ABBS 2008,40(10): SarA influences the sporulation and secondary metabolism in Streptomyces coelicolor M145 -

Original Paper

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 Zhao^1,2,3*, and Xiaoming Ding¹*

State Key Laboratory of

Genetic Engineering, Department of Microbiology and Microbial Engineering,

School of Life Sciences, Fudan University, Shanghai 200433, China

Shanghai-MOST Key

Laboratory of Health and Disease Genomics, Chinese National Human Genome Centre

at Shanghai, Shanghai 201203, China

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 conditionsThe 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 complementationInsertional 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 curvesAct 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. Act and Red were assayed as previously described [21]. The bacteria