STRESS RESPONSES - Cyanobacteria - page 623

weight in case of A . halophila (Yancey et al ., 1982) and up to 2.5 M in case of E . halochloris (Trüper and
Galinski, 1990). Transformants of E . coli with the methyltransferase genes of E . halochloris produced
glycine betaine and exhibited improved salt tolerance (Nyossöla et al ., 2000). Homologues of gene
sequences of GSMT and SDMT of E. halochloris have been found in the halophilic cyanobacterium
A . halophytica but with differing substrate specifi city in case of the latter enzyme. Characaterization of
N-methyltransferases from A . halophytica (ApGSMT and ApDMT) revealed a three step methylation
reactions from glycine to glycine betaine similar to those described in the two distantly related
eubacterial species. The accumulation of glycine betaine up to 2 M concentrations in the cells did not
result in a feed back inhibition of the biosynthetic pathway. The rate of synthesis of these enzymes in
A . halophytica cells linearly increased with an increase of external concentration of sodium chloride.
The binding of methyl acceptors on to these enzymes has been identifi ed due to the amino acids Arg-
169 in ApGSMT and Pro-171 and Met-172 in ApDMT. A change in the amino acids from Arg-169 to
Lys or Glu in ApGSMT and Pro-171 to Gln and/or Met-172 to Arg in ApDMT signifi cantly decreased
V max and increased K m for methyl acceptors (glycine, sarcosine and dimethylglycine). Transformants
of E . coli cells with the two gene sequences for ApGSMT and ApDMT exhibited increased levels of
accumulation of glycine betaine (Waditee et al ., 2003). Gene sequences of ApGSMT and ApDMT were
ligated to E . coli bet -cluster (choline dehydrogenase and betaine aldehyde dehydrogenase) along
with plasmid pUC303-Bm and used to transform freshwater S. elongatus PCC 7942 cells. The growth
performance of the wild-type and the transformants under control conditions was similar but under
salt stress (0.4 M NaCl) only transformants with ApGSMT and ApDMT genes exhibited growth but
the wild-type and control cultures with bet -cluster could not grow. Likewise, transformants with
the potentiality to accumulate glycine betaine could grow in higher concentrations of NaCl (0.5 M
and 0.6 M) as well as in sea water from Mikaway Bay (of the Aichi Prefecture in Japan) with salinity
units 30.4. The performance of the transformants was found to be better than Synechococcus cells
with ApNhaP1gene both in 0.6 M NaCl medium and that of sea water. The accumulation of the
protein levels of ApGSMT and ApDMT in the transformants of Synechococcus increased with the
increasing levels of salinity and corresponded with the levels observed in case of A . halophytica cells.
This is suggestive of the fact that the promoters of these genes from A . halophytica were recognized
in Synechococcus cells. The levels of glycine betaine synthesized in the transformants of Synechococcus
with ApGSMT and ApDMT genes was always higher (~5 fold higher in 0.5 mM NaCl) than those
observed in case of Synechococcus cells with E . coli bet -gene cluster (at 0.3 M NaCl) (Waditee et al .,
2005). Since glycine is the precursor for the three step methylation pathway higher internal levels of
glycine should be available to the organism to produce suffi cient levels of glycine betaine under salt
stress. As serine and glycine are readily interconvertible by serine hydroxymethyltransferase any
attempt to enhance the levels of serine will contribute to the enhancement in the levels of glycine.
Both serine and glycine constitute important intermediates in the photorespiratory pathway and
these are recycled as 3-phosphoglycerate (3-PGA) to the Calvin Cycle. Serine is derived through the
phosphorylation pathway from 3-PGA through the enzyme D-3-phosphoglycerate dehydrogenase
(PGDH, EC 1.1.1.95) that constitutes the fi rst step in this pathway. The identifi cation of a single copy
of the the gene encoding PGDH in the genome of A . halophytica (as revealed by shotgun cloning)
led to a study of metabolic engineering of betaine accumulation in microbes and plants (Waditee et
al ., 2007). The expression of ApPGDH in E . coli led to the production of enhanced levels of glycine
betaine both from serine by choline oxidative pathway as well as glycine via glycine methylation
and contributed to the increased salt tolerance. The levels of glycine betaine in the cells of E . coli
overexpressing the genes ApPGDH, ApGSMT and ApDMT were about 1.2-1.8 fold higher than those
cells expressing ApGSMT and ApDMT only. The levels of serine and glycine likewise were found