86NISR RESEARCH GRANT
2004 Yound Investigator Research Grant

Cloning of rpoZ-dependent Transcriptional Activator Genesof kasT, a Pathway-specific Regulator of Kasugamycin Biosynthesis
Kano KASUGA
Department of Biotechnology, Akita Prefectural University
Introduction
The core enzyme of bacterial RNA polymerase (RNAP)
consists of four subunits with a stoichiometry of a 2bb w
and requires an additional s subunit to form the holoenzyme for promoter-specific initiation of transcription. Of
the five subunits, little has been reported on the biologicalimportance of the subunit w except that a defect in the subunit results in a slow-growth phenotype in Escherichia coli
and that the w subunit of Thermus aquaticus facilitates theassembly of a 2b and b  for formation of the core enzyme.
We have clarified that rpoZ, the gene encoding the subunit w is required for the production of kasugamycin(KSM), an aminoglycoside antibiotic effective against rice
blast disease caused by Pyricularia oryzae, as well as forthe formation of aerial mycelia, an early stage of morphological differentiation, in Streptomyces kasugaensis.1 Indeed, the rpoZ-disrupted mutant produces neither KSM nor
aerial mycelia. Furthermore, transcriptional analysis ofKSM biosynthetic genes revealed that the KSM non-producing phenotype of the rpoZ-defective mutant is due to thelack of transcription of kasT, which encodes KasT, a KSMbiosynthesis specific transcriptional activator. Of particular
relevance is the observation that KasT shows 50% identity
to StrR, a specific transcriptional activator in the streptomycin (SM) biosynthesis.2
As strR transcription is under the control of AdpA, thespecific transcriptional activator, in SM-producing Streptomyces griseus,3 we assumed that kasT might also require anactivator, designated as KasZ, for its transcription and thatrpoZ might be crucial for the kasZ expression as illustratedin Fig. 1. To examine the mechanisms involved in the rpoZdependent KSM biosynthesis, we have established a systemfor cloning kasZ and genes associated with kasT transcription. We present here this cloning system together with several genes of interest subsequently isolated by this system.

the kasT promoter. The reporter gene was generated by fusion of the kasT promoter region (PkasT) to a promoterlessdbfB structural gene that encodes an extradiol dioxygenase
of Terrabacter sp. strain DBF63.4 Subsequently, the gene
cassette was ligated into pSK2162, a vector derived from anS. kasugaensis plasmid, for the formation of pAK557 (Fig.
2). As dbfB is under the control of PkasT , any gene encodinga PkasT -specific transcriptional activator, which is cloned
into the vector, can be identified by the induced DbfB activity that degrades the colorless substance, 2,3-dihydroxybiphenyl (DHB), into a yellow oxidation product (Fig. 3).
To enhance the expression of the genes cloned into theBamHI, HindIII or PstI cloning sites, these sites are locateddownstream of PermE, the strong promoter of an erythromycin resistance gene.
The utility of the pAK557 system was demonstrated byFig. 1. Transcriptional activation of kasT by KasZ and related genes
with reference to rpoZ.

Results and Discussion
Cloning system for kasZ and genes relevant to kasT
expression in S. kasugaensis. As shown in Fig. 1, thereis the possibility that regulators in addition to KasZ are required for kasT transcription. To clone these regulatory
genes, we constructed the cloning vector pAK557, whichharbors a reporter gene that can monitor transcription fromFig. 2.

Cloning vector pAK557.

87transformation of the wild-type S. kasugaensis strain A1R6,
the rpoZ-disrupted S. kasugaensis mutant R6D4 and Streptomyces lividans TK21. Of these transformants, only A1R6
transformants showed the expected yellow coloration, as
shown in Table 1. This suggested that transcriptional activators of PkasT are present only in wild-type S. kasugaensis
and that their expression requires the presence of rpoZ. On
the other hand, despite the presence of rpoZ, the S. lividans
TK21 transformants failed to degrade DHB, probably because the streptomycete lacks the transcriptional activator
genes specific to PkasT . We consequently selected S.
lividansTK21 as the host for cloning these activator genes.
We performed a shotgun cloning experiment as follows.
Genomic DNA of the rpoZ-disrupted mutant R6D4 waspartially digested with Sau3AI and the DNA fragments,
with an average size of 4 kb, size-fractionated by agarose
gel electrophoresis were ligated into BamHI-digested
pAK557. After transformation of S. lividans with the ligation mixture, the resulting 32,000 transformed colonies
were screened with the DHB assay and 37 positive clones
were selected. These cloned DNA fragments were PCR-amplified using primers with XbaI tails and subcloned into theXbaI site of pUC18/19 for nucleotide sequence determination and subsequent BLAST homology analysis.
Cloning of bphC in S. kasugaensis. Of the 37 clones
isolated, transformants of KW6 and KW13, which overlapped in a 3.5-kb region, showed the highest intensity ofyellow coloration after DHB application. As illustrated inFig. 3.

Principle for cloning of kasZ and related genes using pAK557.

Table 1. DHB degradation and KSM production by pAK557-harboring
S. kasugaensis and S. lividans.
Transformant

rpoZ

DHBdegradation

KSM
production

S. kasugaensisAIR6/pAK557
S. kasugaensisR6D4/pAK557
S. lividansTK21/pAK557













Fig. 4.

Fig. 4, three full-length open reading frames (orf1–orf3)
were present in the region. The orf3 was identified as a homolog of bphC, a gene encoding biphenyl-2,3-diol 1,2dioxygenase,5) which also degrades DHB in a manner similar to DbfB (Table 2). Deletion analysis with a dbfB-lessplasmid demonstrated that orf3 was responsible for the observed DHB degradation and that its expression requires
the presence of the promoter region of orf2. We concluded
therefore that orf3 is the bphC ortholog of S. kasugaensis
and is co-transcribed from the orf2 promoter. As wild-type
S. kasugaensis alone fails to degrade DHB, the degradation
activity of these transformants was probably due to a bphC
gene dosage effect. After the remaining positive clones
were PCR amplified with a bphC-specific set of primers,additional 11 clones were found to contain the bphC geneand were therefore eliminated from further analysis.
Classification of positive clones. Based on the yellow
intensity elicited by DHB application in the clone-harboring transformants of wild-type S. lividans TK21 and rpoZdisrupted S. kasugaensis mutant R6D4, the clones wereclassified into three groups, Groups A, B and C, as shownin Table 3. Briefly, clones in Group A developed a dark yellow coloration ( or ) in S. lividans but a faintyellow coloration () in mutant R6D4. Similarly, but oflower intensity, clones in Group C caused a relatively-darkyellow ( or ) in S. lividans but no or a faint yellow coloration ( or ) in mutant R6D4. Meanwhile,clones of Group B gave rise to similar levels of yellow coloration in the both streptomycetes. It is most likely that thecolor intensity correlates with the amount of DbfB produced through an activation of PkasT by protein products ofthe cloned genes and that the far less intensity developed in
the rpoZ-disrupted mutant is due to the need of rpoZ or w containing RNAP for transcription of the genes. From thisviewpoint, the genes classified into Group A, in marked
contrast to those of Group B, are thought to require rpoZ
for their expression. Despite the variation in the DHB
degradation, however, all the clones listed in Table 3 failed
to recover KSM production in mutant R6D4.
A set of genes encoding a putative two-component
system in Clone A23. Clone A23 was found to containtwo full-length genes encoding a putative two-component
system, consisting of a sensor kinase and a response regulaTable 2. Analysis of overlapped region of clone KW6 and KW13.
ORF

Putative function

orf1
orf2
orf3
D orf4NDP-sugar epimerase
HpcE homolog (decarboxylase/isomerase)
BphC homolog (extradiol dioxygenase)
HppA homolog (phenol hydroxylase)

Physical maps of Clone KW6 and KW13.

88
Table 3.

GroupABC

Summary of cloned fragments in this study.

CloneA23A17
KW22
K8K9K33
KW19KW24
KW36
AS6A12M7
K13K14K17K23
KW37
KW40
A18A22A24A26
KW16
KW17

Size of cloned
fragment (kb)
4.5
4.5
5.2
4.3
3.7
3.6
4.2
4.5
5.4
5.0
3.7
5.0
5.2
5.0
4.5
3.7
4.0
3.4
3.6
4.5
5.0
3.0
3.3
4.5

Intensity of yellow induced by:
Putative function of gene(s) encoded
S. lividans TK21

S. kasugaensisR6D4

tor, as a signal transduction system prevalent in prokaryotes. Such systems as absA1/absA2, cutR/cutS andafsQ1/afsQ2 are known to be involved in antibiotic regulation in S. coelicolor.6
bldA in Clones K8 and 9. The bldA gene, encoding aleucyl tRNA for a rarely used UUA codon in streptomycetes, was present in a 0.7-kb overlapping region ofclones K8 and K9. In fact, a subclone of pAK557, harboring a PCR-amplified bldA, induced an intense color reaction in S. lividans. In S. coelicolor and S. griseus, bldA is
associated with antibiotic production by translationally regulating antibiotic-specific activator genes.7,8 It is therefore
most likely that extra copies of bldA enhanced the production of the regulators in S. lividans and that, because of
their low specificity to PkasT, these regulators could only activate the promoter when present in high concentrations. It
should be mentioned that clone KW17 in Group C encodesa putative leucyl-tRNA synthase, an enzyme that adds
leucine to the product of bldA (Table 3).

two-component regulatory systemtransposase
bldA (tRNAUUA-Leu)function unknown
acyl-CoA synthetase
function unknown
asparagine synthetase, methyltransferase
function unknown
1-deoxy-D-xyllulose-5-phosphate synthase
LacI-family trancriptional regulator
glycogen debranching enzyme, trypsin-like protease
regulatory protein
enolase, cytochrome P-450
rrnF (16SrRNA, 5SrRNA, 23SrRNA)
translocase SecA subunit
ABC transporter
zinc protease
ABC transporter
function unknown
DNA polymerase III b subunit
function unknown
leucyl-tRNA synthetasestudy their roles in KSM biosynthesis, we are planning toexamine the expression of these genes in the rpoZ-disrupted mutant R6D4 and to construct gene-disrupted mutants of S. kasugaensis.
References
1.

2.

3.

4.

Concluding Remarks
Initially, we assumed that KasZ, an AdpA homolog,
might be required for kasT transcription in S. kasugaensis
and that kasZ expression may require rpoZ, or more specifically, w -containing RNAP. However, no adpA homologs
were identified among the selected clones. We are now
therefore in the process of cloning an adpA homolog of S.
kasugaensis by PCR amplification with primers specific toadpA and its homologs in S. coelicolor and Streptomyces
avermitilis.
The isolation of a set of genes encoding a putative twocomponent system and also of bldA, whose transcription ishighly dependent on rpoZ presence is of great interest. To

5.

6.

7.

8.

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