RING-FISH and cell sorting of Upland Soil Cluster α

The probes that were designed for application of RING-FISH were first tested with E. coli clones harboring the partial gene of the particulate methane monooxygenase (pmoA) of Upland Soil cluster α from Marburg forest soil as positive control and partial pmoA genes of Methylocapsa acidiphila and Methylocystis sp. strain SC2 as negative controls. RING-FISH for all probes was evaluated under formamide concentrations of 5-55%. The potential of the probes to form secondary structures was analysed in silico (RNAdraw V1.1, www.rnadraw.com, Matzura et al., 1996). It was suggested (Zwirglmaier et al., 2003) that the feature of RNA polynucleotide probes to form a network within and around the cell envelope is decisive for the appearance of halo signals of target bacteria.

III.2.1.1 RING-FISH with long RNA polynucelotide probe MF02

The long RNA polynucleotide probe MF02 (~500 bp), derived from the PCR product of an USCα soil clone (see II.10.1), was analysed in silico using RNAdraw and showed a high potential to form secondary structures at hybridization conditions of 53°C and 15% of formamide (see figure III.2-1).

Figure III.2-1: Secondary structure model (RNAdraw V1.1) of USCα pmoA RNA polynucleotide probe MF02 (~500 bp) with 15% formamide in the hybridization buffer and hybridization temperature of 53°C.

Because USCα has not been isolated so far, no “real” cells of this cluster could be used as positive control for RING-FISH. Therefore, clones harboring the partial gene of the particulate methane monooxygenase of USCα from Marburg forest soil were used as positive control.

These cells showed bright whole cell fluorescence after hybridization. Occasionally, halo signals could also be observed, but only in small, single areas on the well, never in larger areas or as dominating signal type after hybridization (see figure III.2-2). This was

independent from formamide concentration and incubation time. Above a formamide concentration of 35%, all signals completely disappeared.

Figure III.2-2: Halo signals (A) and whole cell fluorescence (C) observed after RING-FISH of particulate methane monooxygenase (pmoA) genes of USCα in clones harboring pmoA of Upland Soil cluster α (positive control) using polynucleotide probe MF02. Fluorescence images (A and C) and respective phase contrast (B and D). Bars = 10 µm.

The negative controls, E. coli clones harboring pmoA of Methylocapsa acidiphila and Methylocystis sp. strain SC2, also continuously showed whole cell fluorescence after RING-FISH, independent from formamide concentration and incubation time (see figure III.2-3).

Above a formamide concentration of 35%, signals completely disappeared. This indicates that hybridizations were not specific enough to target only pmoA of USCα, since successfull discrimination between the control clones could not be achieved with polynucelotide probe MF02.

Figure III.2-3: Whole cell fluorescence observed after RING-FISH of particulate methane monooxygenase (pmoA) genes of USCα in clones harboring pmoA of Methylocapsa acidiphila (A and B) and Methylocystis sp. strain SC2 (C and D) (negative controls) using polynucleotide probe MF02.

Fluorescence images (A and C) and respective phase contrast (B and D). Bars = 10 µm.

To exclude the possibility that these observations resulted from cells damaged by wrong fixation, thawing, or other circumstances, clones were fixed new every month and only fresh aliquots were used for hybridization. Also clones fixed with 2% paraformaldehyde instead of 4% were tested. But all hybridizations resulted in either whole cell fluorescence or no signal of the positive and negative controls.

Another negative control for RING-FISH was represented by Methylocapsa acidiphila. This was done to test whether actual methanotroph cells might behave differently in the hybridization procedure compared to clones harboring the same target gene. Methylocapsa acidiphila cells showed no signal after RING-FISH of pmoA gene of USCα in contrast to the E. coli harboring the same pmoA gene. This observation was independent from formamide concentration and incubation time, suggesting that the different cell morphologies of clones and the methanotroph might have had a significant impact on the hybridization results and therefore clones might not be universally applicable as controls in RING-FISH. Above a formamide concentration of 80%, cells suddenly showed a bright halo signal (see figure III.2-4). To test the specificity of this halo signal, cells of Methylocapsa acidiphila were also hybridized with a probe targeting archaeal amoA as a negative control, also leading to bright halo signals. This indicates that formamide concentrations of 80% and above might have damaged the cell wall and allowed for unspecific formation of the probe network.

Figure III.2-4: Unspecific halos signals observed after RING-FISH of particulate methane monooxygenase (pmoA) genes of USCα in Methylocapsa acidiphila (negative control) at formamide concentration of 80% using polynucleotide probe MF02. Fluorescence image (A) and respective phase contrast (B). Bars = 10 µm.

Hybridizations using RING-FISH probe MF02 with cells extracted from Marburg forest soil using Histodenz density gradient centrifugation (see II.10.3) showed sporadic halo signals when hybridized at 53°C with 15% formamide in the hybridization buffer (see figure III.2-5).

Due to the unspecific results obtained with the clones, however, no statement can be made about the specificity of these signals in soil.

Figure III.2-5: Halo signals observed after RING-FISH of particulate methane monooxygenase (pmoA) genes of USCα in cells extracted from Marburg forest soil using polynucleotide probe MF02.

Fluorescence images (A and C) and respective phase contrast (B and D). Bars = 10 µm.

III.2.1.2 RING-FISH with monospecific oligo-oligonucleotide probes

The monospecific RNA oligo-oligonucleotide probe MF08_25-175 (175 bp) targeting pmoA of USCα (see II.10.5.2), was analysed in silico using RNAdraw and showed a high potential to form secondary structures at hybridization conditions of 53°C and 15% of formamide (see figure III.2-6).

Figure III.2-6: Secondary structure model (RNAdraw V1.1) of monospecific USCα pmoA RNA oligo-oligonucleotide probe MF08_25-175 (175 bp) with 15% formamide in the hybridization buffer and hybridization temperature of 53°C.

Also using this probe, cells from clones harboring pmoA of Upland Soil cluster α (positive control) showed bright whole cell fluorescence and only single partial halos, independent from formamide concentration and incubation time (see figure III.2-7). Above a formamide concentration of 30% and below an incubation time of 12 h, signals completely disappeared.

Figure III.2-7: Partial halo signals (A) and whole cell fluorescence (C) observed after RING-FISH of particulate methane monooxygenase (pmoA) genes of USCα in clones harboring pmoA of Upland Soil cluster α (positive control) using the monospecific oligo-oligonucleotide probe MF08_25-175.

The same was observed with the negative controls, clones harboring pmoA of Methylocapsa acidiphila and Methylocystis sp. strain SC2. Cells consistently showed whole cell fluorescence, independent from formamide concentration and incubation time (see figure III.2-8). Above a formamide concentration of 30%, signals completely disappeared. This shows that also with this probe, no successful discrimination between the control clones to target pmoA of USCα could be achieved.

Figure III.2-8: Whole cell fluorescence observed after RING-FISH of particulate methane monooxygenase (pmoA) genes of USCα in clones harboring pmoA of Methylocapsa acidiphila (A and B) and Methylocystis sp. strain SC2 (C and D) (negative controls) using the monospecific oligo-oligonucleotide probe MF08_25-175. Fluorescence images (A and C) and respective phase contrast (B and D). Bars = 10 µm.

Also with probe MF08_25-175, hybridizations using RING-FISH with cells extracted from Marburg forest soil using Histodenz density gradient centrifugation (see II.10.3) showed sporadic halo signals when hybridized at 53°C with 15% formamide in the hybridization buffer (see figure III.2-9). Due to the unspecific results obtained with the clones, however, no statement can be made about the specificity of these signals in soil.

Figure III.2-9: Halo signals observed after RING-FISH of particulate methane monooxygenase (pmoA) genes of USCα in cells extracted from Marburg forest soil using the monospecific oligo-oligonucleotide probe MF08_25-175. Fluorescence image (A) and respective phase contrast (B). Bars = 10 µm.

The monospecific RNA oligo-oligonucleotide probe RA14_GC121 (121 bp) targeting pmoA of USCα (see II.10.5.2), was analysed in silico using RNAdraw and showed a high potential to form secondary structures at hybridization conditions of 53°C and 10% of formamide (see figure III.2-10).

Figure III.2-10: Secondary structure model (RNAdraw V1.1) of monospecific USCα pmoA RNA oligo-oligonucleotide probe RA14_GC121 (121 bp) with 10% formamide in the hybridization buffer and hybridization temperature of 53°C.

After RING-FISH and detection, cells from clones harboring pmoA of Upland Soil cluster α (positive control) showed bright whole cell fluorescence and also only single partial halos, independent from formamide concentration and incubation time (see figure III.2-11). Above a formamide concentration of 30% and below an incubation time of 12 h, signals completely disappeared.

Figure III.2-11: Partial halo signals and whole cell fluorescence observed after RING-FISH of particulate methane monooxygenase (pmoA) genes of USCα in clones harboring pmoA of Upland Soil cluster α (positive control) using the monospecific oligo-oligonucleotide probe RA14_GC121. Fluorescence images (A and C) and respective phase contrast (B and D). Bars = 10 µm.

Like for the monospecific oligo-oligonucleotide probe RA14_GC121, negative controls, clones harboring pmoA of Methylocapsa acidiphila and Methylocystis sp. strain SC2, continuously showed whole cell fluorescence after RING-FISH, independent from formamide concentration and incubation time (see figure III.2-12). Above a formamide concentration of 30%, signals completely disappeared. These results indicate that there was no difference in the hybridization efficiency and specificity between probe MF08_25-175 with poly-A spacer regions and probe RA14_GC121 containing GC spacers. Also with probe RA14_GC121, specific hybridization of only USCα pmoA was not possible.

Figure III.2-12: Whole cell fluorescence observed after RING-FISH of particulate methane monooxygenase (pmoA) genes of USCα in clones harboring pmoA of Methylocapsa acidiphila (A and B) and Methylocystis sp. strain SC2 (C and D) (negative controls) using the monospecific oligo-oligonucleotide probe RA14_GC121. Fluorescence images (A and C) and respective phase contrast (B and D). Bars = 10 µm.

As for the other probes, hybridizations using RING-FISH with probe RA14_GC121 cells extracted from Marburg forest soil using Histodenz density gradient centrifugation (see II.10.3) resulted in sporadic halo signals when hybridized at 53°C with 15% formamide in the hybridization buffer (see figure III.2-9). But again, due to the unspecific results obtained with the clones, no statement can be made about the specificity of these signals in soil.

Figure III.2-13: Halo signals observed after RING-FISH of particulate methane monooxygenase (pmoA) genes of USCα in cells extracted from Marburg forest soil using the monospecific oligo-oligonucleotide probe RA14_GC121. Fluorescence image (A) and respective phase contrast (B). Bars = 10 µm.