Biology Reference
In-Depth Information
Thiel and Pratte (2001) showed that MM3, an
ntcA
mutant of
A
.
variabilis
ATCC 29413, failed
to grow in nitrate-enriched medium as well as differentiate heterocysts after a nitrogen step-down.
Su
et al
. (2005) employed a bioinformatics approach to fi nd out NtcA-binding sites in the genes of a
genome and concurrently in the orthologues of the other genomes. As
glnA
is known to be transcribed
from different tsps, it is of interest to know which one of the four transcripts is synthesized in the
proheterocysts and heterocysts of
Anabaena
sp. strain PCC 7120. By the use of
lacZ
fusion constructs
of the four tsps in the upstream region of the promoter of
glnA
, Valladares
et al
. (2004) showed that
glnA
is expressed from the P1 region in the proheterocysts and heterocysts of
Anabaena
sp. strain PCC
7120 after a nitrogen step-down. The mutation of NtcA-binding site in the P1 region and the binding
of NtcA to the P1 site appear to govern the expression of
glnA
in vegetative cells and heterocysts.
A comparison of the sequenced genomes (
Gloeobacter violaceus
PCC 7421,
Anabaena
sp. strain PCC
7120,
P
.
marinus
CCMP1375,
P
.
marinus
MED4,
P
.
marinus
MIT9313,
Synechococcus elongatus
PCC
6301,
Synechococcus
sp. strain WH8102,
Synechocystis
sp. strain PCC 6803 and
Thermosynechococcus
elongatus
BP-1) revealed that NtcA-regulated promoters are found not only for genes of nitrogen
assimilation but they are coupled to genes involved in various stages of photosynthesis thus
highlighting the importance of these genes as regulatory points in the two major metabolic processes.
Besides regulating the expression of genes of nitrogen and carbon metabolism, NtcA also is reported
to control the expression of genes related to the maintenance of iron homeostasis. As cyanobacteria
require iron for the synthesis of ferredoxins and other haem-related proteins, the expression of
furA
during heterocyst differentiation has received attention. López-Gomollón
et al
. (2007) found that the
levels of
furA
transcripts and the FurA protein increased in proheterocysts and mature heterocysts as
a result of nitrogen shift-down in the wild-type strain of
Anabaena
sp. strain PCC 7120. The expression
of
furA
in
ntcA
mutant of
Anabaena
sp. strain PCC 7120 was depressed and consequently the levels
of mRNA of
furA
and FurA protein decreased. These differences were attributable to the binding of
NtcA to the promoter region of
furA
in the wild-type and the lack of it in the
ntcA
mutant.
ii) hetR
:
Buikema and Haselkorn (1991a) characterized 140 Fox
-
mutants of
Anabaena
sp. strain PCC
7120 isolated after diethyl sulphate mutagenesis followed by penicillin enrichment. Out of these,
seven mutants exhibited morphologically abnormal phenotypes. Mutant 216, originally suspected to
be Fox
-
and Het
-
phenotype, was unable to fi x nitrogen even under anaerobic conditions and it turned
out to be Fix
-
and Het
-
while the rest of the six had Fox
-
and Het
-
phenotype. A cosmid library of the
genome of
Anabena
sp. strain PCC 7120 was put to use for complementation and a cosmid with 9.5
kb region of the genome could complement mutant 216 as well as two other mutants. Subcloning of
this fragment pinpointed a 2.4 kb region specifi c for complementing mutant 216 and sequencing of
this fragment revealed a single ORF designated by them as
hetR
(Buikema and Haselkorn, 19991b),
a regulatory gene that is essential and controls heterocyst differentiation. The gene product, HetR
(consisting of 299-amino acid residues) is a DNA-binding protein (Huang
et al
., 2004) but it is a
unique protease whose Ser179 is converted to Asn in mutant 216. Directed mutagenesis of
hetR
in
the wild-type
Anabaena
sp. strain PCC 7120 was achieved by transferring
hetR
interrupted gene
[with a neomycin (Nm) resistance cassette carried by plasmid pWB216S2.4] sequence through
conjugation with
E
.
coli
MC1061. This has led to the isolation of single (Nm-resistant) and double
(Nm- and sucrose-resistant, 5%) recombinants with a phenotype similar to that of mutant 216. The
presence of
hetR
in extra copies in the wild-type resulted in multiple-contiguous heterocyst (Mch)
phenotype with enhanced (10-25%) heterocyst frequency than the wild-type (5 to 10%) in a nitrogen
defi cient medium and 5% of cells even differentiated into heterocyst-like cells under repressing
(nitrate-enriched) conditions. But these appeared to be non-functional. The expression of
hetR
at
