Embed Size (px)
Citation preview
Mars-Like Soils in the Atacama Desert, Chile, and La Joya Arequipa
Mars-Like Soils in the Atacama Desert, Chile, and La Joya Arequipa
The Viking missions showed the martian soil to be lifeless and depleted in organic material and indicated the presence of one or more reactive oxidants.
First is the absence of organic materials at the level of parts-per-billion ( ppb), as measured by pyrolysis–gas chromatography–mass-spectrometry (pyr-GC-MS).The second unexpected result is the rapid release of molecular oxygen, at levels of 70 to 770 nmol g–1, when soil samples were exposed to water vapor in the gas exchange experimentThe third puzzling result is that organic material in the labeled-release (LR) experiment was consumed as expected if life were present
Here we report the presence of Mars-like soils in the extreme arid region of the Atacama Desert. Samples from this region had organic species only at trace levels and extremely low levels of culturable bacteria. Two samples from the extreme arid region were tested for DNA and none was recovered. Incubation experiments, patterned after the Viking labeled-release experiment but with separate biological and nonbiological isomers, show active decomposition of organic species in these soils by nonbiological processes.
The Atacama is an extreme, arid, temperate desert that extends from 20°S to 30°S along the Pacific coast of South America
ATACAMA
LA JOYA
MARTE
the stone cover apparently plays an important role in the maintenance of biological activity through its contribution to slope biotope stability
pyrolysis–gas chromatography–mass-spectrometry (pyr-GC-MS).
Peak identification: 1. Formic acid; 2. Sulfur dioxide; 3. 2-Butene; 4. 1,2-Butadiene; 5. Propenenitrile; 6. 1,3- Pentadiene; 7. 2-Methylfuran; 8. Benzene; 9. Methylbenzene; 10. Ethylbenzene; 11. 1, 2-dimethylbenzene; 12. Styrene; 13. Benzenenitrile.
Dr. Christopher P. McKay, Planetary Scientist with the Space Science
Division of NASA Ames.
Rafael Navarro-Gonzalez, Fred A. Rainey,Paola Molina,Danielle R. Bagaley,
Becky J. Hollen, Jose de la Rosa,Alanna M. Small, Richard C. Quinn,
FrankJ. Grunthaner,Luis Caceres, Benito Gomez-Silva, Julio C. Bernabe O.
DISEÑO DE PRIMERS• ClustalW (Alineamiento múltiple de
secuencias).
• Evaluación y selección de secuencias:– Permitir amplificar secuencia parcial del gen
que codifica el ARNr 16S.– No estar muy involucradas en la estructura
secundaria del ARNr 16S. – Altamente conservadas en dominio Bacteria
pero no en Eucariota o Archaea.
• A partir de las secuencias obtenidas se diseñó un primer forward y un primer reverse.
• Tm Calculations for Oligos (Promega)(www.promega.com/biomath/).
Tm=2(A+T)+4(G+C)Wallace, 1979
DISEÑO DE PRIMERS
DIAGRAMA DE LOCALIZACIÓN DE LOS PRIMERS EN ADNr 16S DE E. COLI. Tamaño del gen es de 1542 pb. Primer JO-F1 (forward) reacciona hacia la derecha; JO-R1 reacciona hacia la izquierda (reverse). La secuencia corresponde a E. Coli publicada por Noller y col. en 1978.
• 1 aaattgaaga gtttgatcat ggctcagatt gaacgctggc ggcaggccta acacatgcaa 61 gtcgaacggt aacaggaaga agcttgcttc tttgctgacg agtggcggac gggtgagtaa 121 tgtctgggaa actgcctgat ggagggggat aactactgga aacggtagct aatacctcat 181 aacgtcgcaa gaccaaagag ggggaccttc gggcctcttg ccatcggatg tgcccagatg 241 ggattagcta gtaggtgggg taacggctca cctaggcgac gatccctagc tggtctgaga 301 ggatgaccag ccacactgga actgagacac ggtttagact cctacgggag gcagcagtgg 361 ggaatattgc acaatgggcg caagcctgat gcagccatgc cgcgtgtatg aagaaggcct 421 tcgggttgta aagtactttc agcggggagg aagggagtaa agttaatacc tttgctcatt 481 gacgttaccc gcagaagaag caccggctaa ctccgtgcca gcagccgcgg taatacggag 541 ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcac gcaggcggtt tgttaagtca 601 gatgtgaaat ccccgggctc aacctgggaa ctgcatctga tactggcaag cttgagtctc 661 gtagaggggg gtagaattcc aggtgtagcg gtgaaatgcg tagagatctg gaggaatacc 721 ggtggcgaag gcggccccct ggacgaagac tgacgctcag gtgcgaaagc gtggggagca 781 aacaggatta gataccctgg tagtccacgc cgtaaacgat gtcgacttgg aggttgtgcc 841 cttgaggcgt ggcttccgga gctaacgcgt taagtcgacc gtctggggag tacggccgca 901 aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 961 tcgatgcaac gcgaagaacc ttacctggtc ttgacatcca cggaagtttt cagagatgag1021 aatgtgcctt cgggaaccgt gagacaggtg ctgcatggct gtcgtcagct cgtgttgtga1081 aatgttgggt taagtcccgc aacgagcgca acccttatcc tttgttgcca gcggtccggc1141 cgggaactca aaggagactg ccagtgataa actggaggaa ggtggggatg acgtcaagtc1201 atcatggccc ttacgaccag ggctacacac gtgctacaat ggcgcataca aagagaagcg1261 acctcgcgag agcaagcgga cctcataaag tgcgtcgtag tccggattgg agtctgcaac1321 tcgactccat gaagtcggaa tcgctagtaa tcgtggatca gaatgccacg gtgaatacgt1381 tcccgggcct tgaccacacc gcccgtcaca ccatgggagt gggttgcaaa agaagtaggt1441 agcttaacct tcgggagggc gcttaccact ttgtgattca tgactggggt gaagtcgtaa1501 caaggtaacc gtaggggaac ctgcggttgg atcacctcct ta
SECUENCIAS DEL ADNr de E. COLI, ALTAMENTE CONSERVADAS EN DOMINIO BACTERIA. La secuencia corresponde a E. Coli (Noller y col., 1978).
• 1 aaattgaaga gtttgatcat ggctcagatt gaacgctggc ggcaggccta acacatgcaa 61 gtcgaacggt aacaggaaga agcttgcttc tttgctgacg agtggcggac gggtgagtaa 121 tgtctgggaa actgcctgat ggagggggat aactactgga aacggtagct aatacctcat 181 aacgtcgcaa gaccaaagag ggggaccttc gggcctcttg ccatcggatg tgcccagatg 241 ggattagcta gtaggtgggg taacggctca cctaggcgac gatccctagc tggtctgaga 301 ggatgaccag ccacactgga actgagacac ggtttagact cctacgggag gcagcagtgg 361 ggaatattgc acaatgggcg caagcctgat gcagccatgc cgcgtgtatg aagaaggcct 421 tcgggttgta aagtactttc agcggggagg aagggagtaa agttaatacc tttgctcatt 481 gacgttaccc gcagaagaag caccggctaa ctccgtgcca gcagccgcgg taatacggag 541 ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcac gcaggcggtt tgttaagtca 601 gatgtgaaat ccccgggctc aacctgggaa ctgcatctga tactggcaag cttgagtctc 661 gtagaggggg gtagaattcc aggtgtagcg gtgaaatgcg tagagatctg gaggaatacc 721 ggtggcgaag gcggccccct ggacgaagac tgacgctcag gtgcgaaagc gtggggagca 781 aacaggatta gataccctgg tagtccacgc cgtaaacgat gtcgacttgg aggttgtgcc 841 cttgaggcgt ggcttccgga gctaacgcgt taagtcgacc gtctggggag tacggccgca 901 aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 961 tcgatgcaac gcgaagaacc ttacctggtc ttgacatcca cggaagtttt cagagatgag1021 aatgtgcctt cgggaaccgt gagacaggtg ctgcatggct gtcgtcagct cgtgttgtga1081 aatgttgggt taagtcccgc aacgagcgca acccttatcc tttgttgcca gcggtccggc1141 cgggaactca aaggagactg ccagtgataa actggaggaa ggtggggatg acgtcaagtc1201 atcatggccc ttacgaccag ggctacacac gtgctacaat ggcgcataca aagagaagcg1261 acctcgcgag agcaagcgga cctcataaag tgcgtcgtag tccggattgg agtctgcaac1321 tcgactccat gaagtcggaa tcgctagtaa tcgtggatca gaatgccacg gtgaatacgt1381 tcccgggcct tgaccacacc gcccgtcaca ccatgggagt gggttgcaaa agaagtaggt1441 agcttaacct tcgggagggc gcttaccact ttgtgattca tgactggggt gaagtcgtaa1501 caaggtaacc gtaggggaac ctgcggttgg atcacctcct ta
SECUENCIAS DEL ADNr de E. COLI, ALTAMENTE CONSERVADAS EN DOMINIO BACTERIA. La secuencia corresponde a E. Coli (Noller y col., 1978).
REACCIÓN DE AMPLIFICACIÓN (PCR)
4 μl Primers
40 μlPCR Supermix
3 μlADN
1 ciclo: 95 ºC x 5’ 25 ciclos: 90 ºC x 70’’
35 ºC x 70’’ 70 ºC x 70’’
1 ciclo: 72 ºC x 7 minutos
PCR Supermix (Invitrogen)Taq polimerasa
Anticuerpo Platinum Taq dNTPs 220 µM
Tris-SO4 66 mM, pH 8,9 (NH4)2SO4 19,8 mM
MgSO4 2,4 mM
• Gel de agarosa al 1,3%, teñido con BrEt.
• Marcador 100 bp DNA Ladder(NEB)
• 80V x 60’
• Transiluminador UV, 302 nm
ELECTROFORESIS DE PRODUCTOS DE PCR
AMPLIFICACIÓN DE ADNr 16S BACTERIANO. Bandas corresponden a productos de PCR generados utilizando los primers JO-F1 y JO-R1. Gel de agarosa al 1,3%, teñido con bromuro de etidio. (1) JO06-01, (2) JO06-02, (3) JO06-03, (4) JO06-04, (5) JO06-05, (M) Marcador de peso
molecular 100 bp DNA Ladder.
1 2 3 4 5 M
500
1000
1517
100
700
pb
pb
Fuente: www.neb.com
PURIFICACIÓN DE PRODUCTO DE PCR
• QIAquick PCR Purification Kit (Qiagen) - Columnas de adsorción.
UNIÓN
Buffer PB ó QG
LAVADO
Buffer PE
ELUSIÓN
Buffer EB
Productopurificado
SECUENCIACIÓN DE PRODUCTO DE PCR
Producto de PCR purificado10 μl Primer (Forward),10 μMSequencing Mix• AmpliTaq DNA Polimerasa• dNTPs• ddNTP fluorescentes• Buffer de secuenciación 1X
(Tris-HCl, pH 9; MgCl2)
dH2O
PURIFICACIÓN
ABI 3730XL DNA Analyzer
PCR DE SECUENCIACIÓN
Termociclador (GeneAmp PCR System 9700) 1 ciclo: 95 ºC x 60’’ 25 ciclos: 95 ºC x 30’’
40 ºC x 30’’70 ºC x 4’
Mantenidas a 4 ºC
Formamida
Electroferograma
ANÁLISIS BIOINFORMÁTICO• ALINEAMIENTO Y COMPARACIÓN DE
SECUENCIAS– Secuencias con mayor similitud
(alineamientos significativos con scores más altos)
• ALINEAMIENTO MÚLTIPLE DE SECUENCIAS– Mega 3.1– ClustalW (alineamiento múltiple de
secuencias)– Modelo Kimura-2 parámetros (distancias
genéticas)– Árboles de distancias (método Neighbor-
Joining)
NNNNNNNNANNGCNNNTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGCCGACCGCCTAGGAGACTAGGCCTTCCCTTCGGGGACGGCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCGAACCCGCGAGGGGGAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTATGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGANNNNNNN
1Planococcus2 Dietzia3 Planococcus6c Micrococcus7 PseudomonasLa cepa 4, 5, 6a, 6b, 8, 9 y 10 no estan identificadas
Sequence analysesWe identified the closest available matches to sequences of our samples using
the NCBI BLASTN (i.e. MegaBlast) search tool. For analyses we used the first 100 most similar sequences from the databases for comparison with our sequences. Sequences were edited using the BioEdit Sequence Alignment Editor (Hall, 1999). Initial alignments of sequences were performed using the CLUSTAL X program (Thompson et al., 1997),
and final alignments were adjusted by eye.For phylogenetic analyses we used sequences of Aquifex aeolicus (GenBank:
AJ309733.1) as the outgroup. To estimate the posterior probability of phylogenetic trees we used the Markov Chain Monte Carlo (MCMC) method within a Bayesian framework
(hereafter BMCMC). The MCMC procedure ensures that trees are sampled in proportion to their probabilities of occurrence under a model of gene-sequence evolution. The
analysis was run using the Metropolis-coupled Markov chain, Monte Carlo algorithm, with flat priors. Four separate chains were run for 10,000,000 generations, swapping
chains every 5th generation and sampling trees every 1000th generation to assure that successive samples were independent. Inspection of plots of the natural logarithm of likelihood values (lnL) for the “cold” chain suggests that convergence was reached by approximately the 10,000th generation, but to ensure that trees were sampled after
convergence we discarded the first 2,500,000 generations as burn-in. Posterior probabilities for topologies were then assessed as the proportion of the trees sampled after burn-in, in which that particular topology was observed. BMCMC was performed using MrBayes 3.1 (Huelsenbeck and Ronquist, 2001) and a general time-reversible (GTR) model (see Rodriguez et al. 1990) of gene-sequence evolution was used to
estimate the likelihood of each tree.
ResultThe 60331_G11_9_083 sequence was highly similar to sequences from the division Firmicutes (76%), mainly including Bacillales (i.e. 56 Planococcaceae and 19 Bacillaceae). The phylogenetic tree (Fig. xx9) shows that the 60331_G11_9_083 sequence is included in a basal clade (0.98 posterior probability) very near to three uncultured clones of subsurface water from the Kalahari Shield (South Africa) (Gihring et al., 2006). The most similar classified sequence is Planococcus sp. KRPC10y (GenBank access number DQ375559.1); this information in addition to the high similarity with Firmicutes sequences indicates that 60331_G11_9_083 sequence is a Planococcaceae bacterium and likely a species of Planococcus.
Bayesian inference, 50% consensus trees from the data base. The numbers at the nodes are posterior
probabilities (first 3 numbers), expressed as proportion. Circle indicate taxa whose genes were
sequenced in the present study.
OBJETIVOS
• Caracterizar bioquímica, microbiológica y molecularmente las cepas perilíticas.
• Seleccionar las cepas más aptas de acuerdo a su comportamiento extremófilo.
• Determinar la actividad biotecnológica de dichas cepas.
INTRODUCCIONBacterias periliticasBacterias periliticas
Desierto de La JoyaDesierto de La Joya
Clima desérticoT máx: 26 ºC T mín: 12,1 ºC Alta radiacion UV
RESULTADOSRESULTADOS PRUEBAS BIOQUIMICASPRUEBAS BIOQUIMICAS
CEPA 1: COCOS CEPA 1: COCOS (+)(+)
CEPA 2: BACILOS CEPA 2: BACILOS (-)(-)
CEPA 4: Cocobacilar (-)CEPA 4: Cocobacilar (-)
Prueba SIMPrueba SIM
PRUEBA RMPRUEBA RMRx(+) Rx(-)
ACTIVIDAD CATALASAACTIVIDAD CATALASA
PRUEBA TSIPRUEBA TSI
CONDICIONES DE CRECIMIENTOCONDICIONES DE CRECIMIENTO
T (C) NaCl%
-1 9 25 37 45 60 1 2 4 5 6 8 9 10 11
Cepa 1 - - + + + - + + + + + + + ± -
Cepa 2 - + + + + - + + + + + ± - - -
Cepa 3 - + + + + - + + + + + + + ± -
Cepa 4 - - + + + + + + + + + - - - -
Cepa 5 - + + + ± - + + ± + + ± - - -
Cepa 6a - - + + ± + + + ± + + - - - -
Cepa 6b - + + + ± - + + ± + + ± - - -
Cepa 6c - + + + + - + + - + + + + ± -
Cepa 7 - + + + + + + + + + + - - - -
Cepa 8 - ± + + + + + + + + ± ± - - -
Cepa 9 - + + + ± ± + + + + + ± ± - -
Cepa 10 - + + + + + + + + + + ± - - -
Crecimiento a 25C y 60C
25 C25 C 60 C60 C
Crecimiento a 9%NaCl y 10%NaClCrecimiento a 9%NaCl y 10%NaCl
9%NaCl9%NaCl
9%NaCl9%NaCl
10% 10% NaClNaCl
ANALISIS MOLECULARANALISIS MOLECULAR
Electroforesis Electroforesis de la de la
extracción de extracción de ADNADN
1 2 3 4 5 6a 6b 6c 7 8 9 10 Bandas
de ADN
ACTIVIDAD BIOTECNOLOGICAACTIVIDAD BIOTECNOLOGICA
ACCION BIODEGRADATIVA INDIVIDUAL Y ASOCIATIVA DE ACCION BIODEGRADATIVA INDIVIDUAL Y ASOCIATIVA DE DOS CEPAS AISLADAS Y CARACTERIZADAS DE DOS CEPAS AISLADAS Y CARACTERIZADAS DE
PLANOCOCCUS SP SOBRE XILENO, TOLUENO, BENCENO PLANOCOCCUS SP SOBRE XILENO, TOLUENO, BENCENO Y ACEITES FOSILES. Y ACEITES FOSILES.
• Condiciones óptimas de Condiciones óptimas de crecimientocrecimiento
pH 4 (37C) pH 7 (37C)
PCC1PCC1
PCCCPCCC
PCC2PCC2
PCC1PCC1
PCC2PCC2
PCCCPCCC
pH 9 (37C)
PCC1PCC1
PCCCPCCC
PCC2PCC2
•Curva de crecimientoCurva de crecimiento
•Agar mineral con Xi, To, Be y BTXAgar mineral con Xi, To, Be y BTX (Concentración de inhibición)(Concentración de inhibición)
Benceno 1%– pH9 Benceno 3%– pH9
PCCCPCCCPCCCPCCC
PCC2PCC2
PCC1PCC1
Tolueno 3%- pH 9Tolueno 1% - pH 9
PCC1PCC1
PCC2PCC2
PCCCPCCC
PCC2PCC2
• Caldo mineral con Xi, Be, To, Caldo mineral con Xi, Be, To, BTX y aceites BTX y aceites
Donde: pH7pH7
PCC1PCC1 PCC2PCC2 PCCCPCCC Orden de Orden de CrecimientoCrecimiento
XiXi 22
BeBe 33
ToTo 55
BTXBTX ** **** * * ** * * 11
AceitesAceites * * **** * * ** * * 44
* : poco crecimiento
**: crecimiento regular
***: bastante crecimiento
Concentración contaminante 1%
Incubación: 39 días
• Caldo mineral (1%) pH7Caldo mineral (1%) pH7
Benceno 1%
Tolueno 1%
Xileno 1%
BTX 1%
Aceites 1% (5 días de incubación)
Aceites 1% (17 días de incubación)
Medición de concentración de Medición de concentración de contaminantes por espectrofotometriacontaminantes por espectrofotometria
DíasBE (λ=220 nm) TO (λ=220 nm) XI (λ=221 nm) BTX (λ=220 nm)
PCC1 PCC2 PCCC PCC1 PCC2 PCCC PCC1 PCC2 PCCC PCC1 PCC2 PCCC
0 0.061 0.061 0.061 0.071 0.071 0.071 0.109 0.109 0.109 0.08 0.08 0.08
10 0.061 0.052 0.057 0.065 0.055 0.063 0.061 0.056 0.062 0.062 0.061 0.061
14 0.051 0.051 0.056 0.065 0.065 0.057 0.057 0.055 0.057 0.06 0.057 0.061
17 0.049 0.05 0.055 0.065 0.06 0.055 0.052 0.055 0.051 0.057 0.056 0.06
21 0.047 0.049 0.054 0.064 0.055 0.054 0.05 0.054 0.048 0.056 0.055 0.058
24 0.046 0.047 0.048 0.060 0.054 0.05 0.047 0.053 0.048 0.051 0.053 0.056
28 0.044 0.047 0.047 0.058 0.053 0.049 0.046 0.05 0.055 0.05 0.052 0.052
35 0.042 0.045 0.046 0.057 0.051 0.047 0.045 0.048 0.053 0.048 0.050 0.500
39 0.044 0.046 0.047 0.057 0.052 0.048 0.048 0.052 0.056 0.050 0.050 0.540
CONCLUSIONES
• Se caracterizó bioquímica, microbiológica y molecularmente las doce cepas perilíticas.
• Se determinó la actividad biotecnológica de las cepas 1 y 3 que corresponden al Planococcus sp. ( PCC1 y PCC2) para la degradación de hidrocarburos aromáticos y alifáticos.
AGRADECIMIENTOS
Integrantes del Proyecto:
María Vargas VilcaJohana Pérez
BernalJani Pacheco
Aranibar Julio Cesar
Bernabé Ortiz PhD.
