CIRCADIAN RHYTHMS - Cyanobacteria - page 307

In order to determine the number of genes that control the circadian clock in S . elongatus PCC
7942, Liu et al . (1995) made several fusions of the regulatory regions of known genes to luxAB . All
such resulting mutants showed typical bioluminescence characteristic of the S . elongatus PCC 7942
but at lower amplitude. This confi rmed that all the known genes are under the regulatory control
of circadian clock. In an alternative approach, to assess the percentage of genes under circadian
control throughout the genome, Liu et al . (1995) performed a random fusion of a promoterless luxAB
gene set throughout the chromosome. This was feasible because luxAB can undergo homologous
recombination between a segment on the chromosome and a cloned copy of the same region on
a non-replicating strand. As a result of the single crossover event, the transforming plasmid gets
incorporated into the chromosome at the site of recombination. Rhythmic transcription can easily be
measured by following the luminescence (Andersson et al ., 2000; Mackey et al ., 2007). Most of the S .
elongatus promoters drive luxAB with a bioluminescence with peaks at dusk (in a light-dark cycle) or
subjective dusk (corresponding to dusk in continuous light) or a trough at dawn or subjective dawn.
As a matter of convention, subjective dusk is considered as incubation period of 12 h, 36 h and so on
in LL after a 12-h pulse of darkness. Likewise, subjective dawn is around 24 h, 48 h and so on in LL
after a 12-h pulse of darkness. It has thus been possible to identify dusk peaking genes ( psbAI , psbAII ,
psbAIII , KaiA , KaiBC and rmA ) in S . elongatus PCC 7942 with Class I expression patterns (Liu et al .,
1995; Ishiura et al ., 1998). Plasmid pBR322 carries a kanamycin resistance marker and thus it is easy to
score recombinants for kanamycin resistance. Secondly, due to the presence of bom site, it can easily
be mobilized in a conjugational event from E . coli to S . elongatus PCC 7942. This was considered to be
the most effi cient way of transfer rather than by transformation (Tsinoremas et al ., 1994). Nearly 80
per cent of the recombinant clones showed a circadian rhythm with characteristic bioluminescence,
divisible into two classes. Strains that showed peaks during LL have been included in Class I. In
contrast, those strains with peaks of bioluminescence at other times quite opposite that of AMC149
have been grouped in Class II. AMC287 is a strain in which luxAB gene set has been integrated into
purF region governing a key enzyme in purine biosynthesis. It was also found that purF region lies
immediately adjacent to purL region in S . elongatus PCC 7942 and is co-transcribed with purL region,
the difference being the bioluminescence pattern resembled that of Class 1 group of strains. Liu et
al . (1996) found that a second promoter that is responsible for Class 2 type of bioluminescence of
purF appears to lie within the C-terminal coding region of purL . A Tn5- luxAB transposon construct
developed by Wolk et al . (1991) has been used for creating random luxAB fusions throughout
S. elongatus PCC 7942 genome. A new circadian class 2 gene designated as opcA has been identifi ed
on the basis of transposition of a modifi ed Tn5 element conferring kanamycin resistance into S .
elongatus by triparental conjugation. Although the biochemical properties of the gene product of opcA
are not known, it is very much required for the functioning of glucose-6-phosphate dehydrogenase
(encoded by Zwf ) and for the oxidative pentose phosphate pathway. A mutant, isolated by the
insertion of Tn5 into Zwf upstream of opcA , was able to transcribe opcA independently of Zwf as a
circadian class 2 gene. However, inactivation of opcA gene affected reductant production at night
and caused a limitation in the supply of substrate for luciferase (Min and Golden, 2000).
The main conclusions drawn from the above studies are that (i) there is a global circadian control
of gene expression in S. elongatus PCC 7942, (ii) there is some individuality in the expression of
genes and this can be directly correlated to temporal separation of biochemical processes, and (iii)
the circadian regulation imposes rhythmicity of transcription via a general mechanism that is not
gene specifi c.
A bacterial luciferase gene set derived from Xenorhabdus luminescens (Xl luxAB ) was used as a
reporter gene and fused with the promoter region of psbA1 gene to identify the expression of psbA1