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Disruption of sscR Encoding Butyrolactone Autoregulator

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Disruption of sscR Encoding Butyrolactone Autoregulator Powered By Docstoc
					Folia Microbiol. 53 (2), 115–124 (2008)                                          http://www.biomed.cas.cz/mbu/folia/




Disruption of sscR Encoding a γ-Butyrolactone
Autoregulator Receptor
in Streptomyces scabies NBRC 12914
Affects Production of Secondary Metabolites
S. KITANIa *, M. HOSHIKAa *, T. NIHIRAa,b **
aInternational Center for Biotechnology, Osaka University, Suita, Osaka 565-0871, Japan
bMU–OU Collaborative Research Center for Bioscience and Biotechnology, Faculty of Science, Mahidol University,
 10400 Bangkok, Thailand

                                                                                                          Received 24 July 2007
                                                                                              Revised version 13 November 2007



ABSTRACT. We report the cloning and sequence analysis of a γ-butyrolactone autoregulator regulatory
island that includes an sscR gene encoding the γ-butyrolactone autoregulator receptor from Streptomyces
scabies NBRC 12914, a plant pathogenic strain. γ-Butyrolactone autoregulators trigger secondary metabo-
lism, and sometimes morphological differentiation in the Gram-positive genus Streptomyces through binding
to a specific autoregulator receptor. This gene cluster showed close similarity to other regulatory islands of
Streptomyces origin that are responsible for the control of secondary metabolism. The recombinant SscR pro-
tein expressed in Escherichia coli prefers a γ-butyrolactone autoregulator containing a long C-2 side chain
and β-hydroxyl group at the C-6 position. An inactivation experiment confirmed that this γ-butyrolactone
autoregulator receptor was involved in secondary metabolism but had no effects on the morphological diffe-
rentiation. In the sscR-deleted mutant, the binding activity of the γ-butyrolactone autoregulator was comple-
tely abolished, suggesting that its primary role is to detect the presence of an autoregulator in the environment.
HPLC analysis of the culture broth showed that some peaks disappeared and new peaks that were not pre-
sent in the broth of the wild-type strain appeared.


Abbreviations
ARE       autoregulatory element                                  LB      Luria–Bertani (medium)
DTT       1,4-dithiothreitol                                      ORF     open reading frame
HTH       helix-turn-helix                                        TSB     tryptone soya broth
IPTG      isopropyl β-D-thiogalactopyranoside                     VB      virginiae butanolide

          Members of the genus Streptomyces are soil-dwelling filamentous eubacteria with high G+C geno-
mes that are best known for their ability to produce a large number of biologically active secondary metabo-
lites and to show complex morphological differentiation (Chater and Bibb 1997; Challis and Hopwood 2003).
In this genus, the γ-butyrolactone autoregulators are regarded as microbial hormones that control the pro-
duction of secondary metabolites and/or morphological differentiation (Khokhlov et al. 1967, 1973; Nihira
2003; Takano 2006; Horinouchi 2007). To date, 11 γ-butyrolactone autoregulators have been structurally
characterized, revealing that they have a common 2,3-disubstituted γ-butyrolactone skeleton but differ in the
length, branching and stereochemistry of the acyl side chain at the C-2 position. Their effects are elicited at
nanomolar concentrations via binding to specific cytoplasmic receptor proteins as mediators of the autoregu-
lator signaling. Only a few combinations of an autoregulator and its cognate receptor have been clarified: the
pairing of an A-factor and ArpA, which controls streptomycin production and morphological differentiation
in Streptomyces griseus (Ohnishi et al. 2005); the pairing of VBs and BarA, which controls virginiamycin
production in Streptomyces virginiae (Okamoto et al. 1995; Nakano et al. 1998), the pairing of SCB1 and
ScbR, which controls actinorhodin and undecylprodigiosin production in Streptomyces coelicolor A3(2)
(Takano et al. 2001) and the pairing of IM-2 and FarA, which controls the production of blue pigment,
nucleoside antibiotics and D-cycloserine in Streptomyces lavendulae FRI-5 (Kitani et al. 2001; Waki et al.
1997). In the absence of autoregulators, their cognate receptors recognize and bind to the specific DNA se-
quences called autoregulatory elements (AREs) (Folcher et al. 2001; Bignell et al. 2007), which are located
in the promoter region of target genes, thereby repressing the transcription of the target genes.

 *These authors contributed equally to the work.
**Corresponding author; fax +81 6 6879 7454, e-mail nihira@icb.osaka-u.ac.jp .
116 S. KITANI et al.                                                                                       Vol. 53



          Autoregulators are produced at a specific phase of growth, then reach a threshold concentration in
the cell, bind to the receptor and induce the transcription of target genes as a result of the dissociation of the
receptor–autoregulator complex from AREs, allowing the onset of secondary metabolism and/or morpho-
logical differentiation. On the other hand, a large number of genes encoding an orphan autoregulator re-
ceptor – namely, receptors for which no definite autoregulator has yet been identified – have been found in
the proximity of biosynthetic gene clusters for secondary metabolites (Bate et al. 1999; Mochizuki et al.
2003; Aigle et al. 2005). In all cases, loss-of-function mutation of the orphan receptor genes affected the
production of secondary metabolites, and/or sometimes morphological differentiation, indicating the invol-
vement of autoregulator receptors in these events. Thus, the γ-butyrolactone regulatory systems are thought
to be wide-spread among actinomycetes, including both Streptomyces species and non-Streptomyces species,
such as Kitasatospora setae and Saccharopolyspora erythraea (Choi et al. 2004; Lee et al. 2006).
          Although hundreds of Streptomyces species are known to produce useful secondary metabolites,
only a few Streptomyces species are known to cause plant diseases. Three species, Streptomyces scabies,
S. acidiscabies, and S. turgidscabies, are the causal agents of scab diseases of economically important root
and tuber crops such as potatoes (King et al. 1992; Loria et al. 1997). In the case of potato scab, these strains
penetrate immature potato tubers, resulting in the production of corky lesions on the tuber slice. These les-
ions form large scabby areas, resulting in large financial losses for growers and major technological chal-
lenges for food processors (Loria et al. 2003). The primary pathogenicity determinant is a group of different
phytotoxic 4-nitroindol-3-yl-containing 2,5-dioxopiperazines – thaxtomins, a nitrated dipeptide toxins pro-
duced from the nonribosomal condensation of tryptophan and phenylalanine by a bimodular peptide synthet-
ase, followed by modification with enzymes (Healy et al. 2000, 2002; Kers et al. 2004). These compounds
could be considered as secondary metabolites of the three strains. While the biosynthetic pathway of thax-
tomins has mostly been clarified, it remains obscure how thaxtomins are produced to cause the pathogeni-
city. An understanding of the regulatory mechanism of thaxtomin production would be useful for the ratio-
nal design directed toward the prevention of pathogenicity.
          Here, we isolated a plausible γ-butyrolactone autoregulator-regulatory island that includes a gene
encoding the γ-butyrolactone autoregulator receptor from S. scabies NBRC 12914 and described the proper-
ties of the autoregulator receptor by in vitro and in vivo experiments. The recombinant autoregulator receptor
expressed in Escherichia coli recognizes γ-butyrolactone autoregulators. The phenotypic analysis of the loss-
of-function mutant for the autoregulator receptor demonstrated that the γ-butyrolactone autoregulator recep-
tor of S. scabies NBRC 12914 plays a critical role in secondary metabolism, without having any effects on
morphological differentiation.


MATERIALS AND METHODS

         Bacterial strains, plasmids, and growth conditions. Streptomyces scabies NBRC 12914 was grown
at 30 °C on ISP medium 2 (Becton Dickinson, USA) for solid cultivation, on ISP medium 4 (Becton Dickin-
son) for conjugal transfer of DNA, and in TSB (Oxoid, UK) for isolation of genomic DNA. For sporulation,
S. scabies was cultivated for 7 d at 30 °C on sporulation medium (g/L: maltose 2.5, NZ amine type A 0.5
(Wako Pure Chemical Industries, Japan), Bacto yeast extract 0.25, beef extract 0.25, agar 20; pH 7.3). E. coli
DH5α was routinely employed for DNA cloning and sequencing. The methylation-deficient E. coli strain
ET12567 (dam dcm hsds) harboring pUZ8002 was used for conjugal transfer of DNA from E. coli to Strep-
tomyces (Paget et al. 1999). E. coli strains were grown in LB medium supplemented with appropriate
antibiotics when necessary. E. coli–Streptomyces shuttle vectors pKC1132 and pSET152, which contain
apramycin-resistant gene markers, were used for gene disruption and complementation analysis, respectively
(Bierman et al. 1992). pUC19 was used for construction of a genomic library and DNA sequencing. DNA
manipulations in E. coli and Streptomyces strains were performed according to Sambrook and Russell (2001)
and Kieser et al. (2000), respectively.
         Molecular cloning of sscR and sequence analysis. The degenerate primers AF-L and AR-1 were de-
scribed by Lee et al. (2005), and the AF-V primer (5´-CGC GGA TCC GCS GCS GCS NNN GTS TTC GA-3´)
was also used to clone the helix-turn-helix DNA-binding motif of autoregulator receptors. An internal
segment of sscA was amplified with genomic DNA of S. scabies and the primers XF (5´-CAT GGA TCC GAC
CAC GTS CCS GGS ATG-3´) and XR (5´-CAT GGA TCC CTG GTG SCC SGT SAC SCG SAC-3´). A BamHI
site (underlined) was generated at the 5´ end of each primer for cloning into pUC19. The product was
analyzed by DNA sequencing, and used as a probe for further screening. Partial genomic libraries were con-
structed with size-fractionated SacI fragments (ca. 4.5 kb) or PvuI fragments (ca. 5.0 kb) and pUC19, and
screened by colony hybridization with the 32P-labelled PCR fragment. The DNA sequence of each positive
2008                                            SscR, A γ -BUTYROLACTONE AUTOREGULATOR RECEPTOR              117



plasmid was determined by primer walking at Hitachi High-Tech Science Systems Corp. (Japan). The se-
quence was analyzed with the Genetyx software package (GENETYX Corp., Japan), and protein coding re-
gions were predicted by FramePlot 2.3.2 software (Ishikawa and Hotta 1999). Homology comparisons were
performed with the BLAST program.
          Preparation of cell-free extract and assay of autoregulator-binding activity. Cell-free extract from
E. coli was prepared as follows: A 5.0-kb PvuI fragment was digested with HinfI to recover a 0.7-kb frag-
ment including the entire sscR gene. A 0.7-kb HinfI fragment was treated with T4 DNA polymerase to yield
a blunt end, and cloned into the SmaI site of pUC19 under the lacZ promoter, resulting in pLT102. E. coli
DH5α carrying pLT102 was grown overnight at 37 °C in LB medium containing 50 μg/mL ampicillin. Two
mL of the preculture was inoculated into 200 mL fresh medium in a 500-mL Sakaguchi flask, and the cul-
tivation was continued at 37 °C until the A600 reached 0.5. IPTG was then added to a final concentration of
0.5 mmol/L. Incubation was continued for 3 h at 37 °C. After the cells were harvested, they were washed
with pre-chilled 0.9 % NaCl and collected again. They were resuspended in buffer H (50 mmol/L triethanol-
amine-HCl, pH 7.0, containing 20 % glycerol, 0.3 mol/L KCl, 0.5 mmol/L DTT, and 5 mmol/L 2-sulfanyl-
ethanol) at a concentration of 1 g wet cells per 10 mL buffer, and disrupted by sonication. The cell debris
was removed by centrifugation (17 300 g, 20 min) and cell-free extract was used to assay autoregulator-bind-
ing activity.
          The cell-free extract from Streptomyces strains was prepared as follows: Spores (109) were inocula-
ted into 70 mL of oatmeal broth (20 g oatmeal, pH 7.0) in a 500-mL Sakaguchi flask; after a 4-d of cultivat-
ion (2 Hz, 25 °C) the mycelium was harvested, washed with pre-chilled 0.9 % NaCl, resuspended in buf-
fer H at a concentration of 2 g wet cells per 10 mL buffer, and disrupted by sonication. After centrifugation
to remove cell debris, solid diammonium sulfate was added to the supernatant to 60 % saturation and the
mixture was gently stirred for 1 h at 4 °C. The precipitates were collected by centrifugation (38 900 g, 30 min),
dissolved in buffer H and dialyzed overnight against buffer H. The dialyzed samples were used to assay
autoregulator-binding activity. The activity was measured in the presence of a 3 H-labelled autoregulator
[73 nmol/L 3H-VB-C7 (2.02 TBq/mmol), 100 nmol/L 3H-IM-2-C5 (1.48 TBq/mmol), and 86 nmol/L 3H-SCB1
(1.73 TBq/mmol)] (Choi et al. 2004). The autoregulator-binding assay was performed according to Kim et
al. (1989).
          Targeted disruption of sscR and its complementation. A 3.0-kb HindIII–blunt-ended BglII fragment
of the sscR-upstream region was cloned into the HindIII–SmaI site of pUC19. After this plasmid was clea-
ved by SacI, a 2.2-kb SacI fragment of the sscR-downstream region was inserted, resulting in pLT103.
A 5.2-kb EcoRI–HindIII fragment, recovered from pLT103, was transferred to conjugative plasmid pKC1132
at the EcoRI and HindIII sites, to yield an sscR-disruption vector, pLT104. E. coli ET12567 (pUZ8002)
containing pLT104 was conjugated with S. scabies according to Kieser et al. (2000), except that the ISP4
medium with 10 mmol/L MgCl2 was used in the DNA introduction. Single cross-over exconjugants were
selected on ISP2 medium containing apramycin. After three rounds of nonselective growth on ISP2 medium,
apramycin-sensitive colonies were identified as double cross-over exconjugants and their genotypes were ana-
lyzed by Southern hybridization analysis. The sscR-disrupted strain was designated as S. scabies strain IC51.
For complementation analysis, a 1.0-kb NcoI–SphI fragment containing the entire sscR gene and 0.3-kb
upstream region of sscR was treated with T4 DNA polymerase to give blunt ends, and ligated into the EcoRV
site of the integrative vector pSET152, resulting in pLT105. Plasmid pLT105 was integrated into the chro-
mosomal attB site of S. scabies strain IC51 after conjugal transfer from E. coli, by selecting for apramycin-
resistant exconjugants. Their genotypes were also analyzed by Southern hybridization analysis, to yield an
IC51-complemented strain, S. scabies strain IC52.
          Morphological assessment. For preparation of preculture stocks, spores of S. scabies strains (109)
were inoculated into 100 mL H medium [IFO (Institute for Fermentation, Osaka) medium no. 231 without
agar] in a 500-mL baffled flask, and mycelia were harvested after a 1-d of cultivation (2.34 Hz, 28 °C).
Mycelia were washed with a half volume of H medium, resuspended in a half volume of fresh H medium
and immediately frozen at –80 °C C until use. Preculture of S. scabies strains was grown on ISP medium 2,
ISP medium 4, oatmeal agar, IFO medium no. 231, sporulation medium, MS agar, minimal medium agar
containing 1 % (W/V) glucose as carbon source, R2 agar, R5 agar, TSB agar, modified SMMS agar as de-
scribed by Takano et al. (2001), and oat-bran agar (40 g/L oat bran, 15 g/L agar, pH 7), and were cultivated
for 7 d at 30 °C.
          HPLC analysis of culture broth. Aliquots of the culture supernatant after 3 d of cultivation were col-
lected using oatmeal broth as described above. HPLC analysis of the supernatants was carried out on a CAPCELL
PAK C18 UG80 reverse-phase analytical column (particle size 5 μm, 4.6 × 250 mm; Shiseido Co., Japan).
The column was equilibrated with 5 % acetonitrile and developed with the following programs (0–60 min –
118 S. KITANI et al.                                                                                                                                                                                                      Vol. 53



a linear gradient from 5 to 100 % acetonitrile, 60–80 min – constant 100 % acetonitrile) at a flow rate of
1 mL/min with UV detection at 210 nm.
         Nucleotide sequence accession number. The nucleotide sequence reported in this paper has been
submitted to the DDBJ data bank as accession no. AB304916.


RESULTS AND DISCUSSION

         Cloning and sequence analysis of sscR and its flanking region. The HTH DNA-binding motif of
γ-butyrolactone autoregulator receptors is known to be highly conserved. With primers (AF-L/V, and AR-1)
designed for this region, we have previously succeeded in cloning internal segments of autoregulator recep-
tor genes, such as sngR from Streptomyces natalensis (Lee et al. 2005) and scaR from Streptomyces clavuli-
gerus (Kim et al. 2004). However, initial attempts with these primers failed to amplify any specific products
of the autoregulator receptor gene from S. scabies, suggesting that the HTH part of the S. scabies receptor
may differ from those of known receptors. Thus, we focused on the fact that genes encoding the autoregula-
tor receptor and the AfsA family protein are occasionally present in close proximity, such as in the case of
scbR and scbA from S. coelicolor A3(2), barA and barX from S. virginiae, and farA and farX from S. laven-
dulae FRI-5. During this work, Kato et al. (2007) clarified that AfsA of S. griseus functions as the key enzyme




                                                                                  (BAF93891) from S. scabies. B: ArpA (BAA36282) from S. griseus, BarA (BAA06981) from S. virginiae, FarA (BAA21859) from
                                                                                  Completely conserved amino acids are shown in bold letters and marked with asterisks, strongly conserved amino acids with colons and
                                                                                  Fig. 1. ClustalW alignment of the amino acid sequences of the AfsA family proteins (A) and the γ-butyrolactone autoregulator receptors (B).


                                                                                  numbers indicate amino acid positions within each protein. A: AfsA (BAA32134) from S. griseus, BarX (BAA23611) from S. virginiae, FarX




                                                                                  S. fradiae, ScaR/Brp (BAC66444/CAH55691) from S. clavuligerus, SscR (BAF93892) from S. scabies; the location of the secondary-
                                                                                  partially conserved amino acids with periods. Each arrow represents the region of a designed primer (XF, XR, AF-L/V, and AR-1). The




                                                                                  S. lavendulae FRI-5, ScbR (CAA07628) from S. coelicolor A3(2), SpbR (AAK07686) from S. pristinaespiralis, TylP (AAD40801) from
                                                                                  (BAA21858) from S. lavendulae FRI-5, ScbA (CAA07627) from S. coelicolor A3(2), SngA (AAX97700) from S. natalensis, SscA




                                                                                  structure elements of the DNA-binding motif is shown above the sequence.
2008                                                     SscR, A γ -BUTYROLACTONE AUTOREGULATOR RECEPTOR                         119



for the biosynthesis of γ-butyrolactone autoregulators. We designed two degenerate primers, XF and XR,
based on the C-terminal well-conserved region of the AfsA family proteins (a part of the Pfam03756 do-
main), and a fragment encoding the AfsA family protein was isolated by PCR with the new primers. A 200-bp
PCR product was cloned, and sequence analysis revealed that the fragment encoded the targeted region of
the AfsA family protein with significant homology (Fig. 1A). This PCR-fragment was used as a probe to
screen a S. scabies partial genomic library. Two fragments (a 4.5-kb SacI fragment and a 5.0-kb PvuI frag-
ment) were obtained, and sequence analysis revealed, overall, five complete ORFs (sscD, sscA, sscR, sscB,
sscC) and two incomplete ORFs (sscE and sscF) at the left-most and right-most end, respectively, on the
combined 7.8-kb region (Fig. 2). Each ORF was annotated by comparing the deduced gene products with
proteins in the database; the results are summarized in Table I. In addition to the existence of a gene (sscA)
encoding a homolog of the AfsA family protein, the sscR-product (SscR) showed a significant homology to
a γ-butyrolactone autoregulator receptor (see below), suggesting that this locus could be a γ-butyrolactone
autoregulator-regulatory island.




Fig. 2. Genetic organizations of genes present in the γ-butyrolactone-autoregulator regulatory island in S. scabies NBRC 12914 and
comparison with the virginiamycin regulatory island of S. virginiae (Kawachi et al. 2000). Homologous genes are shown with cross-
hatching (sscR), hatching (sscA), horizontal lines (sscB), and gray (sscF) for comparison. The numbers between the dotted lines are
the percentages of amino acid identity toward the corresponding homologs of S. virginiae.



Table I. Deduced functions of ssc gene products

   Gene       Sizea    Putative function            Protein homology (protein, organism)                 I/Sb    Accession no.

   sscR       209      γ-butyrolactone receptor     BarA, Streptomyces virginiae                        41/58    BAA06981
                          protein
   sscA       311      γ-butyrolactone              NcsR1, Streptomyces carzinostaticus ATCC15944       34/50    AAM78023
                          biosynthesis enzyme
   sscB       257      γ-butyrolactone              SngB, Streptomyces natalensis                       53/66    AAX97701
                          biosynthesis enzyme
   sscC       417      TylR-like transcriptional    ORF54, Streptomyces globisporus                     47/60    AAL06707
                         regulator
   sscD       175      unknown protein              StropDRAFT_4408, Salinispora tropica CNB-440        51/63    ZP_01428642
   sscE      >148c     ABC transporter              SCO6454, Streptomyces coelicolor A3(2)              85/90    CAA22763
                         ATP-binding protein
   sscF      >158c     pseudo γ-butyrolactone       NcsR3, Streptomyces carzinostaticus ATCC15944       42/61    AAM78020
                          receptor protein

aNumbers of amino acids.          b% identity to % similarity.    corf incomplete.
120 S. KITANI et al.                                                                                     Vol. 53



          The sscR-product (SscR) showed significant similarity to γ-butyrolactone autoregulator receptors of
streptomycetes. A region of the HTH DNA-binding motif of SscR corresponding to the primer AF-L/V was
slightly different from other autoregulator receptors, which explained why the initial PCR was unsuccessful
(Fig. 1B). Based on the actual ability to bind autoregulators, the γ-butyrolactone autoregulator receptors and
their close homologs can be classified into two groups: a group of real autoregulator receptors whose bind-
ing activity was verified biochemically, and a group of pseudoreceptors which seem to have no clear binding
activity (Matsuno et al. 2004). The two groups can be distinguished easily by their pI values: the real recep-
tor proteins show acidic to neutral pI values, while the members of the pseudoreceptors show very basic pI
values. Because SscR has a pI value of 6.0, it should belong to the group of real autoregulator receptors.
Moreover, the SscR protein contains the residues Gln-82 and Trp-126, which are well conserved among the
autoregulator receptors and regarded as important residues for the formation of autoregulator-binding pockets
(Natsume et al. 2004), suggesting that the sscR gene encodes the γ-butyrolactone autoregulator receptor of
S. scabies. As one of the unique characteristics of autoregulator receptors, it is known that the transcription
of autoregulator receptor genes is subject to an autoregulatory circuit (transcriptional repression by the auto-
regulator receptor itself, and derepression by the presence of the autoregulator), which serves to sense and
maintain intracellular autoregulator concentrations. In this autoregulatory system, an ARE in the upstream
region of the receptor gene is the key element for binding of the corresponding receptor proteins (Kinoshita et
al. 1997; Kitani et al. 1999; Folcher et al. 2001; Bignell et al. 2007). We found a 28-bp ARE-like sequence
(AAG TAA CAT AGG GAT TAC TAT TTT AAT T) in the 23-bp sequence upstream of the sscR gene, suggest-
ing that transcription of sscR is likely to be autoregulated via the SscR protein.
          The sscA product shows distinct similarity to the AfsA family proteins. AfsA, a representative AfsA
family protein, functions as a principal enzyme for the A-factor biosynthesis, whereas the function of BarX
of S. virginiae and ScbA of S. coelicolor A3(2) has been controversial (Kawachi et al. 2000). In silico analy-
sis of AfsA indicates the presence of two Pfam03756 domains that form an intramolecular dimer-like struc-
ture, and that were estimated to be essential for A-factor biosynthesis. Although the SscA protein also pos-
sesses two Pfam03756 domains in both the N-terminus (position 15 to 95) and the C-terminus (position 178
to 262), its function in the autoregulator biosynthesis remains to be elucidated. The sscB gene encodes a pro-
tein exhibiting high sequence similarity to proteins in the short chain dehydrogenase superfamily, especially
BarS1 from S. virginiae and SngB from S. natalensis (Shikura et al. 2002; Lee et al. 2005). BarS1 is respon-
sible for the conversion of 6-dehydro-VB-A to VB-A, which is the last catalytic step in the VB biosynthesis,
and the barS1 and sngB genes are in close proximity to the barA and sngR genes, respectively. The sequence
similarity and the gene arrangement of sscB in this locus suggest the possibility that SscB is involved in the
biosynthesis of the γ-butyrolactone autoregulator. The deduced gene product of the sscC gene is homologous
to TylR-type transcriptional regulators such as AcyB2 from S. thermotolerans and TylR from S. fradiae, which
are known to regulate the biosynthesis of specific secondary metabolites, such as carbomycin and tylosin,
respectively (Bate et al. 1999; Arisawa et al. 1993). The incomplete ORF, sscF, also encodes a protein simi-
lar to a γ-butyrolactone autoregulator receptor. However, it seems that SscF belongs to a group of pseudo-
receptors because the partial amino acid sequence displays a slightly basic pI value of 7.4 while the corres-
ponding region of SscR shows a highly acidic pI value of 5.1.
          Information on biosynthetic gene clusters for secondary metabolites is rapidly accumulating, and
a number of potential autoregulator-dependent regulatory gene clusters have been found in the middle or in
the vicinity of biosynthetic gene clusters of streptomycetes. Among them, the VB-dependent regulatory
island of S. virginiae, which includes genes encoding VB biosynthetic enzymes, has been the most extensi-
vely studied. In S. scabies, close homologs in the VB-dependent regulatory island (sscA to barX, sscR to barA,
sscB to barS1, and sscF to barB) are clustered similarly (Fig. 2), suggesting that S. scabies may employ a simi-
lar hierarchy of autoregulator and regulatory genes.
          A partial sequence of SscE exhibits significant sequence similarity to a family of ABC transporters
specific for dipeptides, oligopeptides, and nickel. The remaining gene, sscD, encodes a protein that shows
high sequence homology to proteins with unknown functions, such as StropDRAFT_4408 from Salinispora
tropica CNB-440. One unique feature of the genes surrounding sscR is that each of the sscA, sscB, and sscC
genes possesses a single TTA codon. Although this TTA codon would presumably be recognized by a deve-
lopmentally important tRNA encoded by bldA and has never been found in “house-keeping” genes in strepto-
mycetes (Leskiw et al. 1991; Chater and Chandra 2006), the involvement of the bldA-type gene in the secon-
dary metabolism of S. scabies has not yet been clarified and will require further investigation.
          Autoregulator-binding activity of recombinant SscR. Although γ-butyrolactone autoregulator recep-
tors bind to their cognate autoregulators, these receptors often recognize similar chemical structures that
have minor differences from cognate autoregulators, allowing us to predict the chemical structure of the real
autoregulator. In order to determine what type of γ-butyrolactone autoregulators are recognized by SscR, we
2008                                                    SscR, A γ -BUTYROLACTONE AUTOREGULATOR RECEPTOR                         121



examined the autoregulator-binding ability of recombinant SscR protein expressed in E. coli under the con-
trol of the lacZ promoter (Table II). Representatives of the 3H-labelled autoregulators were tested as ligands:
namely, 3 H-VB-C7 – one of the VB-type autoregulators that possesses α-hydroxyl group at the C-6 posi-
tion and a heptyl side chain at the C-2 position,
3H-IM-2-C – one of the IM-2 type autoregula-             Table II. Autoregulator-binding activity of rSscR
             5
tors that possesses a β-hydroxyl group at the C-6
position and a pentyl side chain at the C-2 posi-                          3H-Labeled
                                                                                                Specific binding
tion, and  3H-SCB1 – one of the IM-2 type auto-            Non-labeled                              activitya
                                                           autoregulator autoregulator
regulators that possesses a long and branched side                                            pUC19         pLT102
chain at the C-2 position. Crude cell-free extract
from cells harboring pLT102 showed binding                 SCB1            3H-SCB1               0            5.44
                                                                           3H-VB-C
activity against SCB1 and VB-C7, whereas cell-             VB-C6
                                                                           3H-IM-2-C
                                                                                     7           0            1.64
free extract from cells harboring pUC19 as a nega-         IM-2-C5                     5         0            0.64

tive control showed no binding activity, demon-          apmol/mg protein; average of two independent experiments.
strating clearly that rSscR possessed binding acti-
vity toward the γ-butyrolactone autoregulator. In
view of the ligand specificity, rSscR exhibited higher binding activity toward SCB1 than toward VB-C7, and
no binding activity toward IM-2-C5, suggesting that rSscR may bind an IM-2 type autoregulator with a long
and branched side chain at the C-2 position, although the precise chemical structure of the SscR-ligand
remains to be elucidated.
          Disruption of the sscR gene and phenotypic analysis. In order to clarify the in vivo function of sscR,
a loss-of-function mutant (IC51) was constructed by deleting a 0.6-kb fragment containing a major portion
of the sscR gene (Fig. 3A), as described in the Materials and Methods, resulting in loss of function of SscR




Fig. 3. Disruption of the sscR gene in S. scabies. A: Schematic representation of the strategy for the sscR gene disruption. The gray
arrows represent the entire sscR gene, and the arrowheads indicate the deleted sscR gene (ΔsscR). aac(3)IV shows the apramycin
resistance gene. B: Southern hybridization analysis to confirm the replacement of the sscR gene with ΔsscR and its complementation.
Each genomic DNA was digested with SacII. 1 – genomic DNA of the wild-type strain, 2 – genomic DNA of the ΔsscR strain (IC51),
3 – genomic DNA of the ΔsscR-complemented strain (IC52); the 2.0-kb SacII fragment was used as the probe.
122 S. KITANI et al.                                                                                     Vol. 53



 Table III. Autoregulator-binding activity of the   as a transcriptional regulator. To confirm that the disrup-
 S. scabies wild-type, ΔsscR, and ΔsscR-complemen-  tion of sscR was responsible for the phenotypic differen-
 ted strains
                                                    ces between strain IC51 and the wild-type strain, a 1.0-kb
                                                    NcoI–SphI fragment containing an intact sscR gene and
    Strain                 SCB1-binding activitya
                                                    probable promoter region was re-introduced into the attB
    Wild-type                        0.43
                                                    site of the sscR-disrupted strain IC51, and they were desig-
    ΔsscR                            0.43           nated IC52 (Fig. 3A). The correct genotypes of the result-
    ΔsscR-complemented               0.40           ing strains IC51 and IC52 were confirmed by Southern
                                                    hybridization analysis (Fig. 3B).
 apmol/mg protein; average of two independent expe-       To ascertain whether the functional SscR protein was
  riments.                                          lost in strain IC51, we measured the autoregulator-binding
                                                    activity toward SCB1, because the rSscR protein exhibited
higher binding activity against SCB1 as a ligand. Crude cell-free extract from the wild-type strain showed
apparent SCB1-binding activity (0.43 pmol/mg protein), whereas no binding activity was detected with crude
cell-free extract from strain IC51. The binding activity was restored to the wild-type level by reintroduction
of the sscR gene (Table III). The loss
of the SCB1-binding activity in the
disruptant demonstrated that the func-
tional SscR protein is expressed in
the wild-type strain and plays an im-
portant role in the detection of the
autoregulator in the environment.
           To investigate whether sscR
might participate in the morphologi-
cal differentiation of S. scabies, we
carefully compared morphological
characteristics between the wild-type
strain and strain IC51 on a wide
range of solid media. Because no re-
duction of spore numbers and no dif-
ferences in the process of spore
formation were detected (data not
shown), it was concluded that sscR is
not involved in the morphological
differentiation. Next, to determine the
effects on secondary metabolism,
HPLC profiles of culture broth were
compared between the wild-type
and the sscR-disruptant (strain IC51)
(Fig. 4). The large peak at the elution
time of 19.9 min (Fig. 4; circles)
characteristic for the culture broth of
the wild-type strain was missing in
strain IC51, and new peaks appeared




Fig. 4. HPLC analysis of culture broths of the
wild-type (A), ΔsscR (B), and ΔsscR-comple-
mented (C) strains. For clarity, only portions
of the HPLC profiles are shown. Symbols (cir-
cles, asterisk and diamond) indicate the posi-
tion of different peaks between the wild-type,
ΔsscR-complemented and ΔsscR strains.
2008                                                      SscR, A γ -BUTYROLACTONE AUTOREGULATOR RECEPTOR                          123



at the elution times of 7.8 min and 18.7 min (Fig. 4; asterisk and diamond). Moreover, the observed change
in the HPLC profile was completely restored to the wild-type profile when the intact sscR was reintroduced
(strain IC52) (Fig. 4), indicating clearly that these changes in the product profile were due to the loss of the
functional sscR gene, which is involved in the control of secondary metabolism.
          S. scabies is known to cause scab disease of economically important crops, especially potatoes,
through its production of thaxtomin A and its derivatives, which elicit major symptoms on host tissue. How-
ever, HPLC analysis showed no peak corresponding to thaxtomin A in the culture broth of the wild-type
strain with several kinds of liquid media. Furthermore, we failed to find any visible symptoms on potatoes
using a tuber slice pathogenicity test. As S. scabies NBRC 12914 should originally have exhibited patho-
genicity on potatoes, it seemed that the reproductive ability of thaxtomins became too low or was completely
lost at some point between the deposit procedure and the culture collection. During this study, genomic se-
quence data became available for S. scabies strain 87.22 (http://www.sanger.ac.uk/Projects/
S_scabies/ ), which produces thaxtomins and has been well studied among the plant pathogenic Strepto-
myces species, and our search for homologous sequences revealed that S. scabies strain 87.22 also has
a plausible γ-butyrolactone autoregulator receptor (35 % homology with SscR). In addition, strain 87.22 pos-
sessed four genes, the products of which show similarity to AfsA, BarS1, and BarB (repressor of virginia-
mycin production in S. virginiae), and TylR (activator of tylosin production in S. fradiae), in the proximal
region of the receptor homologue gene. This information indicates that the sscR-regulatory island is well
conserved among S. scabies strains and may participate in the control of secondary metabolism. We are now
on the way to clarifying the involvement of the γ-butyrolactone autoregulator-regulatory cascade in the patho-
genicity of S. scabies strain 87.22.

           We thank Dr. H. Kinoshita for his helpful suggestions and Dr. R.R. King (Agriculture Canada) for providing thaxtomin A.
This study was supported in part by a Grant-in-Aid for Young Scientists of the Ministry of Education, Culture, Sports, Science and
Technology of Japan to the first author and by the “Joint Program in the Field of Biotechnology” under Japan Science and Technology
Agency, National Research Council of Thailand and National Science and Technology Development Agency of Thailand to the corres-
ponding author.

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