Biology Reference
In-Depth Information
of
hetR
in the wild-type cause the formation of Mch-phenotype, it would be interesting to know the
response of an already Mch-phenotype (AMC1286) to the expression of
hetR
on a multicopy plasmid
in it. The introduction of plasmid pWB216S2.4 with Nm resistance marker and a wild-type
hetR
gene
into AMC1286 resulted in the formation of a regular pattern of heterocyst differentiation. This led
them to the conclusion that the mutation in the original S2-45 had been a recessive one in
hetR
gene.
The cloning and sequencing of the
hetR
gene from AMC1285 (the cured strain) revealed a mutation
at position 223 amino acid residue from Arg to Trp due to a C toT transition at 667 nucleotide in the
hetR
gene. Khudyakov and Golden (2004) cloned the mutant gene without its upstream regulator
region and introduced it through a suicide plasmid into wild-type thus enabling it to undergo
homologous recombination and locate itself into
hetR
locus, producing a merodiploid (AMC1287).
The AMC1286 and AMC1287 strains have been shown to be insensitive to the signals of PatS and
HetN because overexpression of
patS
did not cause any inhibition in heterocyst differentiation. The
expression of
patS
gene exclusively in the heterocysts of AMC1286 and AMC1287 as revealed by the
use of
gfp
as reporter gene suggests that the mutant HetR
Arg223Trp
protein did not interfere with
patS
expression. Likewise,
hetN
overexpression in AMC1287 failed to suppress heterocyst differentiation
in nitrogen-free medium. On top of all the above, Khudyakov and Golden (2004) generated a mutant
AMC1288 by the overexpression of
hetR
Arg223Trp
in
hetR
Arg223Trp
background (in AMC1286). After
nitrogen step-down, AMC1288 differentiated all its vegetative cells in to heterocysts thus forming
100% heterocysts where signalling mechanism of
hetR
to and from
patS
and
hetN
was completely
lost. Thus the above studies tell us that HetR is the master regulator of heterocyst differentiation
and has different functions that can be separated by mutation (Khudyakov and Golden, 2004). It
autoregulates its own levels, it induces the expression of
ntcA
, it activates the transcription of early
genes and sends and receives signals to and from PatS and HetN.
In view of the interdependence of
ntcA
and
hetR
on the expression of one another during
heterocyst differentiation in
Anabaena
sp. strain PCC 7120 (Muro-Pastor
et al
., 2002), the effects of
overexpression of
ntcA
were examined by transferring
ntcA
gene into the wild-type
Anabaena
sp.
strain PCC 7120 and its
hetR
mutant (isolated by Buikema and Haselorn, 1991b). The two new strains
generated have been designated as CSEL1 and CSEL2, respectively and constitutive overexpression
of
ntcA
was found in the fi laments grown in ammonium and subjected to nitrogen step-down. The
content of NtcA protein in strains CSEL1 and CSEL2 was at least 25 times the amount of NtcA protein
in the wild-type grown on ammonium medium. When CSEL1 was subjected to nitrogen step-down,
heterocyst differentiation occurred with the expression of NtcA-dependent genes such as
devBCA
operon, or
coxII
operon and
xisA
gene (for excision of 11 kb intervening fragment in
nifD
) but the
organism could not grow in nitrogen-defi cient medium. On the other hand, CSEL2 strain exhibited the
expression of
devBCA
operon and excision of 11 kb element even in presence of ammonium medium.
These results thus emphasize that some of the NtcA-dependent genes can be expressed despite the
absence of
hetR
gene (Olmedo-Verd
et al
., 2005). In this connection, it would be appropriate to mention
here that HetR induces the expression of six regulatory genes in response to growth in nitrogen-
defi cient medium. These are
hetP
(Fernandez-Pinas
et
al
., 1994),
hetC
(Khudyakov and Wolk, 1997),
devA
(Cai and Wolk, 1997),
ntcA
(Muro-Pastor
et al
., 2002),
hetR
(Huang
et al
., 2004) and
patS
(Huang
et al
., 2004). The mutual regulation of
ntcA
and
hetR
during heterocyst differentiation required the
presence of two similar PP2C-type protein phosphatases. PrpJ encoded by
prpJ
(
all1731
) described
by Jang
et al
. (2007) constitutes a new control point for heterocyst maturation in
Anabaena
sp. strain
PCC 7120, since a disruptant mutant (S20) of
prpJ
failed to develop the glycolipid laminated layer.
Jang
et al
. (2009) identifi ed another ORF (
all2470
) and redesignated the two ORFs
all1731
and
all2470
as
prpJ1
and
prpJ2
, respectively and their corresponding gene products PrpJ1 and PrpJ2 together are
