Resultados de las correlaciones entre posiciones de la cadena teniendo en cuenta las propiedades

culture where acetate was found to repress the transcription of sMMO genes of

Methylocella silvestris(Theisenet al.,2005).

5.2.3Methylocella silvestrisBL2 were not the major active acetate-utilizers in peat soil microcosms

To investigate ifMethylocella silvestrisutilized acetate as the carbon source in this peat soils in the absence of CH4,13C-labelled acetate DNA-SIP experiments were

carried out withMethylocella silvestris-spiked Moor House peat soil. The hypothesis to be tested was that ifMethylocella silvestrisutilized acetate rather than methanein situ, Methylocella silvestris16S rRNA gene should be detected in the13C-labelled acetate ‘‘heavy’’ DNA as an enriched band. Bacterial 16S rRNA gene-specific

DGGE carried out with the ‘‘heavy’’ DNA retrieved from theMethylocella silvestris-

spiked13C-labelled acetate soils showed several enriched bands when compared to the banding pattern with the “light” DNA (Figure 5.6).

B1.Burkholderia norimbergensis

B2.Brevundimonas bullata

B3.Acidovorax

B4.Acidocella aluminiidurans

Figure 5.6.DGGE fingerprint profiles of 16S rRNA gene PCR products of ‘‘heavy’’ and “light” DNA retrieved from the

soil incubated with13

re-amplification and sequencing are indicated by arrows.

Acidocella,Acidovorax,

sequences were enriched

be the most dominant bacterial genera actively involved in the utilization of acetate in this peat soil under these incubation conditions

sequences were detected as enriched bands

analyses. The13C-acetate labelled “heavy” DNA retrieved from

Burkholderia norimbergensis(Y09879; 99% identity)

Brevundimonas bullata(EU665637; 99% identity)

Acidovoraxsp. (GQ284468; identity 100%)

Acidocella aluminiidurans(AB362219; 99% identity)

DGGE fingerprint profiles of 16S rRNA gene PCR products of ‘‘heavy’’ and “light” DNA retrieved from theMethylocella silvestris-spiked Moor House peat

13

C-labelled acetate. Enriched DGGE bands that were excised for amplification and sequencing are indicated by arrows.

Acidovorax, BrevundimonasandBurkholderia-like 16S rRNA gene sequences were enriched in the13C acetate labelled heavy DNA, indicating

be the most dominant bacterial genera actively involved in the utilization of acetate under these incubation conditions (Figure 5.6). No

sequences were detected as enriched bands after 16S rRNA gene DGGE fingerprint acetate labelled “heavy” DNA retrieved fromMethylocella

(Y09879; 99% identity) (EU665637; 99% identity)

(AB362219; 99% identity)

DGGE fingerprint profiles of 16S rRNA gene PCR products of ‘‘heavy’’ spiked Moor House peat labelled acetate. Enriched DGGE bands that were excised for

like 16S rRNA gene heavy DNA, indicating them to be the most dominant bacterial genera actively involved in the utilization of acetate

). NoMethylocella-like DGGE fingerprint

clones were analyzed by RFLP and five OTUs were obtained (Figure 5.7).

Figure 5.7. Neighbor-joining phylogenetic tree of 16S rRNA gene sequences (in bold) obtained from the “heavy” DNA retrieved from the Methylocella silvestris-

spiked Moor House peat soil incubated with 13C-labelled acetate and the closely related sequences available in the public database (accession numbers are in parentheses). The tree was generated with a final data set of 1043 nucleotide positions. The scale bar represents 5% sequence divergence. The percentages of clones obtained from the same OTU are shown. The numbers at the nodes represent bootstrap values (≥70) observed with 1000 replicates. The tree is rooted with the 16S rRNA gene sequences fromThermodesulfatator indicus(NR025171). The 16S rRNA gene sequences retrieved from the Moor House peat soil incubated with 13C-acetate are named as Acetate2, Acetate8 etc.

This clone library analysis identifiedBrevundimonasspp. as the most dominant bacteria (67% of clones in the library) involved in utilization of acetate in the Moor House peat soil. In addition, 16S rRNA genes phylogenetically related to the 16S rRNA genes ofPandoraea(15% ),Acetobacter(9%),Pseudomonas(5%) andClostridium(4%)were also identified in this clone library. Several studies have used 13C-labelled acetate to identify bacterial species actively involved in the utilization of acetate in soil and sediment (Scholten & Stams, 2000; Chauhan & Ogram, 2006; Schwarzet al., 2007; Longneckeret al., 2009).Acetobacterhas been identified in Florida soil as a major acetate-utilizing bacterium in Florida soil (Chauhan & Ogram, 2006). Schwarzet al.(2007) identifiedAcetobacterand

Burkholderiaas active genera involved in acetate-utilization in the sediment of Lake Kinneret (Israel). Therefore, our 16S rRNA gene clone library analyses, as well as DGGE fingerprinting analyses, suggested thatMethylocellaspp. were not significant acetate-utilizers even when added to the peat soil microcosm under these present experimental conditions. ProbablyMethylocellaspp. were outcompeted by more efficient acetate-utilizers such asBrevundimonasandBurkholderia. This observation is further supported by the acetate uptake data obtained from the non-spiked and

Methylocella silvestris-spiked soil. Virtually no difference was observed in the ability of theMethylocella silvestris-spiked soil to take up acetate when compared to non-spiked soil in microcosms(Figure 5.8).

Figure 5.8. The ability of the non-spiked and Methylocella silvestris-spiked Moor House peat to take up acetate (0.5 mM final concentration). Error bars indicate the standard error of the mean of triplicate acetate concentration measurements.

In conclusion,13CH4DNA-SIP experiments indicated thatMethylocellaspp. were

not the major methane-utilizers in Moor House peat soil in microcosms under the experimental conditions tested. Spiking of the peat soil withMethylocella silvestris

increased the ability of the soil to oxidize methane in microcosms. However, more importantly, it was evident that 0.5 mM acetate reduced the ability of the

Methylocella silvestris-spiked peat soil to oxidize methane probably due to acetate inhibiting methane oxidation byMethylocella silvestris. Furthermore, the13C- labelled acetate DNA-SIP analyses showed thatMethylocellaspp., were not acetate- utilizers in this peat soil microcosm under the conditions used in this study and that they seem to have been outcompeted by more efficient acetate-utilizers. Further experiments are required to investigate the potential competition for acetate between

0 10 20 30 40 50 60 70 80 90 100

Day 0 Day 5 Day 8 Day 11 Day 14 Day 17 Day 20 Day 23 Day 26 Day 29

Pe rc en ta ge of ac et at e re m ai ni ng Time (days)

Methylocellaand other acetate-utilizers, such asBrevundimonasandBurkholderia, and the consequences of this for methane oxidation by facultativeMethylocellaspp. in peat soils. In addition, the recently identified facultative methanotrophs

Methylocapsa aurea(Dunfieldet al.,2010) andMethylocystissp. H2s (Belova et al.,

2010) isolated from forest soil and peat soil of Germany respectively can also be tested in the similar way to determine if acetate represses their potential to oxidize methane in soil.

Chapter 6

Methylocella silvestris

whole genome