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Peer-reviewed

Development of a Gene Replacement Method for the Filamentous Bacterium Leptothrix cholodnii SP-6

丝状菌Leptothrix cholodnii SP-6的基因替换方法的开发

Tatsuki Kunoh Tatsuki Kunoh
EO Erika Ono
TY Tatsuya Yamamoto
IS Ichiro Suzuki
MT Minoru Takeda
NN Nobuhiko Nomura

发布: 2023年04月20日第13卷第8期 DOI: 10.21769/BioProtoc.4652 浏览次数: 1624

评审: Alberto J. Martin-RodriguezGonzalo Durante-RodríguezAnonymous reviewer(s)

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Cover of Applied and Environmental Microbiology, featuring study using the protocol.

Dec 2022

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Abstract

Genetic strategies such as gene disruption and fluorescent protein tagging largely contribute to understanding the molecular mechanisms of biological functions in bacteria. However, the methods for gene replacement remain underdeveloped for the filamentous bacteria Leptothrix cholodnii SP-6. Their cell chains are encased in sheath composed of entangled nanofibrils, which may prevent the conjugation for gene transfer. Here, we describe a protocol optimized for gene disruption through gene transfer mediated by conjugation with Escherichia coli S17-1 with details on cell ratio, sheath removal, and loci validation. The obtained deletion mutants for specific genes can be used to clarify the biological functions of the proteins encoded by the target genes.


Graphical overview


Keywords: Leptothrix cholodnii SP-6 (Leptothrix cholodnii SP-6) search Gene transfer (基因转移) search Conjugation (共轭) search Escherichia coli S17-1 (大肠杆菌S17-1) search Calcium depletion (钙消耗) search

Background

Leptothrix species are microorganisms with a long history as research targets. The type strain, L. ochracea, was reported in the late 18th century (Spring, 2006) and, since then, has attracted much attention from researchers in the fields of microbiology and environmental science. Leptothrix ubiquitously inhabits seeps, streams of iron-rich groundwater, and activated sludge. Leptothrix divides to produce cell chains encased with entangled nanofibrils termed sheaths and eventually generates mats composed of porous networks of cell chains (Spring, 2006). Over the time of mat aging, sheaths are encrusted with inorganic particles, especially iron oxide, resulting in the production of rigid microtubes (Furutani et al., 2011). Focusing on this innate characteristic, Leptothrix has been utilized for the removal of metal contaminations such as iron and manganese from groundwater in water purification plants (Kunoh et al., 2015a). However, overgrowth of filamentous bacteria, including Leptothrix, often results in clogging of water distribution pipes and bulking of activated sludge in wastewater treatment plants (van der Waarde et al., 2002). Therefore, understanding the functions of key proteins responsible for nanofibril formation and entanglement is significant for the artificial control of their filamentous growth.

The next-generation sequencing technologies that have been developed recently enable us to freely access significant data of the whole genome sequences existing in a wide range of organisms, from prokaryotes to eukaryotes. For Leptothrix species, the complete genome sequence of L. cholodnii SP-6 (hereafter SP-6) was published by the US DOE Joint Genome Institute (Copeland et al., 2008) [GenBank (accession: GCA_000019785.1)], which opened new doors for understanding the gene functions and interactions of this strain.

To date, a gene replacement system for Leptothrix species remains unpublished, except for the sheath-less strain L. discophora SS1 (Bocioaga et al., 2014). Although we attempted to perform gene transfer of SP-6 mediated by conjugation with Escherichia coli S17-1 by following their report, the efficiency of gene replacement was quite low relative to that of SS1. We therefore considered the possibility that the sheath structure of SP-6 might interfere with the conjugation with E. coli cells, and eventually the incorporation of extracellular DNA into SP-6 cells.

We previously reported that sheath structure plays a critical role in holding aligned cell chains of SP-6 (Kunoh et al., 2020), and that calcium depletion frequently results in breakage of cell filaments of SP-6 due to failure in the production of sheath structures encasing the cell chains (Kunoh et al., 2021). From these findings, we hypothesized that preculturing SP-6 cells in a calcium-lacking medium might enhance the accessibility of E. coli cells to SP-6 cells for conjugation, which results in efficient gene transfer and subsequent gene replacement of SP-6 cells.

Here, we describe a protocol for gene replacement in SP-6 covered with sheaths (Graphic abstract), which was modified from that reported previously (Yoshihara, 2002). This protocol is expected to contribute to efficient systematic genetic analyses of SP-6, including genes responsible for sheath formation (Kunoh et al., 2022).

Materials and Reagents

Chemical compounds

  1. Kanamycin sulfate (FUJIFILM Wako Pure Chemical Co., catalog number: 117-00341)

  2. Ampicillin sodium salt (FUJIFILM Wako Pure Chemical Co., catalog number: 012-23303)

  3. Rifampicin (Nacalai Tesque Inc., catalog number: 30259-94)

  4. Ammonium sulfate [(NH4)2SO4] (Nacalai Tesque Inc., catalog number: 02633-15)

  5. Magnesium sulfate heptahydrate (MgSO4·7H2O) (FUJIFILM Wako Pure Chemical Co., catalog number: 131-00405)

  6. Calcium chloride dihydrate (CaCl2·2H2O) (Nacalai Tesque Inc., catalog number: 031-00435)

  7. Potassium dihydrogen phosphate (KH2PO4) (Nacalai Tesque Inc., catalog number: 28721-55)

  8. Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) (Nacalai Tesque Inc., catalog number: 31725-15)

  9. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Nacalai Tesque Inc., catalog number: 17514-86)

  10. Iron(II) sulfate (FeSO4) (FUJIFILM Wako Pure Chemical Co., catalog number: 094-02942)

  11. Sodium pyruvate (FUJIFILM Wako Pure Chemical Co., catalog number: 191-03061)

  12. Sodium hydroxide (NaOH) (Nacalai Tesque Inc., catalog number: 31511-05)

  13. Sodium chloride (NaCl) (Nacalai Tesque Inc., catalog number: 31319-45)

  14. Hexadecyltrimethylammonium bromide (CTAB) (Nacalai Tesque Inc., catalog number: 07906-82)

  15. Sodium dodecyl sulfate (SDS) (Nacalai Tesque Inc., catalog number: 30400-85)

  16. Phenol/chloroform/isoamyl alcohol (25:24:1) (FUJIFILM Wako Pure Chemical Co., catalog number: 311-90151)

  17. Chloroform (Nacalai Tesque Inc., catalog number: 08401-65)

  18. Isopropanol (Nacalai Tesque Inc., catalog number: 29113-95)

  19. Ethanol (Nacalai Tesque Inc., catalog number: 14713-95)

  20. Sodium acetate trihydrate (CH3COONa·3H2O) (Nacalai Tesque Inc., catalog number: 31115-05)

  21. Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) (Nacalai Tesque Inc., catalog number: 35434-21)

  22. Ethylenediaminetetraacetic acid tetrasodium salt (EDTA-4Na) (FUJIFILM Wako Pure Chemical Co., catalog number: 349-01885)

  23. 35% hydrochloric acid (HCl) (Nacalai Tesque Inc., catalog number: 18321-05)

  24. Acetic acid (Nacalai Tesque Inc., catalog number: 00212-85)

  25. Polyethylene glycol 8000 (PEG8000) (MP Biomedicals LLC., catalog number: 195445)

  26. Glycerol (Nacalai Tesque Inc., catalog number: 17017-35)

  27. Dimethyl sulfoxide (DMSO) (Nacalai Tesque Inc., catalog number: 13407-45)

  28. Potassium chloride (KCl) (Nacalai Tesque Inc., catalog number: 28514-75)

  29. Magnesium chloride (MgCl2) (Nacalai Tesque Inc., catalog number: 20935-05)

  30. d-glucose (Nacalai Tesque Inc., catalog number: 16805-35)

  31. Agarose S (FUJIFILM Wako Pure Chemical Co., catalog number: 312-01193)

  32. 1 kb Plus DNA ladder (Thermo Fisher Scientific, catalog number: 10787018)

  33. Ethidium bromide solution (10 mg/mL) (Nacalai Tesque Inc., catalog number: 14631-94)

  34. Green nucleic acid stain (Midori Green Advance, Nippon Genetics Co. Ltd., catalog number: NE-MG04)

  35. Liquid nitrogen (Air Water Inc., YJ code number: 799070CX1020)


Culture media

  1. BD Difco LB broth, Lennox (Thermo Fisher Scientific, catalog number: 240230)

  2. BD Bacto agar (Thermo Fisher Scientific, catalog number: 214010)

  3. BD Bacto yeast extract (Thermo Fisher Scientific, catalog number: 212750)

  4. BD Bacto tryptone (Thermo Fisher Scientific, catalog number: 211705)

  5. BD Difco nutrient broth (Thermo Fisher Scientific, catalog number: 234000)


Vitamins

  1. d-biotin (Nacalai Tesque Inc., catalog number: 04822-04)

  2. Folic acid (Nacalai Tesque Inc., catalog number: 16221-91)

  3. Thiamine hydrochloride (HCl) (Nacalai Tesque Inc., catalog number: 36319-24)

  4. d-calcium pantothenate (Nacalai Tesque Inc., catalog number: 26004-44)

  5. Vitamin B12 (Nacalai Tesque Inc., catalog number: 36323-96)

  6. Riboflavin (Nacalai Tesque Inc., catalog number: 36322-51)

  7. Nicotinic acid (Nacalai Tesque Inc., catalog number: 24326-52)

  8. Pyridoxine hydrochloride (Nacalai Tesque Inc., catalog number: 29611-81)

  9. p-aminobenzoic acid (Nacalai Tesque Inc., catalog number: 01930-32)


Enzymes

  1. Proteinase K (FUJIFILM Wako Pure Chemical Co., catalog number: 166-28913)

  2. Ribonuclease A (FUJIFILM Wako Pure Chemical Co., catalog number: 182-01493)

  3. Lysozyme from egg white (FUJIFILM Wako Pure Chemical Co., catalog number: 122-02673)

  4. Tks Gflex DNA polymerase (Takara Bio Inc., catalog number: R060A)

  5. PrimeStar Max DNA polymerase (Takara Bio Inc., catalog number: R045A)

  6. FastDigest restriction enzymes BamHI, HindIII, and SmaI (Thermo Fisher Scientific, catalog numbers: FD0054, FD0504, FD0663)


Kits

  1. Gel Loading Buffer (10×) (Thermo Fisher Scientific, catalog number: 10816015)

  2. Gel Extraction kit (Promega Co., Wizard® SV Gel and PCR Clean-Up System, catalog number: A9282)

  3. Plasmid Extraction kit (Merck KGaA, GenEluteTM Plasmid Miniprep Kit, catalog number: PLN350-1KT)

  4. DNA Ligation kit <Mighty Mix> (Takara Bio Inc., catalog number: 6023)


Materials

  1. 50 mL polypropylene tube (IWAKI AGC Techno Glass Co., Ltd., catalog number: 2345-050)

  2. 15 mL polypropylene tube (IWAKI AGC Techno Glass Co., Ltd., catalog number: 2425-015)

  3. 1.5 mL centrifuge tube (Midland Scientific, catalog number: 311-08-052)

  4. PCR microtube (Bio Regenerations Co., Ltd., catalog number: BRG-DC-01)

  5. ϕ 90 × 15 mm sterilized dish (RIKAKEN Holdings Co., Ltd, catalog number: RSU-SP915-2)

  6. 10 and 30 mL syringes (TERUMO Co., Ltd., catalog numbers: SS-10ELZ, SS-30ESZ)

  7. 25 G × 1 inch injection needle (TERUMO Co., Ltd., catalog number: NN-2525R)

  8. Stericup quick release-HV sterile vacuum filtration system (Merck Millipore, catalog number: S2HVU05RE)

  9. MF-Millipore membrane filter, 0.22 µm pore size (Merck Millipore, catalog number: GSWP04700)

  10. Toothpicks (e.g., Yanagi Products Co., Ltd., DN Tsumayouji L)

  11. 0.22 μm syringe filter (Sartorius AG, catalog number: S7597-FXOSK)

  12. Vacuum filtration device (Merck Millipore, catalog number: SCGPS02RE)

  13. 2.0 mL stock tube (Sarstedt AG, catalog number: 72.694.107.03)

  14. IWAKI 20 mL glass test tube (ϕ 16.5 × 165 mm) (AGC Techno Glass Co., Ltd., catalog number: TEST16.5NP)

  15. Test-tube closures (ϕ 17–18 mm) (LÜDI SWISS AG, catalog number: 08418)

  16. Erlenmeyer flask (200 mL) (AGC Techno Glass Co., Ltd., catalog number: 4980FK200)

  17. Silicon stopper (Shin-Etsu Polymer Co., Ltd., catalog number: T-38)

  18. 100–1,000 mL medium bottles (AGC Techno Glass Co., Ltd., catalog numbers: 1516/04D, 1516/08D, 1516/10D)

  19. Cuvette for electroporation (2 mm gap) (NEPA GENE Co. Ltd., catalog number: EC-002S)


Bacterial strains

  1. Leptothrix cholodnii SP-6 (ATCC strain 51168)

  2. Bacillus subtilis 168 (Lab stock)

  3. Escherichia coli S17-1 (Lab stock)

  4. Escherichia coli DH5α (Lab stock)

  5. Escherichia coli SN1187 [National BioResource Project – E. coli Strain (National Institute of Genetics, Mishima, Japan)]


Plasmids

  1. pSUP5011::Tn5-mob (Lab stock, DSMZ collection 5167)

  2. pUC18 (Takara Bio Inc., catalog number: 3218)


Prepared recipes (see Recipes below)

  1. MSVP (ATCC 1917) medium

  2. LB medium

  3. SOC medium

  4. NB medium

  5. 1 M MgSO4 and 1 M MgCl2 solutions

  6. 1 M glucose solution

  7. NaCl solutions (0.9%, 5 M)

  8. TE buffer

  9. TEN buffer

  10. 10% SDS solution

  11. CTAB/NaCl solution

  12. 3 M sodium acetate (CH3COONa) solution

  13. 50× TAE buffer

  14. 2× TSS solution

  15. TSS solution

  16. 50% glycerol solution

  17. Rifampicin solution

  18. Kanamycin solution

  19. Ampicillin solution

Equipment

  1. Reciprocating shakers (TAITEC Co., Ltd., models: personal-11 SM, BR-41FM)

  2. Centrifuges (Eppendorf AG., models: 5430R, 5425R)

  3. Thermostat (Yamato Scientific Co., Ltd., model: DG400)

  4. Spectrophotometer (ERMA INC., model: MP-1200)

  5. Water purification systems (ELGA LabWater, model: PURELAB Chorus 1 Complete, PURELAB Chorus 2+)

  6. pH meter (HORIBA, Ltd., model: F-74)

  7. Electronic scale (A&D Co., Ltd., model: HR-202i)

  8. Magnetic stirrer with hot plate (Kenis, model: MI10 33220962)

  9. Autoclave (TOMY SEIKO Co., Ltd., model: LBS-245)

  10. Safety cabinet (DALTON Co., Ltd., model: Class 2 A1)

  11. Thermal cycler (BIO-RAD, model: T-100)

  12. Microwave (for example, Sharp Co., Ltd., model: RE-21G)

  13. Submarine electrophoresis system (Mupid.com, model: Mupid-2plus)

  14. Gel documentation system (ATTO Co., model: WSE-5400 Printgraph Classic)

  15. Heat block incubator (Anatech Co., Ltd., model: 5520a)

  16. NanoDrop UVVis spectrophotometer (Thermo Fisher Scientific, model: 2000c)

  17. Mixer (Vortex Co., Ltd., model: VORTEX-GENIE 2)

  18. Adjustable air displacement pipette (Gilson Inc. model: PIPETMAN P series)

  19. Electroporation system (Bio-Rad Laboratories Inc., model: Genepluser Xcell)

  20. -30 freezer (for example Nihon Freezer Co., Ltd., model: GS-5210HC)

  21. -80 freezer (for example Thermo Fisher Scientific, model: TSX600G)

  22. LED illuminator (Optocode Co., Ltd., model: LED-EXTA/GFP＋RFP)

  23. Ceramic tweezer (ASONE, catalog number: 7-166-03)

  24. Microscope (for example Carl Zeiss, model: Axio observer Z1)

Software

  1. ApE (Davis and Jorgensen, 2022) used as a DNA plasmid sequence editor

  2. R software version 4.2.2 [R Core Team (2022). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. (https://www.R-project.org/)] used for statistics

Procedure

English
中文翻译

文章信息

版权信息

© 2023 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).

如何引用

Kunoh, T., Ono, E., Yamamoto, T., Suzuki, I., Takeda, M. and Nomura, N. (2023). Development of a Gene Replacement Method for the Filamentous Bacterium Leptothrix cholodnii SP-6. Bio-protocol 13(8): e4652. DOI: 10.21769/BioProtoc.4652.

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分类

微生物学 > 微生物遗传学 > 基因组编辑

细胞生物学 > 细胞工程

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