least one pair (SEQ ID NO. 1 and 6) was tested in two host species, there are two features that indicates broad applicability of the invention. The first feature is tunability, which is particularly important for sensing nitrate because the biological ranges for levels of nitrate in humans has not been studied much. Because this system is tunable, once that range is known the sensor can be easily tuned to sense and provide output at the needed levels.

[0180] The second feature piggybacks on the tunability function but also relies on the fact that the inventors have engineered and characterized a suite of DBD, promoters, and reporters for use in this system (described in 62/157,293). When combined, these features allow the inventors to transfer the system to a broad range of microbial species and strains.

[0181] Each of the following is incorporated by reference herein in its entirety for all purposes: [0182] Claesen J. & Fischbach M. A., Synthetic Microbes As Drug Delivery Systems, ACS Synthetic Biology 2015 4 (4), 358-364. [0183] Stewart V., Nitrate- and nitrite-responsive sensors NarX and NarQ of proteobacteria, Biochemical Society Transactions February 2003, 31 (1) 1-10; [0184] DeAngelis, Kristen M., Pingsheng Ji, Mary K. Firestone, and Steven E. Lindow. Two Novel Bacterial Biosensors for Detection of Nitrate Availability in the Rhizosphere. Applied and Environmental Microbiology 71, no. 12 [0185] 62/157,293, IDENTIFYING LIGANDS FROM BACTERIAL SENSORS, May 5, 2015