CIRCADIAN RHYTHMS - Cyanobacteria - page 306

later came to be known as Synechococcus R-2), an amenable experimental material for a mutational
analysis of genes governing circadian rhythms. The choice for such studies was made on the basis
of its (i) natural transformability, (ii) ability to receive DNA by conjugation from E. coli at high
effi ciency and (iii) potential to express reporter genes. Synechococcus sp. strain PCC 7942 which
is not diazotrophic meets all the above requirements and was chosen as the experimental system
(Golden et al ., 1997). In many of the communications that followed, this organism has been referred
to as Synechococcus sp. strain PCC 7942 but a redesignation of this strain as S. elongatus PCC 7942
settled the ambiguity.
Li and Golden (1993) employed a promoterless luxAB gene set that encodes the luciferase from
Vibrio harveyi and fused it to the promoter of a PSII gene, psbAI (that encodes the photosystem II
reaction center D1 protein). This fusion product has a strong expression in S . elongatus PCC 7924
because of its integration at a site in the chromosome that has been used previously for harbouring
heterologous genes (Kondo et al ., 1993). The resulting strain, designated as AMC149, emitted light,
showed a persistent 24 h rhythm of bioluminescence when the cells are shifted from LD to LL. By
using this strain it was possible to demonstrate the phase resetting cues at different times during
circadian cycle that is characteristic of circadian clock controlled phenomena as seen in eukaryotes.
Exposure of cultures to opposite LD cycles resulted in opposite peaks of light production after transfer
to LL. Furthermore, single pulses of 4 h of darkness in otherwise LL conditions shifted the peaks
sooner or later. Another important feature is that the period remained close to 24 h when measured
at 25ºC, 30ºC and 35ºC signifying that the bioluminescence rhythm is also temperature compensated
(Kondo et al ., 1993). The rhythmic expression of bioluminescence from luxAB reporter has also been
demonstrated in two other cyanobacteria ( Synechocystis sp. strain PCC 6803 and Anabaena sp. strain
PCC 7120) that have been widely manipulated at the level of molecular genetics.
Liu et al . (1995) concluded that bioluminescence is an accurate parameter to measure the activity
of psbAI gene in AMC149 because of rhythmic variation in mRNA of psbAI gene and the luciferase
enzyme. The bacterial luciferase enzyme uses oxygen and reduced fl avin mononucleotide as substrate
and these are expected to vary in a circadian fashion as a consequence of rhythmic photosynthesis.
A long chain aldehyde, N-decanal has been used as a third substrate for luciferase at 3% (v/v)
dissolved in vacuum pump oil. Colonies of AMC149 were formed in 10 days in LL condition at 30°C.
After subjecting the plates for 12 h darkness to reset the circadian clocks, the bioluminescence was
monitored at various times during LL by exposing the colonies to decanal vapours. Bioluminescence
reached a stable level within 15 min and continued longer than a week.
After having chosen the ideal experimental organism as S . elongatus PCC 7942 and its genetically
transformed strain (with bioluminescence luxAB reporter gene) AMC149, the next hurdle to overcome
is the rapid screening of thousands of colonies for circadian phenotypes. Kondo and Ishiura (1994)
developed a device that can screen thousands of colonies with the help of a charge-coupled device
(CCD) camera and a photomultiplier tube device. A turntable device that can hold a dozen petri-
plates (each with as many as 1000 widely separated colonies) helped in screening the individual
colonies for the light emission. Circadian profi les of bioluminescence of individual colonies could
be recorded after several days of monitoring with this automated screening. Genetic analysis of
800 insertion mutant strains revealed that none of them was clearly arrhythmic. On the basis of
waveforms of bioluminescence rhythms, the genes were grouped into fi ve classes. Class 1 to class
3 rhythms though possess symmetrical waveforms but peaked in different phases. Strains grouped
in class 4 rhythms are with asymmetric, saw-toothed waveforms whereas those grouped in class
5 show binodal peaks per cycle. These point towards the existence of several pathways of output
from the circadian oscillator in S . elongatus PCC 7942 (Liu et al ., 1995).