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SUPPLEMENTARY MATERIAL
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Optimization of the microarrays hybridization and selection of parameters
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Prior to hybridization with the NS sample and to ascertain the fluorescence values that
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could reflect unspecific hybridization, the viral microarray was hybridized with
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cDNACy3-labelled from 2 g of total RNA from Leptospirillum ferrooxidans, a gram-
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negative chemolithoautotrophic bacterium highly abundant in acid environments
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(González-Toril et al., 2003) that should be absent from CR30 crystallizer. After this
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hybridization under the same conditions used with the CR30 viral metatranscriptome,
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the signal intensity obtained with the L. ferrooxidans RNA was used as a base-line
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control
to
estimate
unspecific
hybridization
in
our
experiments.
In
the
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metatranscriptome studies we considered as positive only those spots having signal
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intensities 9-fold the background fluorescence in each hybridization, which were always
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higher than the unspecific hybridization determined as described above.
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Total RNA from the May 2007 NS was extracted, amplified, reversely transcribed and
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labelled as described in the “experimental procedures” section and 2 g of cDNA-Cy3
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were hybridized against the constructed viral microarray. The amplification step was
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necessary since, as previously described (Moreno-Paz and Parro, 2006; Moreno-Paz et
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al, 2010), the nucleic acids extraction yielded low amounts of environmental RNA.
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After hybridization with this sample, the viral microarray was analysed and 340 spots
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(57%) showed fluorescence signals over 9-fold the background. It is probable that we
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missed spots with intensities below this restrictive threshold, but by using the 9-fold
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background threshold, we ensure that we only include specific and highly expressed
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viral transcripts in the analysis. Two categories for the level of expression were
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established: (i) highly expressed, between 9 and 15 fold background fluorescence, and
1
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(ii) very highly expressed, above 15 fold the background fluorescence. Prior to double
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hybridizations between C and stressed samples (UV and D), the NS was hybridized
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against C1 to see if maintaining the sample in the laboratory might cause an over-
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expression of viral genes in the sample. After the analysis, we could not detect over-
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expression in any spot (data not shown), which indicated that the over-expression
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observed in the stressed samples was due to applied treatments. For all the double
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hybridizations, data were filtered and normalized and spots that showed a total intensity
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(the sum of both Cy5 and Cy3 channels) equal to or higher than 9 fold background
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fluorescence (see above) were selected. The ratios between control samples versus
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stressed samples were compared and the spots that showed a fluorescence at least two
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times higher in the treated samples, were selected for the final analysis.
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FISH analysis in the May 2009 sample
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The NS taken in May 2009 had 34% salinity. Archaea and Bacteria accounted for
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79.35% and 10.31% of the DAPI counts, respectively. UV, D and C subsamples were
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analysed as described in the “experimental procedures” section of the manuscript.
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Data showed that Bacteria were very sensitive to UV treatment, with reductions of up to
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30% while Archaea suffered more dramatically the change in the salinity (with losses of
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up to 23%, compared to losses in Bacteria, that accounted for 10%). Results are shown
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in supplementary Figure 1 and are in agreement with data obtained in the stress
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experiments carried out in 2007: members of Bacteria recovered after the radiation
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treatment, while Archaea showed a higher decrease with reductions of up to 50% and
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33% under dilution and radiation conditions, respectively.
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2
1
Legends
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Fig Sup 1. Changes in the number of total cells (DAPI), Archaea and Bacteria with
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time after the application of the stress treatmens, compared to values in the natural
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sample (NS) taken in May 2009. Green lines are the values of the control samples. In
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red (UV), the number of cells after the UV-radiation treatment. In blue (D), the number
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of cells under osmotic shock. Black lines (DT) represent the theoretical values after the
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dilution of the sample without considering lysis.
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3