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Acta Biochim Biophys Sin 2008, 40: 877-882

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 struc�turally� 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 pleio�tropic control of anti�biotic� 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 anti�biotics 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 �C. 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 �C for 30 s, 60 �C for 30 s and 72 �C 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 trans�membrane 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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