NISR Research GRANT
2007 Research Grant

Studies on regulation of secondary metabolism and morphogenesis of Streptomyces
by DNA microarray analysis
Yasuo OHNISHI
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo

Research aims

Results

The Gram-positive, soil-inhabiting, filamentous bacterial
genus Streptomyces is characterized by its ability to produce awide variety of secondary metabolites represented byantibiotics and by its complex multicellular development. InStreptomyces griseus, both secondary metabolism andmorphological development are triggered by a low molecularweight chemical signaling molecule, A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone).1) A-factor switches on thetranscription of adpA by binding to ArpA, the A-factor receptor
protein, which has bound to the adpA promoter, anddissociating the ArpA from the DNA. AdpA then activates anumber of genes of various functions required for morphological development and secondary metabolism including
streptomycin (Sm) production.2) In this study, the effects ofA-factor on global gene expression were determined by DNA
microarray analysis of transcriptomes obtained with theA-factor-deficient mutant ∆afsA. The purposes of this study
were (i) to confirm the A-factor regulatory cascade proposed byour previous studies, (ii) to show the direct effect of A-factor on
genome-wide gene expression, (iii) to find novel
A-factor-inducible genes, and (iv) to find possible AdpA-target
genes among them.

Exogenously added A-factor affected transcriptomes of the∆afsA mutant.
In the DNA microarray analysis, we employed the following
criteria to discriminate A-factor-responsive genes: those thatshowed an increase or decrease in expression of more than
two-fold and a P value of < 0.05 when analyzed with a t-test (n= 3). When these criteria or thresholds were used, the following
numbers of genes were transcriptionally up- and downregulatedby A-factor addition: (i) at 5 min, 14 and 4 genes, respectively;(ii) at 15 min, 12 and 4; (iii) at 30 min, 13 and 14; (iv) at 1 h,
32 and eight; (v) at 2 h, 72 and 26; (vi) at 4 h, 118 and 32; (vii)
at 8 h, 229 and 33; and (viii) at 12 h, 275 and 57. adpA wasidentified as a gene upregulated by A-factor at all eight time
points. Furthermore, adpA proved to be the only gene that wascontinuously activated by A-factor immediately after its
addition. Thus, we confirmed that AdpA plays an absolute role
as a transmitter and amplifier of the A-factor signal as we hadexpected.

Methods
Transcriptome analysis was performed using the S. griseus
mutant ∆afsA, which produces no A-factor due to the absenceof the key A-factor biosynthetic enzyme AfsA,3) but stillresponds to exogenously supplemented A-factor. A-factor (final100 nM) was added to the ∆afsA mutant grown in a liquidmedium to the mid-log phase and RNA samples were preparedfrom the mycelium at different time points (5 min, 15 min, 30min, 1 h, 2 h, 4 h, 8 h, and 12 h after A-factor addition). The
transcriptomes obtained with the RNA samples were comparedto those of a control culture without A-factor addition bycompetitive hybridization using DNA microarray analysis.
Microarray preparation and the method for microarrayhybriddization, including synthesis and labeling of cDNA, weredescribed previously.4) Transcripts of all 7,138 ORFs on the S.
griseus chromosome could be theoretically monitored by theoligonucleotide microarray we prepared. Details in statisticalanalyses were described in our recent publication.5) For
competitive electrophoretic mobility shift assay (EMSA), anapproximately 800-bp DNA fragment, which probably included
an A-factor-inducible promoter and its upstream regulatory
sequence, was prepared by PCR. Recombinant AdpA wasincubated with a DIG-labeled DNA fragment containing theAdpA-binding site of sgmA or sgiA in the presence of 10-foldexcess of a competitor DNA fragment, and the reaction mixture
was applied to polyacrylamide gel electrophoresis. Details incompetitive EMSA were also described in our recentpublication.5)

A gradual increase in the A-factor effect on the transcriptome.
At 1 h after A-factor addition, strR was detected as a genethat was upregulated by A-factor (2.6-fold), indicating that
AdpA began to activate the transcription of its target genes
between 30 and 60 min in response to exogenously
supplemented A-factor. In accordance with this, the number of
upregulated genes increased gradually for 1 to 12 h afterA-factor addition. Although activation of some of these genes
was apparently transient, many were successively upregulated,
especially from 4 to 12 h after A-factor addition. These data
show that the effect of exogenous A-factor on gene expression
in the mutant ∆afsA was gradually increased through thefunction of AdpA as an amplifier of the A-factor signal.
Quality threshold (QT)-clustering analyses of DNA microarraydata.
To determine temporal changes in gene expression, weanalyzed variance of the DNA microarray data that wereobtained with the RNAs prepared at the eight time points. Thisvariance analysis (ANOVA) suggested that 477 genes weredifferentially expressed after addition of A-factor (expressionratio [with A-factor/without A-factor] of either > 2.0 or < 0.5 at
at least one time point and ANOVA P of < 0.05). We analyzed
these 477 genes by QT-clustering analysis to classify them into
groups according to their expression patterns. They weregrouped into 15 clusters, although 24 genes were not assigned
to any of these 15 clusters. In total, 345 genes (72%) wereassigned to two major groups, Clusters 1 and 2. Cluster 1
contained 281 genes (59%), the expression level of whichalmost continuously increased after exogenous addition ofA-factor. On the other hand, Cluster 2 contained 64 genes(13%), the expression level of which almost continuouslydecreased after A-factor addition. The number of genes in
Cluster 2 was much smaller than that in Cluster 1. Furthermore,the expression ratio (without A-factor/with A-factor) of almost
all genes classified in Cluster 2 was less than 3.0. These results
suggested that the negative effect of A-factor on gene
expression was smaller than the positive effect. We assumed
that transcription of genes in Cluster 2 decreased in response tochanges in some physiological conditions triggered by A-factor.
However, we could not completely exclude the possibility thatthese genes were repressed directly by AdpA, because AdpA
acts as a transcriptional repressor on its own promoter.6) Among
64 genes in Cluster 2, 27 genes encode proteins that areprobably involved in the transport of metabolites. Whenmorphological and physiological differentiation proceeds,
biosynthesis of a variety of permeases and ABC transporters,
which are probably required for vegetative growth, maydecline.
Selection of probable A-factor-inducible genes (PAGs).
Among the 27 genes in the Sm biosynthetic gene cluster, 24
genes were classified into Cluster 1, suggesting that Cluster 1
was composed of A-factor-inducible genes. Therefore, we
further analyzed each gene in Cluster 1 to select PAGs. We
identified 152 genes (that belong to 74 transcriptional units) asPAGs, taking into consideration a putative transcriptional unit
containing the gene in question and levels of upregulation.
In addition to the Sm biosynthesis genes, several genes thatwere probably involved in secondary metabolism were selected
as PAGs: two putative non-ribosomal peptide synthetase(NRPS)
geneclusters(SGR452-SGR448
and
SGR6714-SGR6717), a lantibiotic biosynthesis gene cluster(SGR3851-SGR3845), putative melanin biosynthesis genes(SGR2446-2447), and a putative terpene cyclase gene
(SGR2079).
Only three genes (SGR3340, SGR3902, andSGR299) that were probably involved in morphologicaldevelopment were selected as PAGs. SGR3340 (wblA) has animportant role in aerial mycelium formation in S. griseus (ourunpublished result). SGR3902 (ORF1590) was cloned andcharacterized as a gene that could suppress a morphologicaldefect of bald mutants of S. griseus.7) SGR299 encodes a
Spo0M family protein. Although Spo0M in Bacillus subtilis
exerts certain negative effects on sporulation,8) the functions ofSGR299 and its orthologs in Streptomyces remain to becharacterized. We assume that the small number of PAGs
involved in morphological development can be ascribed todifferences in the mechanism or pattern of gene expression
between liquid and solid medium. Several PAGs probably havesome regulatory functions. The in vivo functions of theA-factor-dependent
DNA-binding
proteins(SGR2064,SGR3226, SGR4070, SGR4257, SGR5049, SGR6383, and(SGR4396-4392
andSGR6385)
andconservons9)
SGR6126-6129) are worth characterizing. Several putative
enzyme genes and putative genes for transport and stressresponses were also selected as PAGs. In addition to thesegenes, many function-unknown gene clusters and genes wereselected as PAGs.
Putative members of the AdpA regulon.
As described above, we selected 152 genes belonging to 74
transcriptional units as PAGs. To estimate how many promoters
are directly activated by AdpA, binding of AdpA toapproximately 800-bp DNA fragments, which probablyincluded a promoter and its upstream regulatory sequence foreach A-factor-inducible gene, was determined by competitiveEMSA. The competitive EMSA showed that AdpA bound to 38
DNA fragments, two of which contained two divergent
promoters of neighboring PAGs, among the 74 transcriptional
units of 152 PAGs. The binding of AdpA to the intergenic
region between SGR5930 (strD) and SGR5931 (strR)undoubtedly activates the latter but not the former.10)

Unexpectedly, AdpA bound to a DNA fragment containing theintergenic region between SGR5914 (strU) and SGR5915 (strV),
which should contain two divergent promoters of the two genes.
Because an StrR-binding sequence was found in this region,and the promoters were inactive in an strR-deleted mutant,10)
the importance of the AdpA-binding to the region wasquestionable. Therefore, we excluded the region and estimatedthat AdpA should bind directly to 37 regions to activate 72
genes. Thus, approximately half of the promoters of PAGs
appear to be activated directly by AdpA. Furthermore, theremaining promoters of PAGs appear to be activatedsecondarily by some AdpA-dependent transcription factor.
Alternatively, these promoters may be activated in response to achange in some physiological conditions triggered by A-factor.
Conclusion
In this study, a direct influence of A-factor on thegenome-wide gene expression was detected for the first time.
Analysis of variance among the transcriptomes revealed that477 genes, which were dispersed throughout the chromosome,were differentially expressed during the 12 h after addition of
A-factor, when evaluated with specific criteria. QT-clustering
analysis with regard to putative polycistronic transcriptional
units and levels of upregulation predicted that 152 genes
belonging to 74 transcriptional units were PAGs. Competitiveelectrophoretic mobility shift assays using DNA fragments
including putative promoter regions of these 74 transcriptionalunits suggested that AdpA bound 37 regions to activate 72
genes in total. Many of these A-factor-inducible genes encoded
proteins of unknown function, suggesting that the A-factor
regulatory cascade of S. griseus affects gene expression at aspecific time point more profoundly than expected.
Characterization of novel PAGs will provide many pieces of
information about secondary metabolism and morphologicaldifferentiation in Streptomyces. Transcriptome analysis of cells
grown on solid medium and ChIP-chip analysis of in vivo
AdpA-binding sequences are in progress in our laboratory,
which should reveal several new aspects of global gene
regulation by the A-factor regulatory cascade.
References
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