Biology Reference
In-Depth Information
iii)
Genes for
heterocyst envelope polysaccharides
:
During heterocyst maturation, after the deposition
of Hgl laminated layer, the formation of a new layer of heterocyst envelope polysaccharide layer
(Hep) appears to be a pre-requisite for acquisition of Fox
+
and Fix
+
phenotype. Any defi ciency in the
formation of Hep layer leads to Fox
-
phenotype. The genes involved in the regulation of Hep layer
are classifi ed under
hep
genes. Holland and Wolk (1990) recognized
hetA
gene (2,555 bp) that encodes
a protein (of 601 amino acid residues) necessary for improving the cohesiveness of the heterocyst
envelope polysaccharides. Mutant EF116 of
Anabaena
sp. strain PCC 7120 defective in
hetA
showed
a reduction in the cohesiveness of the polysaccharides. The expression of
hetA
has been found to
be prominent around 7 h after nitrogen step-down. An unexplained but signifi cant feature of this
gene is the occurrence of four identical repeats of the sequence 5'-TTCAAAA-3' situated at the 3'-
end and 12 identical repeats of the sequence 5'-CCCCAAT-3' that extend into 5'-end of a second
ORF located downstream of
hetA
. Such direct repeats were reported earlier in between
petA
and
petC
of
Nostoc
sp. strain PCC 7906 (Kallas
et al
., 1988). The gene identity of
hetA
was shown to be in
fact
hepA
subsequently.
Zhu
et al
. (1998) identifi ed the essential nature of
hepA
gene (
alr2835
) of
Anabaena
sp. strain
PCC 7120 for the deposition of Hep layer. The gene product of
hepA
is a member of the family of
ATP-binding proteins of ATP-binding cassette transporters. The expression of
hepA
in response
to nitrogen deprivation has been reported (Holland and Wolk, 1990; Wolk
et al
., 1993; Ehira
et al
.,
2003). In an attempt to fi nd out the genes that regulate the expression of
hepA
, Zhu
et al
. (2001)
subjected strain DR 1069 (that possesses a chromosomal
hepA
::
luxAB
fusion construct) to Tn5-1058
induced mutagenesis. The inactivation of a gene designated as
hcwA
in this mutant HNL3 resulted
in the down-regulation of
hepA
but the expression of
hepA
was restored to normal levels only after
complementation of the mutant with wild-type
hcwA
gene. The gene product of
hcwA
resembles the
N-acetylmuramoyl-1-alanine amidase that has degradative effects on the cell wall. It was suggested
by Zhu
et al
. (2001) that degradation of peptidoglycan wall layer is a prerequisite for heterocyst
differentiation. Another gene,
hepC
(
alr2834
) is localized upstream of
hepA
. The predicted product of
hepC
is a UDP-galactose-lipid carrier transferase. In the intervening region of
hepC
and
hepA
, DNA
sequences required for induction of
hepA
are localized in between -574 and -440 bp and -340 and
-169 bp relative to the tsp of
hepA
. The regulation of
hepA
by
hepK
(
all4496
) has been found to be
signifi cant for two reasons. The fi rst one is that inactivation of
hepK
resulted in repression of
hepA
and secondly the synthesis of Hep layer also is inhibited. Functionally, HepK is predicted to be a
sensor protein-histidine kinase belonging to two-component regulatory systems and DevR protein
constitutes the response regulator (receiver domain) described earlier by Campbell
et al
. (1996) in
N
.
punctiforme
ATCC 29133. However, the corresponding ortholog of
devR
of
Anabaena
devR
A
, located
1.4 Mb away from
hepK
has been identifi ed in the genome
Anabaena
sp. strain PCC 7120 (Kaneko
et
al
., 2001). Zhou and Wolk (2003) cloned and sequenced
devR
A
(
alr0442
) and conducted
in vitro
studies
on recombinant proteins produced in
E.
coli
BL21(DE3) that have been tagged with a hexahistidine
tag (H
6
) at their N-terminal end. In presence of ATP, autophosphorylation of cytoplasmic portion
of HepK (a truncated portion of HepK with amino acid residues 267 to 575) took place but not of
HepK and in presence of ADP dephosphorylation of phosphorylated cytoplasmic part of HepK
took place. The phosphoryl group from cytoplasmic part of HepK has been readily transferred to
the receiver domains H
6
DevR or H
6
DevR
A
. The phosphorylation site on HepK has been found to be
His348 and the receiver of phosphoryl group on DevR
A
has been detected to be Asp53 residue. Site-
directed mutagenesis of His348 to Ala348 of HepK resulted in a loss of
in vitro
autophosphorylation
activity and caused the production of heterocysts without the Hep layer but with HGL laminated
layer. So this constitutes the fi rst two-component regulatory system known to regulate heterocyst
