Procesos

To experimentally establish the localisation of the putative muticopper oxidase

protein on theSynechococcuscell surface, two outer membrane isolation methods

were used. Method 1 is based on differential centrifugation (Resch and Gibson, 1983).

peptidoglycan network, but bound primarily by physical forces, i.e. ionic and/or

hydrophobic. As a result, treatments such as incubation with a chelating agent like

EDTA cause release of the OMF, a complex of LPS, protein, and phospholipid into

the surrounding medium (Lindberg, 1973). The detergent Trition X-100 was used in

the method 1 to remove cytoplasmic membrane (Schnaitm, 1971).

90μg of the OMFs fromSynechococcussp. WH7803 and WH7803RS-PM2 obtained using the method 1 showed a profile with three predominant bands at approximate 98,

64 and 50 kDa (indicated by solid arrows) (Figure 5.10). No protein bands

corresponding to the MCO (~180 kDa) were identified, which may be due to

limitation of the isolation method. One faint protein band with an apparent molecular

mass lower than 16 kDa (indicated by a dashed arrow) seemed to be only present in

the OMFs of mutant cells (Figure 5.10) (identical results were obtained from three

biological replicates). This difference was confirmed using the silver staining of the

OMFs obtained using method 2 (lower part of lane 3, Figure 5.13). However this

protein band with an apparent molecular mass lower than 16 kDa was found both in

the mutant and wild-type cells using whole-cell SDS-PAGE analysis (Figure 5.3), it

1 2 3

Figure 5.10 SDS-PAGE profiles of OMFs obtained by method 1 from Synechococcus sp. WH7803 and WH7803RS-PM2 cells.

Lane 1 SeeBlue®Plus2 Pre-Stained protein standard (Invitrogen) that was used to estimate protein molecular weight, Lane 2Synechococcussp. WH7803RS-PM2, Lane 3Synechococcussp. WH7803.

Since SDS-PAGE analysis of the OMFs obtained using method 1 did not reveal the

MCO band, method 2 was tried. The resulting pellet obtained after centrifuging the

cell-free fraction of EDTA-treated WH7803 had a green base covered with a yellow

layer (Figure 5.11). Since the OMF contains no chlorophyll and phycobilins, the

yellow layer should be the OMF and the green pellet should be a membrane fraction

containing chlorophyll. A similar finding was observed previously (Resch and

Gibson, 1983).

Figure 5.11 Diagram to indicate the pellets obtained after centrifuging the supernatant of EDTA- treated WH7803. Green pellet Yellow layer KDa 250 148 98 64 50 36 22 16

To find out the protein composition of the pellet obtained by the second method, 600

μg of the yellow layer and green pellet were analysed using SDS-PAGE alongside whole-cell protein extracts. As seen in Figure 5.12, the whole-cell protein profiles of

Synechococcussp. WH7803 and EDTA-treated WH7803 appear to be the same,

which indicated that EDTA treatment was mild enough to preserve most of the cell

structures. A well-defined protein band was revealed in the yellow fraction

(highlighted by a yellow rectangle), but almost invisible in the green material. This

band was analysed by MALDI-TOF and demonstrated to be the putative MCO (this

was performed and analysed by Samuel J.H. Clokie using the same methodology as

described in Section 5.3.3)

.

From now on the yellow fraction will be referred to as the OMF. This band can also be visualised as faint bands in the whole cells (due to the

limitation of resolution in printing, they can’t be seen in the printed format, but can be

see on the electronic version of Figure 5.12 stored on the CD accompanying this

thesis). Additionally, the OMF and the green material displayed marked difference. A

band of approximately 98 kDa (highlighted by a black rectangle) that was observed

using method 1 was almost indistinguishable from the background in the OMF, but

prominent in the green material as well as in the whole cells. The 50 kDa band

(highlighted by a blue rectangle) that was observed using method 1 was also revealed

in the OMF obtained by method 2 (Figure 5.12). In addition, the 64 kDa band

(highlighted by a red rectangle) that was observed using the methods 1 was only

present in the green material. Both the OMF and the green material were devoid of

phycobiliproteins, which were the most noticeable bands in the whole-cell extracts.

Figure 5.12 SDS-PAGE profiles of OMFs obtained by the use of method 2.

Lane 1 wild-type WH7803 cells, Lane 2 EDTA-treated WH7803 cells, Lane 3 the yellow pellet obtained from the supernatant of EDTA-treated WH7803 cells, Lane 4 the green pellet obtained from the supernatant of EDTA-treated WH7803 cells, Lane 5 the SeeBlue®Plus2 Pre-Stained protein standard (Invitrogen) in kDa.αandβsubunits of phycobiliproteins were labelled. The band highlighted by a yellow rectangle was the putative multicopper oxidase. The band highlighted by a black rectangle was the protein band that was only revealed using method 1 to prepare the OMF. The band highlighted by a red rectangle was only present in the OMF prepared using method 1 and the green material

1 2 3 4 5 kDa 250 148 98 64 50 36 22 16 αandβsubunits of phycobiliproteins

prepared using method 2. The band highlighted by a blue rectangle was revealed using both method 1 and 2.

5.3.5.3 Comparison of the OMFs of Synechococcus sp. WH7803 and

In document Gestión estratégica de Empresas familiares y su influencia en elsector Restaurante Picanterías 2018 (página 84-105)