PROCESOS DE SOLDADURA

(Carried out by Elizabeth Hirst, NIMR, according to Hayat, 1978) 2.19.1 Fixation

Samples o f Synechocystis cells were washed in 1 x PBS and dried on cover slips coated with 2pl poly-L-lysine (Sigma) at a concentration o f lOOpg/ml. Following fixation at room temperature by incubation for 45 minutes in O.IM SCB/2% gluteraldehyde, the slides were washed with 2% cacodylate buffer, pH7.2.

2.19.2 Post fixation

Post fixation, the slides were dehydrated in ethanol: 5 minutes with 50% EtOH, 10 minutes with 70% EtOH, 10 minutes with 95% EtOH and finally for 30 minutes with 100% EtOH. The slides were then immersed for 15 minutes in HMDS (heXamethyl disilazine) and allowed to air dry at room temperature. The cover slips were then mounted onto SEM stubs and desiccated overnight before being sputter coated with 20nM gold (Sputter Coater, EMscope).

The samples were viewed in a JEOL CF35 scanning electron microscope.

2.20 DAPI staining

Nucleoids were stained with DAPI (Sigma) at O.Olpg/ml, following a protocol similar to that described by Hiraga et a l (1989). 2pl o f poly-L-lysine (Sigma), at a concentration o f lOOpg/ml, was spotted onto glass slides and allowed to dry. Cell samples o f either E. coli or Synechocystis were washed twice in PBS and 2pl spotted onto the poly-L-lysine and air dried. The slides were then fixed with methanol and washed with (IH2O. 2pl o f DAPI stain was placed onto the fixed cell samples and incubated at room temperature for approximately 20 seconds before being washed off with dH20. Localization o f the nucleoids under Vectashield mounting medium (Vector Laboratories Inc.) was detected under UV light and analysed using conventional UV-light microscopy (Zeiss) and Ectachrome P I 600 film.

2.21 Giemsa staining

Slides were prepared as described above using poly-L-lysine and fixed in methanol. The samples were stained with Giemsa’s staining solution (BDH) diluted to 10% in water. Following air drying, samples were analysed using light microscopy (Zeiss) and photographed using P I 600 film (Fuji).

2.22 Autofiuorescence of Synechocystis (Carried out by Iain Wilson, NIMR)

Slides were prepared as described above with washed WT and mutant Synechocystis cells. Following fixation, the samples were observed directly under UV light and pictures taken with Ectachrome P I 600 film. Cells with normal amounts of chlorophyll showed bright orange fluorescence.

2.23 Statistical analysis of SEM data - The Chi-square test.

The statistical significance (P value) of the proportion o f cells undergoing division in Synechocystis y c f 24 mutants when compared to wild type (WT) cells also in division, was determined using the chi-square test (Croxden and Cowden, 1962). The value was determined following the formula below:

^ = 1 (£z£i

/c

Where: / = Observed frequency, i.e. the number o f mutants undergoing division. f = Computed frequency, i.e. the number of cells expected to be undergoing

division, in this case the number o f WT cell in division.

As only two criteria were analysed, either dividing or not-dividing, the n (degrees o f freedom) value for the experiment was 1. This value, with that calculated for were used to look up the specified value o f P (Appendix J, Croxden and Cowden, 1962). P values <0.001 were assumed to be significant.

2.24 Analysis of Synechocystis cell cycle (Carried out by Conrad Mullineaux, UCL)

Live Synechocystis cells growing on BGl 1 plates were monitored by a video linked phase contrast microscope (Zeiss Axiophot) with images taken over 5-6 generations to monitor cell cycle length. Images were recorded at 3 hour intervals for the first 12 hours and at time points 23hrs, 30hrs and 47hrs thereafter. The state o f dividing cells within a colony was then analysed. In between time points, the plates were returned to the light incubator at 30°C (10-20pE light). Whole field images were analysed by A. Law at the cellular level.

2.25 PAGE

Samples were solubilized in SDS sample buffer by heating at 100°C for 5 minutes and analysed by SDS-PAGE on homogenous 10% SDS/polyacrylamide (w/v) gels according to the method o f Laemmli, 1970.

Soluble proteins were analysed by electrophoretic separation in native (non­ denaturing) polyacrylamide (12% w/v) gels using native loading buffer.

Electrophoresis was carried out at 25-40mA using a Mighty Small II vertical slab gel unit (Hoeffer Scientific Instruments) or using the Xcell IF ^ Mini-Cell (NOVEX^^) according to the manufacturer’s instructions. Pre-cast gels were used in conjunction with the Xcell Il’^’^^ini-C ell. For native applications, 10% Tris-Glycine gels were used with the supplied running buffer. For denaturing applications, 10% NuPAGE® Bis-Tris gels were used, with MOPS running buffer, (both supplied by

NOVEXTM).

Following electrophoresis, proteins were stained under agitation (Orbital Mixer, Denley) for 1 hour at room temperature with 0.1% (w/v) Coomassie Brilliant Blue R- 250 (Sigma). This was followed by several incubations in destaining solution until the separated proteins and markers were easily visualized.

Molecular mass markers

Prestained Protein Marker, Broad Range (New England Biolabs) with marker sizes in kDa of: 6.5, 16.5, 25, 32.5,47.5, 62, 83, and 175.

Unstained Wide Range Protein Standard Marker (NOVEX^'^) with marker sizes in kDa o f : 2.5, 3.5, 6.0, 14.4, 21.5, 31.0, 36.5, 55.4, 66.3, 97.4, 116.3 and 200.

Unstained Protein marker. Low Range (Pharmacia) with marker sizes in kDa of: 20, 30, 45, 68 and 90.

2.26 W estern blotting (Burnette, 1981)

2.26.1 Protein transfer to nitrocellulose m em brane

Protein samples were run on a 10% SDS or native polyacrylamide gel and electrophoretically transferred to Hybond'^'^-C Pure nitrocellulose membrane (Amersham) using the Sartoblot®II-S semi-dry blotting apparatus (Sartorius), according to the manufacturer’s instructions. Transfer o f the proteins was carried out at 25-50mA (depending on the size o f the blot) for three hours.

If unstained protein marker was used, blots were subsequently stained with Ponceau-S stain (Sigma) and washed with dH20 until the marker bands were easily

observed. These were marked with pencil and the remaining stain removed by further washing before continuing with the protocol.

2.26.2 Probing and developing of the membrane

Western blots to be probed with antibody were blocked with 5% (w/v) non-fat milk powder (Marvel) in 1 x PBS/0.1% Tween 20 (v/v) (Sigma) with agitation for 1 hour at room temperature. The membrane was then washed three times, for 10 minutes in PBS/0.1% Tween 20 (v/v), before incubating for 1 hour at room temperature (or overnight at 4°C) with agitation in solutions containing various primary antibodies. Those used were murine monoclonal anti-GST (Zymed) diluted 1/1,000 in PBS/Tween, rabbit polyclonal anti-MBP diluted 1/10,000 in PBS/Tween (New England Biolabs) and a mouse penta-His Antibody (QIAGEN) diluted 1/1,000 in PBS/ Tween. I also used anti-FtsZ kindly provided by Dr H. Erikson (Duke University, North Carolina, USA.), diluted 1/1,000 in PBS/Tween. Unbound primary antibody was removed by 3x 10 minute washes in PBS/Tween. This was followed by incubation with agitation for 1 hour at room temperature with various second antibodies conjugated to horseradish peroxidase. Rabbit anti-mouse IgG (Zymed) diluted 1 in 1,000 in PBS/Tween was used for anti-GST and anti-His probed western blots. Goat anti-rabbit IgG (BIO-RAD) diluted 1 in 3,000 in PBS/Tween was used for anti-MBP and antibody-probed western blots. The presence o f peroxidase was detected by chemiluminescence using ECL^^ Western Blotting Detection Reagents (Amersham) according to the manufacturer’s instructions.