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GENETICS AND MOLECULAR BIOLOGY

Cloning, Sequencing, and Phenotypic Characterization of the rpoS Gene from Pseudomonas putida KT2440

María Isabel Ramos-González, Søren Molin
María Isabel Ramos-González
Department of Microbiology, The Technical University of Denmark, DK-2800 Lyngby, Denmark
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Søren Molin
Department of Microbiology, The Technical University of Denmark, DK-2800 Lyngby, Denmark
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DOI: 
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  • Fig. 1.
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    Fig. 1.

    Restriction maps of plasmids harboring the P. putida rpoS gene. Plasmids pMIR2-9 and pMIR1-34 are two cosmids from a P. putida library carrying a gene homologous to the rpoS gene of E. coli. Plasmid pMIR61 contains the 2.5-kb BamHI/NheI fragment of pMIR11 (Table 1) inserted in the BamHI and XbaI sites of pUNØ19A (Table 1). Plasmid pMIR13450 carries the 3.4-kbEcoRI fragment of pMIR1-34 inserted in pUNØ19. Cosmid pMIR13415 harbors a mini-Tn5/′Sm element in the 3.4-kbEcoRI fragment of pMIR1-34 (Table 1). Plasmid pMIR492 is the result of inserting a luxAB cassette at position 43 inside the ORF rpoS. (The SalI/BamHI fragment from pUJ20 [Table 1], carrying the genes luxAB fromVibrio harveyi, after filling in the single-strand ends, was inserted in the unique AatII site of pMIR61 after removal of the single-strand protruding ends.) Plasmid pMIR592 was the result of inserting the fusion rpoSp::luxAB as a filled-in KpnI/SphI fragment from pMIR492 in the unique SmaI site of pKNG101 (Table 1). The plasmids listed on the right represent the relevant cloning vectors omitted from the maps. A, AatII; B, BamHI;E, EcoRI; K, KpnI; N,NheI; P, PstI; S,SphI.

  • Fig. 2.
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    Fig. 2.

    Multiple alignments of the P. putidaORF2 sequence with homologous sequences derived from the nonredundant GenBank CDS translations +PDB+SwissPot+PIR. An alignment with RpoS proteins from members of the pseudomonads and enteric bacteria is shown. The sequences of the RpoS proteins of P. fluorescens (P.f.), P. aeruginosa(P.a.), E. coli (E.c.), and S. typhimurium (S.t.) were derived from Samiguet et al. (37), Tanaka and Takahashi (43), Swiss-Prot entry 13445, and Swiss-Prot entry 37400, respectively. TheP. putida (P.p.) RpoS sequence (ORF2) was determined in this study. ∗, amino acid conserved in all sequences; ., residue that belonged to the same group in all sequences (neutral changes). Changes of amino acids in the following groups were considered neutral: (i) A, G, P, S, and T; (ii) D, N, E, and Q; (iii) R, H, and K; (iv) I, L, M, and V; (v) F, Y, and W; and (vi) C. For consensus 1, pseudomonad RpoS was used as reference; for consensus 2, both pseudomonad RpoS and enteric RpoS were used.

  • Fig. 3.
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    Fig. 3.

    Replacements of the rpoS gene with theluxAB insertion mutant rpoS gene. A single homologous recombination event between the functional rpoSpresent on the chromosome of P. putida KT2440 and the inactivated rpoS present on pMIR592 was isolated by selection for resistance to streptomycin (pMIR592 [Fig. 1]). One of the Smr (see the footnote to Table 1 for abbreviations) transconjugants was selected and named R6C1; this merodiploid strain contained the entire plasmid pMIR592 integrated in the genome. A second crossover event at the rpoS locus was selected by cultivating R6C1 overnight in LB without streptomycin (about 10 generations) and subsequent plating on LB medium supplemented with 10% sucrose. Sucr colonies were analyzed by replica plating. One of the Sucr Sms Lux+ colonies was called C1R1. The genomes of three merodiploid isolates (from three independent matings) obtained as the result of the first recombination event and the genomes of six clones (two from each merodiploid) obtained as the result of the resolution of the merodiploids after the second recombination event were examined by Southern blot analysis, which revealed correct single and double recombination events, respectively, at the rpoS locus (not shown).

  • Fig. 4.
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    Fig. 4.

    Growth-dependent expression of the rpoS genes of P. putida R6C1 (○, •), and P. putida C1R1 (□, ▪). Exponentially growing cells in 10 mM citrate-supplemented AB minimal medium were diluted in the same medium, and growth (open symbols) and emission of light (solid symbols) were measured. Measurement of luminescence in liquid culture was carried out as described previously (34). LU values are not normalized per cell. Notice that the same range of log units, four, is plotted in both y axes, and therefore the curve slopes of light emission and optical density are comparable. Experiments were repeated with three cultures; results of a typical experiment from a single culture are presented. Duplicate measurements of LU in a single experiment yielded an average standard deviation of 10%.

  • Fig. 5.
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    Fig. 5.

    Long-term survival of P. putida strains. Carbon starvation of P. putida KT2440 (○), R6C1 (▵), and C1R1 (□) cells was accomplished through exhaustion of 1 mM citrate present in AB minimal medium as described in Materials and Methods. Exponentially growing cells in 10 mM citrate-supplemented AB minimal medium were centrifuged, the supernatant was discarded, and the cells were resuspended in 1 mM citrate-supplemented AB medium up to 3 × 107 to 4 × 107 cells per ml. Time zero was defined in day 0 as cultures reached stationary phase. Survival of the starved cultures was monitored by determination of viable counts on LB plates, supplemented with streptomycin in the case of R6C1. Each starvation condition was repeated at least twice with two cultures each time. Means and standard deviations of duplicate experiments with the same cultures are plotted. Some of the error bars are too small to be distinguished.

  • Fig. 6.
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    Fig. 6.

    Stress challenges of carbon-starved cultures ofP. putida KT2440 (○), R6C1 (▵), and C1R1 (□). Starvation (for 48 h) was accomplished by exhaustion of 1 mM citrate from minimal medium M9. (A) Challenge with 18% (vol/vol) ethanol; (B) challenge with 200 μM H2O2; (C) challenge with 2.4 M NaCl. Viable counts present in each of the prechallenge samples (time zero) were normalized to 1. Survival was determined as relative viable counts. Each challenge experiment was repeated at least twice with two cultures each time. Means and standard deviations of duplicate experiments with the same cultures are plotted. Some of the error bars are too small to be distinguished.

  • Fig. 7.
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    Fig. 7.

    2D-PAGE autoradiograms of carbon starvation-induced proteins of P. putida KT2440 (A) and P. putida C1R1 (B) 60 min after removal of the carbon source. Cells were cultivated in 10 mM citrate-supplemented AB minimal media. Spots positively (not present in panel B) and negatively (not present in panel A) dependent on rpoS are enclosed in boxes and stars, respectively. Molecular mass decreases from top to bottom (100 to 5 kDa), and pH decreases from left to right.

  • Fig. 8.
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    Fig. 8.

    Expression from bolAp 1 andficp in P. putida KT2440 (A), P. putida R6C1 (B), and C1R1 (C) bearing either pGM112 (▵, ▴), pGM115 (□, ▪), or pGM118 (○, •). P. putidacells were electrotransformed with plasmids pGM112 (bolAp 1), pGM115 (ficp), and pGM118 (control vector, no promoter), and expression from the plasmids carrying bolAp 1 and ficp was studied in growing cultures. Exponentially growing LB cultures were used as inoculates, and cells were grown in the same LB medium supplemented with the appropriate antibiotics. OD600 (open symbols) and specific β-galactosidase (B∼gal) activities (solid symbols) were monitored. β-Galactosidase activities are not the result of subtracting the background levels that are synthesized from the vector pGM118 alone. Duplicate measurements of β-galactosidase activities in a single experiment yielded an average standard deviation of 5%.

  • Fig. 9.
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    Fig. 9.

    Survival of P. putida strains in soil. (A) P. putida KT2440 (○), itsrpoS-deficient derivative strain P. putidaC1R1 (▵), P. putida KT2440(pWWO) (□), andP. putida C1R1(pWWO) (◊) were introduced in unamended sterile soil in independent jars; (B) symbols as in panel A, but the microcosms were amended with 0.1% (wt/wt) m-methylbenzoate. For each determination, five different dilutions were plated by the drop-plating technique (20-μl drops were laid on selective plates). Mean values are shown, and maximum and minimum values are presented with error bars. Data are from a single experiment, although microcosms were run in duplicate, and typically the same result was observed. Selective medium for P. putida strains bearing the TOL plasmid was 5 mM m-methylbenzoate-supplemented M9; for strains without the TOL plasmid the same medium was supplemented with 5 mM benzoate.

Tables

  • Figures
  • Table 1.

    Bacteria and plasmids used

    Strain or plasmidRelevant characteristicsaReference and/ or source
    P. putida
     KT2440 hsdMR 10
     KT2442 P. putida KT2440 Rifr V. de Lorenzo
     R6C1Smr SucsLux+; P. putida KT2440 cointegrate containing pMIR592This study
     C1R1Lux+; RpoS− derivative from KT2440This study
    E. coli
     ZK918W3110 ΔlacU169 tna-2 λMAV103rpoS::kan 3; G. W. Huisman
     HB101F−Δ(gpt-proA)62 leuB6 supE44 ara-14 galK2 lacY1Δ(mcrC-mrr) rpsL20 (Smr)xyl-5 mtl-1 recA13 4
     DH5αF′F′/endA1 hsdR17(rK − mK +) supE44 thi-1 recA1 gyrA (Nalr) relA1Δ(lacIZYA-argF)U169 deoR[φ80dlacΔ(lacZ)M15] 48
     CC118(λpir)λpirRifr 16
    Plasmids
     pRK600Cmr; mob tra V. de Lorenzo
     pLAFR3Tcr derivative from the cosmid pLAFR1 (11) modified to include multiple cloning sites and the Plac promoter fused to the α peptide of ′LacZJ. L. Ramos
     pUTSmSmrApr; mini-Tn5/′Sm element inserted in pUT 16
     pUC18 and pUC19Apr, multiple cloning site, Plac promoter fused to the α peptide of ′LacZ 46
     pUNØ18 and pUNØ19Apr; identical to pUC18 and pUC19 except that the NheI site was changed to a NotI site andoriT was inserted as a 0.7-kb fragment in the newNotI siteSilvia Marqués
     pUNØ19AApr; pUNØ19 after removing its uniqueAatII siteThis study
     pUJ20TcrApr; mini-Tn5/′luxABTc element inserted in pUTV. de Lorenzo
     pMIR0-1Tcr; chimeric cosmid of P. putida library bearing a gene homologous to therpoS gene of E. coli This study
     pMIR1-2Tcr; chimeric cosmid of P. putida library bearing a gene homologous to the rpoSgene of E. coli This study
     pMIR1-34Tcr; chimeric cosmid of P. putida library bearing a gene homologous to the rpoSgene of E. coli This study
     pMIR2-9Tcr; chimeric cosmid of P. putida library bearing a gene homologous to the rpoSgene of E. coli This study
     pMIR13415Tcr Smr; mini-Tn5/′Sm element inserted in the P. putida rpoS gene on the 3.4-kb EcoRI fragment of pMIR1-34This study
     pMIR13450Apr; 3.4-kbEcoRI fragment of pMIR1-34 was inserted in pUN19ØThis study
     pMIR11Apr; 7-kb BamHI fragment of pMIR2-9 was inserted in pUN19ØThis study
     pMIR61Apr; rpoS gene as a 2.5-kbBamHI/NheI P. putida DNA fragment inserted at the BamHI and XbaI sites of pUN19ØThis study
     pKNG101 strAB mobRK2 oriR6K sacBR 19
     pMIR492Apr;luxAB cassette as a SalI/BamHI fragment from pUJ20 inserted at AatII unique site of pMIR61This study
     pMIR592Smr; PrpoS::luxAB fusion asKpnI/SphI fragment from pMIR492 inserted at theSmaI site of pKNG101This study
     pGM112Kmr Cmr bolAp 1::lacZ 26
     pGM115Kmr Cmr ficp::lacZ 26
     pGM118Kmr Cmr lacZpromoterless 26
    • ↵a Ap, ampicillin; Cm, chloramphenicol; Km, kanamycin; Lux, light emission; Nal, nalidixic acid; Rif, rifampin; Sm, streptomycin; Suc, sucrose; Tc, tetracycline.

  • Table 2.

    Identities and similarities of P. putidaKT2440 rpoS gene and RpoS sigma factor sequences with otherrpoS gene and RpoS amino acid sequences from the database

    Organism% DNA identityProtein
    % Identity% Similarity
    P. fluorescens 81 (1,009a)90 (335)96 (335)
    P. aeruginosa 84 (1,007)85 (335)95 (335)
    E. coli 70 (834)65 (327)84 (327)
    S. typhimurium 70 (834)65 (328)85 (328)
    S. entomophila 71 (824)65 (328)83 (328)
    S. flexneri 69 (828)64 (326)82 (326)
    Y. enterocolitica 66 (820)66 (325)81 (325)
    • ↵a Extent of overlap (in base pairs).

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Cloning, Sequencing, and Phenotypic Characterization of the rpoS Gene from Pseudomonas putida KT2440
María Isabel Ramos-González, Søren Molin
Journal of Bacteriology Jul 1998, 180 (13) 3421-3431; DOI:

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Cloning, Sequencing, and Phenotypic Characterization of the rpoS Gene from Pseudomonas putida KT2440
María Isabel Ramos-González, Søren Molin
Journal of Bacteriology Jul 1998, 180 (13) 3421-3431; DOI:
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