Original Paper |
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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2008.00466.x |
SarA influences the sporulation and secondary metabolism in Streptomyces
coelicolor M145
Xijun Ou1, Bo Zhang1, Lin Zhang1, Kai Dong1, Chun Liu1, Guoping Zhao1,2,3*, and Xiaoming Ding1*
1
State Key Laboratory of
Genetic Engineering, Department of Microbiology and Microbial Engineering,
School of Life Sciences, Fudan University, Shanghai 200433, China
2
Shanghai-MOST Key
Laboratory of Health and Disease Genomics, Chinese National Human Genome Centre
at Shanghai, Shanghai 201203, China
3
Laboratory of Molecular
Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes
for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China
Received: June 26,
2008
Accepted: August 6, 2008
This work was
supported by a grant from the National Natural Science Foundation of China (No.
30600009)
*Corresponding
authors:
Guoping Zhao: Tel,
86-21-50801919; Fax, 86-21-50801922; E-mail, [email protected]
Xiaoming Ding: Tel,
86-21-65643616; Fax, 86-21-65650149; E-mail, [email protected]
The filamentous bacteria Streptomyces
exhibit a complex life cycle involving morphological differentiation and secondary
metabolism. A putative membrane protein gene sarA (sco4069),
sporulation and antibiotic production related gene A, was partially
characterized in Streptomyces coelicolor M145. The gene product had no
characterized functional domains and was highly conserved in Streptomyces.
Compared with the wild-type M145, the sarA mutant accelerated sporulation
and dramatically decreased the production of actinorhodin and
undecylprodigiosin. Reverse transcription-polymerase chain reaction analysis
showed that SarA influenced antibiotic production by controlling the
abundance of actII-orf4 and redZ messenger RNA.
Keywords Streptomyces
coelicolor; sporulation; antibiotic production; sarA
The life cycle of streptomycetes is remarkably intriguing for a
prokaryote, as it encompasses a series of structurally differentiated states
and physiological changes [1]. Colonies germinate from spores and continue to
grow by forming a mat of branched hyphae called substrate mycelium. In response
to some signals, including A factor, ppGpp, SapB, SapT and chaplins, the
substrate hyphae cease and aerial hyphae begin to form [2–5]. These aerial
hyphae then undergo synchronous septation leading to the formation of
unigenomic spores [6]. Coinciding with the onset of aerial mycelium formation
is the production of secondary metabolites, which have many important
commercial medical applications, such as antibacterial, antitumor and
immunosuppression activities [7].
Sporulation of Streptomyces coelicolor (S. coelicolor),
a well-studied model for the actinomycetes genus, is probably affected by
metabolite, morphological, homeostatic and stress-related checkpoints. Sigma
factors and the regulators encoded by the whi and bld genes are
known to be implicated [8]. Secondary metabolism is typically affected by the
nature and levels of the carbon and nitrogen source as well as by the
availability of phosphate and small signaling molecules, such as ppGpp and
r-butyrolactone [9]. It has also been shown that certain regulators are
involved in the pleiotropic control of antibiotic production including AbsA1/A2,
AfsR/K, PhoR/P and regulators encoded by bld genes [10–14]. Although
there has been limited understanding of the regulatory mechanism involved in
the production of actinorhodin (Act) and undecylprodigiosin (Red) in S. coelicolor,
it has been established that these antibiotics are regulated directly by the
pathway-specific transcriptional regulators ActII-ORF4, RedD and RedZ [11,15–19].
In this study we characterized a new putative membrane protein,
SarA (SCO4069), which negatively regulates sporulation in S. coelicolor
M145. The sporulation and antibiotic production related gene A (sarA)
mutant decreased the production of Act and Red by influencing the
pathway-specific activators ActII-ORF4 and RedZ at the mRNA level.
Materials and Methods
Bacterial strains, plasmids, and growth conditions
The bacterial strains, plasmids and primers used in this study are
listed in Table 1. Escherichia coli (E. coli) DH5a [20] was used
for plasmid propagation. Mannitol Soya flour medium (MS) [21] agar was used to generate spores
and for selection of Streptomycete exoconjugants. YBP medium agar (2 g
yeast extract, 2 g beef extract, 4 g Bacto-peptone, 1 g MgSO4, 5 g NaCl, 15 g agar and 10 g glucose combined with 1 l water) was
used to screen for phenotypes. Yeast extract-malt extract medium (YEME) [21]
was used to cultivate mycelia to prepare genomic DNA and supplemented
minimal medium solid (SMMS) liquid medium [21] was used to prepare RNA.
The conjugation of E. coli ET12567/pUZ8002 with Streptomycetes was
performed as described [21]. Antibiotics were added, whenever necessary, at
following final concentrations: 50 mg/ml ampicillin, 33 mg/ml chloramphenicol, 30 mg/ml kanamycin
and 25 mg/ml thiostrepton.
Mutagenesis of S. coelicolor M145 and gene complementation
Insertional mutagenesis of M145 was conducted by in vivo
transposition with plasmid pDZY101, a derivative transponson from IS204 which
was first identified in Nocardia asteroids YP21 [22], through
conjugation from E. coli ET12567/pUZ8002 to S. coelicolor M145.
The exconjugants were selected by growth on MS media flooded with 30 mg/ml kanamycin.
The pDZY101 carrying the replication region of pUC serial plasmids is capable
of causing highly efficient random and stable mutagenesis with a single copy
number in S. coelicolor M145. The chromosomal locations of the pDZY101
insertions were determined by sequencing the insertion plasmid flanking DNA
through plasmid rescue.
sarA and its upstream DNA fragment was
amplified by PCR using primer sets of Oxj138/139 (Table 1). It was then
inserted into the SacI/HindIII-digested pFDZ16, a Steptomycete/E.
coli shuttle single integrate vector carrying genes encoding thiostrepton,
kanamycin and ampicillin resistance, to give rise to plasmid pFDZ16-sarA for
genetic complementation of sarA mutant K66. The plasmid was conjugated
into the K66 from the donor E. coli ET12567/pUZ8002. The
thiostrepton-resistant Streptomyces exoconjugant was designated as
K66-sarA.
Quantification of antibiotics and assay of growth curves
Act and Red were assayed as previously described [21]. The bacteria
grew in 30 ml SMMS liquid medium and was filtered to separate the supernatant
from the pellet. For Act, KOH was added to the supernatant to a 1 M final concentration,
and was then assayed at an optical density of 640 nm. For Red, the mycelia
pellet was dried under vacuum conditions and extracted with 10 ml methanol
(adjusted to pH 2) overnight at room temperature and the optical density was
measured at 530 nm. Measurements were always taken from triplicate cultures.
Growth curves of the prototype, the mutant K66 and the revertant strain
K66-sarA were determined as described by Kieser et al [21]. Cultivation
was performed by using 25-ml test tubes each containing 3 ml of YBP liquid
medium with the inoculation of 2×107 spores per ml and incubated on a reciprocal shaker (200 rpm) at 30
ºC. Cultures were taken at each time point and weight.
Reverse transcription-polymerase chain reaction analysis
Methods for RNA isolation were performed according to the manual of
Bacterial RNA Kit (Omega, Norcross GA, USA). Reverse transcription (RT) was
performed according to the manual of High fidelity RNA PCR kit (TaKaRa, Otsu
Shiga, Japan). The primers used for RT-PCR are shown in Table 1. PCR
conditions were 94 ºC for 30 s, 60 ºC for 30 s and 72 ºC for 30 s in a total of
26 cycles. For redD, there were 32 cycles. Controls were performed using
the RNA from the parent strain M145 or K66 without RT, and the results were negative.
Results
Identification of sarA in S. coelicolor M145
We used an in vivo transposition system to generate a
collection of mutants with abnormalities in aerial mycelium differentiation
and secondary metabolite production by conjugation plasmid pDZY101 from E.
coli ET12567/pUZ8002 to S. coelicolor M145. Insertion mutant K66
showed accelerated sporulation and decreased antibiotic production. By
sequencing the DNA flanking the pDZY101 insertion in K66, we identified a gene,
sarA (sco4069), that was disrupted in K66 [Fig. 1(A)]. The
sarA gene in S. coelicolor encodes a 664 amino acid protein with
a calculated molecular mass of 69,158 Da without any characterized functional
motif except for the transmembrane domain. The proteins SAV4148 in Streptomyces
avermitilis MA-4680, SGR3860 in Streptomyces griseus NBRC 13350 and
SCAB47711 in Streptomyces scabies 87.22 have, respectively, a 77%, 68%
and 66% similarity to the SarA protein [Fig. 1(B)]. BLAST results
revealed that members of this type of protein are highly conserved and have
only been identified in Streptomcyes thus far. Genes located immediately
upstream and downstream of sarA are purD (or sco4068), sco4070
and purC (or sco4071) in M145. Homologs of these genes are
arranged in the same order in Streptomyces avermitilis, Streptomyces
griseus and Streptomyces scabies. Because purD– and purC-encoded
proteins participate in the biosynthesis of de novo purine nucleotide,
we wondered if SarA also participated in this metabolic pathway. By testing the
growth of sarA mutant on minimal medium agar, we found that the mutation
of this gene does not cause auxotrophy and the mutant strain could grow well on
this medium without any growth factor (data not shown). This result indicated
that SarA was not essential for the purine nucleotide biosynthesis.
SarA influences the morphogenesis and secondary metabolism in a
divergent way
The morphological phenotype of the sarA mutant was firstly
screened on YBP medium (Fig. 2). The results showed that the sarA
mutant sporulated earlier and better than the M145 strain, while the production
of Act and Red dramatically decreased to a level that was hardly visible from
the bottom of the plates. We also screened the phenotype on YBP with 1%
mannitol instead of glucose and the results were the same (data not shown). To
investigate whether the phenotype of antibiotic production in liquid medium
is the same as that on solid medium, we tested the antibiotic production in SMMS
liquid medium; the experiments showed that sarA mutant’s production of
Act and Red were lower when compared to M145’s [Fig. 3(A)]. The
phenotype was complemented by an integrative plasmid containing only sarA+ with its 0.4 kb upstream probable promoter sequence. The growth
curves of M145, sarA mutant and K66-sarA in liquid YBP medium were
tested, and the results showed that there was no difference in their
respective growth rates [Fig. 3(B)]. These data highlight the fact that
SarA negatively regulates sporulation, though it has a positive influence on
Act and Red production.
SarA regulates the antibiotic production by controlling the
abundance of the ActII-ORF4 and RedZ mRNA
The expression of antibiotic biosynthesis clusters is normally regulated
by pathway-specific activators [17–19]. In S. coelicolor, Act and Red
biosynthesis have been shown to depend on the transcriptional activation of the
Act and Red biosynthesis clusters by ActII-ORF4, RedD and RedZ proteins
respectively. RedD, the direct transcriptional activator for the biosynthesis
Red cluster, is RedZ dependant [11,15]. Down-regulated expression of these
proteins results in the decreased production of Act or Red. The transcription
of actII-orf4, redD and redZ in the sarA mutant K66 were
therefore analyzed by RT-PCR. Total RNA was isolated from two developmental
stages of M145 and K66 grown on SMMS liquid medium cultured for 36 h and 80 h.
As shown in Fig. 4, the transcription of actII-orf4, redD
and redZ decreased markedly in the later stage in K66 compared to that
in M145, suggesting that SarA regulated the Act and Red production by
controlling the mRNA abundance of the ActII-ORF4 and RedZ.
Discussion
In this study sarA (sco4069) in S. coelicolor
was identified by gene disruption as a gene negatively affecting sporulation
but positively influencing the production of Act and Red. SarA belongs to a
putative membrane protein family that has so far been only found in Streptomyces.
Levels of actII-orf4, redZ and redD mRNA decreased
dramatically at a late time point in the sarA mutant, suggesting
exerted either by the activated genes that were regulated by SarA over long
time periods or by the effects on mRNA half-life. Though the disruption of sarA
dramatically decreased the production of antibiotics in S. coelicolor
M145, the sporulation of the strain was accelerated rather than delayed. The
cause of this paradox remains unknown. One possible explanation is that SarA
exists as a membrane protein, senses the extracellular or intracellular
signals, and balances the nutrients and energy between aerial mycelium
morphogenesis and antibiotic production. Since the growth rate of sarA
mutant in liquid culture and aerial mycelium formation on solid medium were not
changed, and this mutant maintained the prototrophic phenotype, it seems that
the effect of SarA on sporulation and antibiotics production of S.
coelicolor M145 is not correlated with primary metabolism.
In conclusion, sarA encodes a putative membrane protein, which
is a representative of a new family of Streptomyces-specific proteins.
The presence of SarA and its homolog exclusively in Streptomyces could
imply that this type of protein plays an important role in controlling the
development of these streptomycetes.
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