Estructura de las proteínas

2.11.1pmoAmicroarrays

pmoAmicroarray experiments were performed as described in Stralis-Paveseet al.,

(2004).pmoAgenes were amplified from environmental DNA in triplicate using primer set A189f and T7mb650. Amplified PCR products were combined and purified using the QIAquick Spin column (Qiagen) according to manufacturer’s instruction.In vitrotranscription was carried out in a 1.5 ml Eppendorf tube to generate RNA. The final reaction volume was 20 µl containing 1T7 RNA polymerase buffer, 10 mM DTT, 20 U RNAsin (Promega), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 0.25 mM UTP, 1 mM Cy3-UTP, 40 U T7 RNA polymerase (Gibco, BRL) and 400 ng of purifiedpmoAproduct. The reactions were incubated at 37° C for 4 hours. The RNA produced was immediately purified using the Qiagen RNeasy Kit and eluted in 50 µl of nuclease-free water (Ambion). The concentration of the RNA was determined using a NanoDrop spectrophotometer (ND-1000; NanoDrop™, USA).

(Promega) was added to the fragmented target and the labelled RNA was stored at -20 °C.

Hybridisation was performed overnight on an aluminium block placed on a temperature-controlled Belly Dancer shaker (Stovall Life Sciences, Greensboro, USA). The hybridisation block was preheated to 55° C. Slides that had been pre-spotted with thepmoAprobes were assembled with 200 µl HybriWell stick on hybridisation chambers and preheated on the hybridisation block for 1 to 2 min. Meanwhile an Eppendorf incubator was also heated to 65° C and hybridisation mixtures were preheated for 1 min. 200 µl hybridisation mixtures were made in 1.5 ml Eppendorf tubes containing 0.01% (w/v) SDS, 1Denhardt’s reagent (Sigma), 6 SSC, 124 µl DEPC-treated water and 10 µl target RNA. Preheated hybridisation mixture was added to each slideviathe open port and incubated at 55° C at 30-40 rpm circulation and maximum bending overnight, in the Belly Dancer shaker. After hybridization, the array chambers were removed and the slides were immediately washed once for 5 min in 2SSC, 0.1% (w/v) SDS at room

temperature. The slides were then washed twice for 5 min using 0.2SSC, 0.1% (w/v) SDS and finally with 0.1SSC for 5 min at room temperature. Slides were dried individually using an airgun before storing in the dark at room temperature. Hybridisation slides were scanned at 10 µm resolution with a Agilent G2565CA Scanner (Agilent technologies, USA) at a wavelength of 532 nm. Results of individual microarrays were normalised and displayed using GeneSpring software (Agilent Technologies, USA). Microarray data were analyzed as described by Bodrossyet al.,(2003). List of oligonucleotide probes spotted on the microarray and their intended specificity is presented inAppendix 1.

2.11.2Methylocella silvestriswhole genome microarray experiments

Methylocella silvestriswhole genome differential gene expression microarray experiments were carried out to identify genes transcribed differentially under methane and acetate grown conditions. The Agilent custom-designed microarray platform (single colour) was adopted for the micrioarray experiments. Microarray probes (60-mer oligonucleotide probe) covering the whole genome ofMethylocella silvestriswere designed using the eArray software available at the Agilent

Technology web site (https://earray.chem.agilent.com/earray/). A total of 3,917 candidate protein-encoding gene has been identified in the genome ofMethylocella silvestrsi(Genome size 4.3 Mbp; accession number CP001280)

(http://genome.ornl.gov/microbial/msil/). For each gene, three probes were designed (total 11751). The microarray experiments were carried out with RNA extracted from

Methylocella silvestrisgrown on a 1:10 dilution of NMS medium (pH 5.5) either in presence of CH4(10% (v/v) head space) or acetate (final concentration 5 mM) in

batch culture harvested at exponential phase (OD5400.30). The cultures were

incubated at 25C on a shaker (150 rpm). Three biological replicates of RNA were used for each condition. In addition, one batch culture from each methane and acetate grown conditions were split into two groups to extract RNA separately and was considered as the technical replicate. Total bacterial RNA was extracted as described earlier. Microarray target preparation, hybridization, slide washing, scanning and data acquisition were done by the University of Warwick Molecular Biology Facility. Cy-3 labelled cDNA was synthesized from 200 ng total DNA free RNA using the One-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling) kit

MultiStart Primers for the T7 IVT RNA Amplification kit (SBI, Systems Biosciences, USA) as described by Suet al.,(2007). The slides were hybridization with Cy-3 labeled cRNA at 65° C for 17 hours, followed by washing in GE Wash Buffer (Agilent Technologies, USA) at room temperature in an Agilent platform as

described by the manufacturers. Finally the slides were allowed to dry and scanned in an Agilent G2565CA Scanner (Agilent Technologies, USA). Feature extraction software provided by Agilent (Version 7.5, Agilent Technologies, USA) was used to quantify the intensity of the fluorescent images and to normalize initially the results by subtracting local background fluorescence, according to the manufacturer’s instructions. Microarray data were further normalized using GeneSpring software at the 75th percentile level using the GeneSpring GX 11 software package (Agilent Technologies, USA). Thepvalue derived by unpaired t test using the GeneSpring software was used to assess the statistical significance of that estimate. The value ranges from 0 to 1, with a smallerpvalue providing more confidence in the regulation pattern. For this study, data withpvalues of0.05 were generally considered confident and reliable. The biological significance for each comparison was analyzed using the assigned cut-off expression ratio of 2.0 fold-changes and the direction of regulation (e.g.an upregulation or downregulation in expression). The functional grouping of the genes were developed based on proposed functions of each gene in the genome annotation page ofMethylocella silvestris.

Chapter 3

Distribution of

Methylocella

spp. in the