Genome Sequence of Kitasatospora setae NBRC 14216

14 Pages·2010·0.58 MB·English

DNA RESEARCH 17, 393–406, (2010) doi:10.1093/dnares/dsq026
Advance Access publication on November 8, 2010
Genome Sequence of Kitasatospora setae NBRC 14216T: An Evolutionary
Snapshot of the Family Streptomycetaceae
NATSUKOIchikawa1, AKIOOguchi1, HARUOIkeda2, JUNIshikawa3, SHIGERUKitani4, YUMIWatanabe1,
SANAENakamura1, YOKOKatano1, EMIKishi1, MACHISasagawa1, AKIHOAnkai5, SHIGEHIROFukui5,
YOSHIMIHashimoto1, SACHIKamata1, MISAOtoguro6, SATOSHITanikawa1, TAKUYANihira4,
SUEHARUHorinouchi7, YASUOOhnishi7, MASAYUKIHayakawa8, TOMOHISAKuzuyama9, AKIRA Arisawa10,
D
FUMIKINomoto11, HIROMIMiura12, YOKOTakahashi12, and NOBUYUKIFujita1,* ow
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NITEBioresourceInformationCenter,DepartmentofBiotechnology,NationalInstituteofTechnologyandEvaluation, ad
e
2-49-10 Nishihara, Shibuya-ku, Tokyo 151-0066, Japan1; Kitasato Institute for Life Sciences, Kitasato University, d
1-15-1Kitasato,Sagamihara,Kanagawa252-0373,Japan2;DepartmentofBioactiveMolecules,NationalInstitute from
of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan3; International Center for h
Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan4; Tohoku Regional Office, ttps
National Institute of Technology and Evaluation, 4-5-18 Higashisendai, Miyagino-ku, Sendai, Miyagi 983-0833, ://a
c
Japan5;NITEBiologicalResourceCenter(NBRC),DepartmentofBiotechnology,NationalInstituteofTechnologyand ad
e
Evaluation, 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan6; Department of Biotechnology, Graduate m
ic
SchoolofAgricultureandLifeSciences,TheUniversityofTokyo,1-1-1Yayoi,Bunkyo-ku,Tokyo113-8657,Japan7; .o
u
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DivisionofAppliedBiologicalSciences,InterdisciplinaryGraduateSchoolofMedicineandEngineering,Universityof .c
Yamanashi,Takeda-4,Kofu,Yamanashi400-8511,Japan8;BiotechnologyResearchCenter,TheUniversityofTokyo, om
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan9; Bioresource Laboratories, Mercian Corp., 1808 Nakaizumi, /dn
a
Iwata,Shizuoka438-0078,Japan10;Nagase&Co.Ltd,ResearchandDevelopmentCenter,2-2-3Murotani,Nishi- re
s
ku, Kobe, Hyogo 651-2241, Japan11 and Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, ea
Minato-ku, Tokyo 108-8641, Japan12 rch
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*To whom correspondence should be addressed. Tel. þ81 3-3481-1933. Fax. þ81 3-3481-8424. le
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E-mail: [email protected] b
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Edited by Katsumi Isono t/1
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(Received 15July2010;accepted 30September 2010;publishedonline 8November 2010) /6
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Abstract /4
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Kitasatospora setae NBRC 14216T (5KM-6054T) is known to producesetamycin (bafilomycinB1) pos- 47
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sessing antitrichomonal activity. The genus Kitasatospora is morphologically similar to the genus 3 b
y
Streptomyces, although they are distinguishable from each other on the basis of cell wall composition g
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and the 16S rDNA sequence. We have determined the complete genome sequence of K. setae NBRC e
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14216T as the first Streptomycetaceae genome other than Streptomyces. The genome is a single linear t o
n
chromosome of 8783278bp with terminal inverted repeats of 127148bp, predicted to encode 7569 1
0
protein-coding genes, 9 rRNA operons, 1 tmRNA and 74 tRNA genes. Although these features resemble A
p
those of Streptomyces, genome-wide comparison of orthologous genes between K. setae and ril 2
Streptomyces revealed smaller extent of synteny. Multilocus phylogenetic analysis based on amino acid 01
9
sequences unequivocally placed K. setae outside the Streptomyces genus. Although many of the genes
related to morphological differentiation identified in Streptomyces were highly conserved in K. setae,
there were some differences such as the apparent absence of the AmfS (SapB) class of surfactant
protein and differences in the copy number and variation of paralogous components involved in cell
wall synthesis.
Key words: Kitasatospora setae; complete genome; Streptomyces
#TheAuthor2010.PublishedbyOxfordUniversityPressonbehalfofKazusaDNAResearchInstitute.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium,
providedtheoriginalworkisproperlycited.
394 Complete Genome Sequence of Kitasatospora setae [Vol. 17,
1. Introduction of Kitasatospora, in contrast, contains both LL- and
meso-DAP.16 In K. setae NBRC 14216T, aerial spores
Amongmanyfilamentousbacteriabelongingtothe on solid culture and submerged spores in liquid
phylumActinobacteria,Streptomycesspecieshavebeen culture both contain exclusively LL-DAP, whereas
extensively studied because of the ability to produce mycelia in both cultures contain mainly meso-
various bioactive secondary metabolites and the DAP.14,15,17 This suggests that the composition of
complex process of morphological differentiation. cell wall peptidoglycan would be regulated in
ThelifecycleofStreptomycesisinitiatedby thegermi- Kitasatospora depending on differentiation stages.
nation of spores that develop into vegetative mycelia. Many of the genes involved in the formation of
In response to environmental signals, aerial mycelia aerial mycelium (bld genes and others) and sporula-
emerge from the colony surface and differentiate tion (whi genes and others) have been identified by D
into chains of spores. Many pioneering works about genetic analysis of Streptomyces species,1,2 but the ow
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theregulationofsecondarymetabolismanddifferen- genetic and molecular basis for the differential lo
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tiationofStreptomycesspeciesweredoneusingmodel incorporation of DAP isomers in Kitasatospora raises de
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organismssuchasS.coelicolorA3(2)andS.griseusIFO new questions. Considering the unique taxonomic fro
13350.1,2 The complete genome sequences of three position of Kitasatospora, as well as the common and m
Streptomyces species, S. coelicolor A3(2),3 S. avermitilis distinct features of Kitasatospora and Streptomyces, http
MA-4680T4 and S. griseus IFO 13350,5 have been Kitasatospora would be a key microorganism for s://a
reported and further accelerated the studies. further understanding of the evolution of not only c
a
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Kitasatosporasetaeisasoil-habitingmycelialbacterium actinobacteria within the family Streptomycetaceae e
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belongingtothesamefamilyStreptomycetaceae.All23 but also other mycelial actinobacteria. ic
.o
validly published species belonging to the genus We determined the complete genome sequence of u
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Kitasatospora exhibit a similar life style and mor- K. setae NBRC 14216T (¼KM-6054T) and compared .c
o
m
phology with Streptomyces species. Kitasatospora may it with Streptomyces genomes. Although the overall /d
also be comparable with Streptomyces in its capacity topology and gene organization of the K. setae NBRC na
to produce bioactive secondary metabolites. The type 14216T genome showed close resemblance to res
e
strain of K. setae, NBRC 14216T, is known to produce Streptomyces genomes, there are discriminative dis- arc
h
setamycin (bafilomycin B1) and bafilomycin A1, tances between them. We established a robust phylo- /a
specific inhibitors of vacuolar ATPase and commonly genetic position of the genus Kitasatospora by rtic
le
used as biochemical reagents for investigation of mol- multilocus phylogenetic analysis and confirmed the -a
b
ecular transport in eukaryotic cells. This genus also previous results based on 16S rDNA sequences. We s
tra
includes several other strains reported as producers also describe theconservation and variation ofdiffer- c
ofbioactivecompoundsincludingaproteasomeinhibi- entiation-related genes predicted from the annota- t/17
torand an anti-fungal agent.6,7 tion of K. setae NBRC 14216T genome, as well as /6/3
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After the first proposal of the genus Kitasatospora the possible coding capacity of the genome for bio- 3
(originally Kitasatosporia) by Omura et al. in 1982,8 active secondary metabolites. /48
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the taxonomic position of Kitasatospora had been 7
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under a debate. It was once reclassified as a b
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synonym of Streptomyces based on morphology and 2. Materials and methods g
u
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partial 16S rDNA analysis reported by Wellington s
et al.9 and Ochi and Hiranuma.10 Afterward, Zhang 2.1. Genome sequencing, assembly and validation t on
et al.11 reported that Streptomyces and Kitasatospora DNA shotgun libraries with average insert sizes of 10
A
form distinct phyletic groups in the detailed inspec- 1.6 and 6.5kb were constructed in pUC118 p
tionofthe16SrDNAandthe16S–23SrDNAinternal (TaKaRa), whereas a fosmid library with average ril 2
0
spacer region and proposed the revival of the genus insert size of 37kb was constructed in pCC1FOS 19
Kitasatospora. Such a history made Kitasatospora a (EPICENTRE) as described previously.18,19 A total of
suitable model for the development of methods dis- 50400 clones (34560, 10752 and 5088 clones
tinguishing closely related species and genera based fromlibrarieswith1.6,6.5and37kbinserts,respect-
on the DNA sequence.12,13 Besides the molecular ively)weresubjectedtosequencingfrombothendsof
phylogeneticapproach,thereareclearphenotypiccri- the inserts on either ABI 3730xl DNA Analyzer
teriatodistinguishKitasatosporafromStreptomyces,of (Applied Biosystems) or Base Station DNA Fragment
which most notable is the chemical composition of Analyzer BST-0100 (MJ Research, Inc.). Sequence
cell wall peptidoglycan.14,15 Cell wall peptidoglycan reads were trimmed at a threshold quality value of
of streptomycetes contains the LL isomer of diamino- 20 by Phred and assembled by Phrap and CONSED
pimelic acid (DAP), whereas manyother sporoactino- assembly tools.20,21 For alignment and validation
mycetes contain the meso isomer. The peptidoglycan of contigs, Optical Mapping (OpGen) was used. Gaps
No. 6] N. Ichikawa et al. 395
between contigs were closed by sequencing PCR pro- 2.3. Data and strain submission
ducts, which bridge two neighboring contigs. Finally, The nucleotide sequence of the K. setae NBRC
each base of K. setae NBRC 14216T genome was 14216T genome has been deposited in the DDBJ/
ensured to be sequenced from multiple clones and EMBL/GenBank databases under accession number
from both directions with Phrap quality score (cid:2)70 AP010968. The annotated genome sequence is
or from one direction with Phrap quality score (cid:2)40. also available at the genome database DOGAN
Chromosomal terminus wasdetermined afterattach- (http://www.bio.nite.go.jp/dogan/Top).Themicrobial
ingadenineandthyminehomopolymerstothenaked strain and genomic DNA clones used for the sequen-
30 ends of the chromosome as described previously.5 cing are available through the NBRC (NITE Biological
Resource Center, Chiba, Japan, http://www.nbrc.nite.
2.2. Data analysis and annotation go.jp/e/index.html). D
o
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The prediction of open reading frames (ORFs) was n
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performed using Glimmer3.22 The initial set of ORFs a
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was manually selected from the prediction result in 3. Results and discussion ed
combination with BLASTP23 and FramePlot24 results. fro
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Each ORF was annotated manually using in-house 3.1. General features of the K. setae NBRC 14216T h
genome annotation system OCSS (unpublished). genome ttps
Similarity search results against Uniprot,25 Interpro26 TheK.setaegenomewascomposedofasinglelinear ://a
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and HAMAP27 databases were used for functional chromosome of 8783278bp with 127148bp of ad
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prediction.TheKEGG28databasewasusedfortherecon- terminal inverted repeats (TIRs). Thesecharacteristics m
ic
struction of metabolic pathways. If necessary, annota- in the genome topology were similar to those of .o
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tionwasconfirmedbymolecularphylogeneticanalysis Streptomyces,3–5 although the microorganism does p.c
usingClustalW,NJplotorGARLI(http://www.bio.utexas. not harbor a linear or circular plasmid. The general om
edu/faculty/antisense/garli/Garli.html).Putativetrans- features of the K. setae genome are summarized in /dn
a
porters and peptidases were independently evaluated Table1andFig.1.Thechromosomewaspredictedto re
s
using TransportDB29 and MEROPS30 databases, res- encode 74 tRNA genes, 9 copies of ribosomal RNA e
a
pectively. Non-coding genes were predicted using the operon and 7569 protein-coding genes. Among the rch
RPufatmat,i3ve1 toRrNiCAscreagni-oSnE32waansdloAcRatAeGdORuNsi3n3g pororiggrianmx3s4. pwreerdeicatsesdigpnreodtepinu-tcaotidveingfugnecntieosn,s5. 3T.h5e%a(v4e0ra4g9e OGRþFsC) /article
program. Putative ORFs related to mobile genetic content of the chromosome was 74.2%, which is -ab
s
elements were predicted and their boundaries were among the highest in actinobacteria, being nearly tra
inferredwiththeassistanceofGenomeMatcher35soft- equal to that of Kineococcus radiotolerans (http:// ct/1
7
ware.Foraccurateassignmentoforthologsfromactino- genome.jgi-psf.org/finished_microbes/kinra/). The /6
bacterial genomes, comparative data compiled in the putative replication origin containing 20 DnaA box- /39
3
MBGD36 database were used with further molecular likesequenceswaslocatedatthecenterofthechromo- /4
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phylogeneticevaluation,ifnecessary. some. This putative origin was flanked by dnaA and 47
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Table1. GeneralfeaturesofK.setaeNBRC14216TgenomeandStreptomycesgenomes g
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Length(bp) TIR(bp) GþC CDS rRNA tRNA AverageCDS Coding ReciprocalBLAST t o
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Content (no.) operons genes length(bp) density best-hitpair 1
(%) (no.) (no.) (%) (no.)a 0 A
K1.4s2e1ta6eTNBRC 8783278 127148 74.2 7569 9 74 1012 87.0 — pril 2
0
1
S.coelicolor 8667507 21653 72.1 7825 6 63 991 88.9 3550 9
A3(2)
S.avermitilis 9025608 49 70.7 7582 6 68 1027 86.3 3498
MA-4680Tb
S.griseusIFO 8545929 132910 72.2 7138 6 66 1055 88.1 3513
13350
S.scabies 10148695 18488 71.5 8746 6 75 1005 86.2 3534
87.22
aNumbers of best-hit pair between K. setae NBRC 14216T and each Streptomyces are calculated using BLASTP program
with athreshold E-valueof 1e220.
bOnthebasisofthelatestannotationdataofS.avermitilisMA-4680TmaintainedbyH.Ikeda(http://avermitilis.ls.kitasato-
u.ac.jp).
396 Complete Genome Sequence of Kitasatospora setae [Vol. 17,
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Figure 1. Schematic representation of the K. setae NBRC 14216T chromosome. (i) GþC content. (ii) GC skew. (iii) ORFs encoded in n
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forward (upper) and reverse (lower) strand. Each ORF is colored on the basis of the predicted function. (iv) RNA encoding genes. a
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rRNA operons are colored in red and tRNAs are colored in blue. (v) Putative insertion sequences. (vi) Secondary metabolism gene e
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clusters.(vii)RedbarsindicateORFsconservedcommonlyinallfourStreptomycesgenomes. fro
m
h
ttp
dStnraepNtoamsyincesthspeeccaieses.s37of almost all bacteria, including 3.2. oTafxKo.nsoemtaiec NreBevRaClu1a4ti2o1n6aTnd comparative analysis s://ac
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The finished sequence consisted of a big contig From morphological similarity and rDNA related- de
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representing the major part of the chromosome con- ness to Streptomyces species, genus Kitasatospora ic
nected to the same small contig at both termini. As was once regarded as a synonym of Streptomyces.9 .ou
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neither sequence variation nor assembly inconsis- Although phylogenetic analysis based on the 16S .c
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tency was found in the terminal contig, we con- rDNA sequence usually separates Streptomyces and m/d
cluded that the chromosome has inverted identical Kitasatospora species into distinct sister groups, the na
sequences at both extremities. Terminal sequences results sometimes depend on the choice of the out- res
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of linear chromosomes and plasmids of group11 or the region used for the alignment, depict- arc
Streptomyces and Rhodococcus can be classified into ing the difficulties in determining correct taxonomic h/a
at least six groups. The terminal sequence of the K. relationships only from the nucleotide sequences of rtic
setae chromosome was distinct from any of these rDNA. In order to obtain a more robust measure of le-a
groups; while the first 13bp sequence exactly the taxonomic position of Kitasatospora by taking bs
matched with that of major Streptomyces groups I advantage of its genomic information, we performed trac
and II (Supplementary Fig. S1), the subsequent amultilocusphylogeneticanalysisusing31conserved t/17
region containing palindrome structures and loops, amino acid sequences. Ciccarelli et al.40 reported the /6/3
which are known in Streptomyces to be required for construction of a tree of life across all three domains 93
binding of telomere-associated protein (Tap), was using 191 species including 14 actinobacteria. We /48
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not conserved.38,39 Tap and terminal protein (Tpg) adopted the same method by utilizing 58 actinobac- 77
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encoding genes (KSE_73020 and KSE_73030) were terial genomes with the Escherichia coli genome as b
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detected in the K. setae chromosome with lower an outgroup (Supplementary Tables S1 and S2). g
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similarity (42–45% amino acid identity) to those Amino acid sequences were aligned for each of the es
of Streptomyces. Kitasatospora setae thus seems to 31 conserved protein genes, and then all 31 align- t on
have similar mechanisms for chromosome mainten- ments were concatenated and ambiguous portions 10
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ance; it possesses a linear chromosome with TIRs, were deleted before performing phylogenetic recon- p
replicates bidirectionally from a centrally located struction.Figure2showsaphylogenetictreeobtained ril 2
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oriC region and maintains terminal sequences by by the neighbor-joining method (a phylogenetic tree 19
Tap and Tpg. However, the binding specificity of obtained by the maximum-likelihood method is
Tap might be different even from those recognizing shown in Supplementary Fig. S2). Relationships
group I and II replicons of Streptomyces. between major taxonomic groups were broadly con-
No critical difference was found between K. setae sistent with the previous results obtained by 16S
and Streptomyces species in the predicted primary rDNA sequences. Within the phylogenetic tree, 82%
metabolism such as carbohydrate metabolism, ofallpredictedbranchesweresupportedbybootstrap
amino acid metabolism, nucleic acid metabolism proportionsgreaterthan90%(i.e.900of1000).Four
and respiration. The numbers of other ubiquitous Streptomyces species were grouped in the same
components, such as membrane transporters, pepti- branch, with S. griseus IFO 13350 branching out at
dases, transcriptional regulators and sigma factors, the deepest position. Kitasatospora setae NBRC
were also almost equivalent to those of Streptomyces. 14216T was placed within the same clan as
No. 6] N. Ichikawa et al. 397
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Figure 2. Phylogenetic tree based on amino acid sequences of 31 protein-coding genes analyzed by the neighbor-joining method. Ap
Branches with less than 90% bootstrap support are represented in dashed lines. Lists of organisms and genes used for the analysis ril 2
areshowninSupplementaryTablesS1andS2,respectively.Namesoftheorganismsmentionedinthetextareshowninboldtype. 0
1
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Streptomyces creating the outermost branch. All proteins of K. setae and four Streptomyces species.
theseresultsweresupportedbybootstrapproportions More than half of ORFs predicted in K. setae had
of 100%, reinforcing the idea that the genera orthologs (reciprocal best-hit pairs) in each of
Kitasatospora and Streptomyces were generated from four Streptomyces species using a BLASTP threshold
acommon progenitorand have diverged into distinct of E,10220 (Table 1). About 34% of K. setae ORFs
sister groups. The closest to this group was had orthologs in all four Streptomyces genomes. The
Catenulispora acidiphila DSM 44928,41 a mycelial average amino acid identity between orthologous
actinobacterium with a circular chromosome. pairs from K. setae and Streptomyces species was
For further analysis of the relationship between around 60%. Despite such high similarities observed
K. setae and Streptomyces, we compared all annotated between orthologs, genome-wide comparison using
398 Complete Genome Sequence of Kitasatospora setae [Vol. 17,
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Figure 3. Synteny between the genomes of K. setae NBRC14216Tand S. coelicolor A3(2) (A), K. setae NBRC 14216Tand S. avermitilis tra
MA-4680T (B), K. setae NBRC 14216T and S. griseus IFO 13350 (C) and K. setae NBRC 14216T and C. acidiphila DSM 44928 ct/1
(D). Reciprocal BLAST best-hit pairs with a threshold value of E,10220 were plotted. The direction of each chromosome was 7
/6
adjusted so that the dnaA gene faces the same direction. Green bar in each panel represents the conserved core region on the K. /3
setaechromosome. 93
/4
8
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orthologplotsdemonstratedsmallerextentofsynteny 3.3. Conservation and variation of genes related to 77
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between K. setae and Streptomyces genome (Fig. 3A– developmental regulation b
y
C). Many short synteny blocks were observed along Streptomycesiswellcharacterizedasamodelorgan- g
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either of the diagonal lines, suggesting that frequent ism by its complex life cycle; it grows as a thread-like es
inversions around the replication origin have had mycelium and forms aerial mycelium in the air t on
occurred. This is in contrast to the higher extent of under nutrient-limited conditions.1,2,42,43 A series of 10
A
colinearities with only 2–4 inversions observed in genes whose mutations cause defects in aerial p
comparativeanalysisamongStreptomycesgenomes.3–5 growth(‘bld’genes) were characterized in S. coelicolor ril 2
0
ConservedcoreregionoftheK.setaegenomepredicted A3(2).Acomplexinteractioncascadeamongbldgene 19
bythreadingmajorsyntenyblockswas(cid:2)5Mbinlength products results in the secretion of surfactant pro-
ranging from KSE_13770 to KSE_57600, which is teins, which assist Streptomyces in extending aerial
(cid:2)1Mb smaller than the core regions deduced from mycelium upward and in differentiating into spores.
comparison among Streptomyces genomes.5 Long- Anumberofgenesin‘whi’lociwerealsocharacterized
range synteny between K. setae and C. acidiphila mainly in S. coelicolor A3(2) as causing sporulation
genomes was much less obvious compared with that deficiency. Most of these classical differentiation
between K. setae and Streptomyces genomes (Fig. 3D), genes were found to have orthologs in K. setae. The
although the number of orthologous gene pairs number of conserved bld and whi genes and their
between K. setae and C. acidiphila (3185) was only similarity (amino acid identity) to S. coelicolor A3(2)
10% smaller than that between K. setae and counterpartswerehigherthanthoseinothermycelial
Streptomyces(3498–3550,Table1). actinobacteria, such as C. acidiphila DSM 44928,
No. 6] N. Ichikawa et al. 399
Saccharopolyspora erythraea NRRL 2333844 and setae with remarkably high amino acid identities
Salinispora arenicola CNS 205 (Table 2), consistent (69–90%). Despite such close similarities in genetic
with the closer morphological similarity of K. setae background, preliminary experimental results
to Streptomyces. Notably, bld gene pairs orthologous suggest that K. setae produces seemingly less mature
betweenK.setaeandStreptomycesspeciesrepresented spores compared with Streptomyces; (i) although
80–97% amino acid identities, which are much K. setae genome encodes full set of whiE gene
higher than the average (60%) of all orthologous cluster(encodingbiosyntheticenzymesforpolyketide
pairs and comparable with those between spore pigment), no expression of whiE genes nor the
Streptomyces species. Higher than the average production of spore pigment was observed in any
conservation of bld genes was also observed culture conditions for sporulation, (ii) both aerial
between C. acidiphila and Streptomyces (58–95% and submerged spores produced by K. setae are D
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amino acid identities). The bldA tRNA gene was also much more sensitive to freeze-thaw cycles than w
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highly conserved in K. setae, with its cognate codon those of Streptomyces (S. Kitani and H. Ikeda, unpub- lo
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UUA being the rarest in K. setae; used only in 69 pre- lished observations). Further studies are needed to de
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dicted ORFs, most of which were either of unknown elucidate mechanisms underlying such differences. fro
function or with predicted regulatory functions. In The initiation of secondary metabolite synthesis is m
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four completely sequenced Streptomyces genomes, known to be linked with morphological differen- ttp
s
oUnUlAy cthodreoengaetntehsewsaemreefpoousnitdiontoinhaevaechaocrtohnosleorgv.e4d5 taiuattoiorneguinlatSo.rgcraissecuasdev.iaInththeisgaremgmarad-,buittywroolualcdtonbee ://ac
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One of these, the bldH gene, shared a UUA codon at noteworthy that K. setae possesses three homologs e
m
an equivalent position also in K. setae. The high con- of the autoregulator receptor: KsbA (KSE_58650), ic
.o
servation of these regulatory components may KsbB (KSE_01050t and KSE_75690t; identical genes u
p
further imply that each component in the regulatory encoded in the TIR) and KsbC (KSE_44580). .c
o
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cascadeundergoesahigherthantheusualnumberof However, KsbA in K. setae was experimentally /d
interactions with other bld gene products or with confirmed to be involved only in secondary metab- na
other cellular components. olism,48 but not in morphological differentiation, res
e
On the contrary, we could not identify most of the whereas the involvement of other two remains to be arc
h
amf genes in K. setae (Table 2). The amf genes are clarified. In addition, the AfsA family protein, which /a
highly conserved among Streptomyces species and contains two A-factor biosynthesis repeat motifs, rtic
le
are necessary for the synthesis of AmfS [SapB in has been reported to be a crucial enzyme to syn- -a
b
S. coelicolor A3(2)] surfactant protein, which is thesize the gamma-butyrolactone autoregulator in s
known in Streptomyces to be secreted before the Streptomyces.49 Interestingly, three AfsA family pro- trac
initiation of aerial growth. The presence of amfAB teins were encoded on the genome of K. setae: t/17
homologs in C. acidiphila41 and S. erythraea,44 myce- KsbS2 (KSE_01060t and KSE_75680t; identical and /6/3
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lial actinobacteria more distantly related to present in the TIR, similar to the case of KsbB), 3
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Streptomyces, may suggest that the pre-existing AmfS KsbS3 (KSE_22970) and KsbS4 (KSE_44600). These 8
4
7
system was eliminated in the Kitasatospora lineage, findings suggested that there might be a more com- 7
3
although other possibilities such as the horizontal plicated signaling network for secondary metabolism b
y
acquisition of these genes in each lineage cannot be and/or morphological differentiation in K. setae. g
u
e
excluded. In addition to the lack of the amf gene s
t o
cluster, differences from Streptomyces were also n
suggested in the variation of paralogous components 3.4. Genes involved in peptidoglycan biosynthesis 10
A
(Supplementary Table S3) such as SsgA-like proteins One of the most important features of the p
involved in peptidoglycan synthesis in sporogenic genus Kitasatospora from the chemotaxonomic ril 2
0
cell division, WhiB family transcriptional regulators viewpoint is that the cell wall peptidoglycan 19
and chaplins, another class of surfactant protein contains both LL-DAPand meso-DAP.14,15 DAPanalysis
showninS.coelicolortoplayapartinaerialmycelium of Kitasatospora strains showed that spores
formation.46,47 contain only LL-DAP, whereas mycelia contain mostly
A numberof whi genes control the process of spor- meso-DAP.16,17 This observation suggests that
ulation septation and spore maturation in Kitasatospora incorporates different DAP isomers into
Streptomyces. Of the classical whi genes, whiA, whiB the cell wall depending on differentiation stage. We
and whiD are commonly found in actinobacteria, can speculate two steps which might be responsible
andtheirexactfunctionsinsimpler(non-sporulating) for the differential incorporation of DAP isomers.
actinobacteria need to be elucidated.1 All other whi (i) DAP biosynthesis and isomerization: LL-DAP is iso-
genes, which had been considered specific to merized to meso-DAP by dapF gene product in the
Streptomyces species,1,42 were also conserved in K. course of lysine biosynthesis.50 (ii) Incorporation of
4
Table2. Conservationofaerialmyceliumandsporeformation-relatedgenesinK.setaeNBRC14216Tandothermycelialactinobacteria 0
0
Kitasatosporasetae Streptomyces Streptomyces Streptomyces Streptomyces Catenulispora Saccharopolyspora Salinisporaarenicola
NBRC14216Ta coelicolorA3(2) avermitilisMA- griseusIFO scabies87.22 acidiphilaDSM erythraeaNRRL CNS205
4680T 13350 44928 23338 D
o
Aerialmycelium wn
lo
bldA KSE_t0069 SCOt24 SAV_t57 SGR_tRNA42 SCAB_t50 Caci_R0007 SACE_8016 Sare_R0014 a
d
bldB (KSE_16220)b(64) SCO5723 SAV_2529 SGR_1796 SCAB_25271 ND SACE_6156 Sare_3856 ed
(SCO7246) (SAV_1241) (SCAB_84941) fro
m
bldC KSE_41620(97) SCO4091 SAV_4130 SGR_3882 SCAB_47901 Caci_0301 SACE_6926 Sare_0170 h
bldD KSE_13950(92) SCO1489 SAV_6861 SGR_6045 SCAB_75171 Caci_2399 SACE_2077 Sare_1844 ttps
bldG KSE_35840(96) SCO3549 SAV_4614 SGR_3307 SCAB_40861 Caci_8450 SACE_4194 Sare_4413 C ://ac
(¼rsbV) om ad
e
bldH KSE_26930(80) SCO2792 SAV_5261 SGR_4742 SCAB_57831 Caci_5972 SACE_4523 ND p m
(¼adpA) let ic.o
e u
bldKA- KSE_48250– SCO5112– SAV_3152– SGR_2414– SCAB_31501– Caci_7147– ND ND G p.c
bldKE KSE_48290(41–78) SCO5116 SAV_3156; SGR_2418 SCAB_31541 Caci_7151 e o
n m
SAV_3172– o /d
SAV_3176 m na
e re
bldM KSE_31060(97) SCO4768 SAV_4998 SGR_2759 SCAB_36231 Caci_1011 SACE_6712 Sare_4229 S se
(¼whiK) e a
q rc
bldN KSE_33690(92) SCO3323 SAV_4735 SGR_4151 SCAB_39121 Caci_8330 SACE_6951 Sare_0420 ue h/a
(¼adsA) nc rtic
amfC KSE_43740(54) SCO4184 SAV_4026 SGR_3974 SCAB_49711 Caci_8239 SACE_7115 ND eo le-a
f b
Laa(n¼mtirbfaRiomtRic)-likeNsuDrfactant SCO6685 SAV_7499 SGR_2393 SCAB_8642c ND ND ND Kitasa stract/17
amfS ND SCO6682 SAV_7502 SGR_2396 SCAB_8621 Caci_4240 SACE_4231 ND tos /6/3
(¼ramS) po 93
amfB ND SCO6683 SAV_7501 SGR_2395 SCAB_8631 Caci_4242 SACE_4232 ND ra /48
(¼ramA) se 47
t 7
a 3
amfA ND SCO6684 SAV_7500 SGR_2394 SCAB_8641 Caci_4243 SACE_4233 ND e b
(¼ramB) y g
u
amfT KSE_63000(44) SCO6681 SAV_7503 SGR_2397 SCAB_8611 Caci_4239 SACE_4230 ND es
(¼ramC) t o
n
Sporulation 1
0
whiA KSE_55390(89) SCO1950 SAV_6294 SGR_5572 SCAB_69681 Caci_5609 SACE_2141 Sare_3326 Ap
whiB KSE_29410(83) SCO3034 SAV_5042 SGR_4503 SCAB_55081 Caci_7609 SACE_6464 ND ril 2
0
whiD KSE_31070(90) SCO4767 SAV_4997 SGR_2760 SCAB_36241 Caci_1009 SACE_5583 ND 19
(¼wblB)
[
V
whiE KSE_72410– SCO5314– SAV_2837– ND SCAB_43281– Caci1083– ND Sare_2682– o
KSE_72480(46–67) SCO5321 SAV_2844 SCAB43341 Caci1090 Sare_2690 l.
1
whiG KSE_52540(79) SCO5621 SAV_2630 SGR_1866 SCAB_26051 Caci_1526 SACE_6040 ND 7
,
No. 6] N. Ichikawa et al. 401
s - DAP into peptidoglycan: the addition of DAP to UDP-
a b
w su N-acetylmuramyl-L-alanyl-D-glutamate precursor is
ND ND ND ND Sare_0058 oacididentity bldBparalog. poratedinthe otcdhanoeAtgeasllycymicenzaenuntdrthEebeirsygcoecaafnonseemaeopn(lfKoozSsSynetEmgrd_ee2pcot1eoof2nnmc8LsLyeo0-crdDe)ve,sAeddPsw,pbhedKyicc.cimwhessuetir(wsaEdeh.i5lvoo1ihsscaieaodtnpeedopcnetilinly-l
n r
mi he co wall) cluster. Amino acid residues known to be
ofa oft enin rfreosmpomnseisbol-eDfAoPrcsounbtsatriantiengrebcaocgtenritiaioanreinaltshoecoennszeyrmveeds
_1833 _0037 range tholog yetbe iinncltuhdeingenSztyrmepetsomfyrocems. TLLh-DeAMPucroEntpariontienign bpaucrtiefireida Dow
Caci_1760ND Caci_6781SACE Caci_7708ND NDND Caci_0043SACE wninparentheses.The 20correspondstoanor mentarystrand)hasnot fmTtgrhKrehlhaSoyegeEnumscu_odsadl3-,na,iDfE2ttfKehA.e6sd.ePry3c,enos0nMtetlbhihttuayiaeeacernstaEniidunsoha,pnnatKriuk-udnSodnrnEctaeo_telolpehw5irnsesrp3sneuno7embdtr5sheafatdatn0eryeacat)tptnsoaeLFwuoLrl-tb(htiDgesnsoe)rtbAa.srntePeatheitOosirepisnennr(ssobsKppadaioStndceuEhntcdc_eepsitifi3irtesbiic2puolosiet6tmnhtiydh0af.5ooter0ioe2srr-, nloaded from https://academ
24461 20881 47291 47281 45631 wassho SE_162 comple hatwnigaohlyloysfisctbohanesseeDdravopendF apamrcotitinveoeinassciti(deensrceeoqsduideeudnecsbe.ysPsKhhSyoElwo_eg3de2n6teh0tai0ct ic.oup.co
SCAB_ SCAB_ SCAB_ SCAB_ SCAB_ ortholog cluded.K 2onthe SafrtnordemptoKmLSLEy-_Dc5eAs3P7sp5ec0oc)inetsawianenrinedgNcloaoccsaterilndyioobirdaeeclsatetserpida. BtAsouAc-ht4h9o9sae/s m/dnarese
2) in 80 JS614 (Fig. 4A). In contrast, the third DapF protein arc
SAV_2445SGR_1702 SAV_2230SGR_1475 SAV_4185SGR_3551 SAV_4186SGR_3552 SAV_4331SGR_3718 TS.coelicolor6ORFandA3( TvermitilisMA-4680arealso gtoposition975194–975 i(bmCapdoonKnafbealecoSpcpdmssrsEetFotete_imorr-adrivD3grsdieoepea2eAsigdnuhlo6Pl,me-oy5g3iDsnc2lr.o,0oaAcgIo)nusPnoLiurp.cLnwp-gaDstttDa,gohlahiseAfobdeiefnsPluieiptsfirdrftinoneeawpgscirngpnioroetFtoinnuiottatLttlhmhendlaiiaaieseirnutnttaedeitvfiograpnfitiauenchnargipcolet,namtaditsptrtiuirtyotLhgeercanheaplftoacrsittfonttoc,ipoidomfvpiblonoemartagofhytctpltsheiyEhaeiloeczl.neimusenaicsaintscuomicooollftitfuprni,awinopornaLosmaolrLndees---f h/article-abstract/17/6/393/48477
SCO5819 SCO6029 SCO4035 SCO4034 SCO3854 K.setaeNBRC1421assigned.colorS.aA3(2)and 8642(correspondin tkDossappncunSoacorttcarwiruensreindolgpoaltntecotiraonmivonlgelsyyhgstc-aheeswvseter,eapaeactltsilhtauvrsweae.ltpnIoainnrgreegegdssprieiosondtnwirientnputapcehltoatpi,wvtstiieminitddceogooeedfdgmllmlyelyiysnyccdcaaeoeinnalvfildiaicsac,cioleoiomnblinlorarcprcdedootigoinevsnuicritstalircaiiaatoour,esnnrlidtyss.,. 3 by guest on 10 April 2
KSE_54050(74) KSE_55090(77) KSE_37310(69) KSE_37320(80) KSE_39340(50) noacididentitybetweenheremultiplegeneswereS.coelieparalogfoundin tentativelynamedSCAB_notation. laPdHemKgatxBeoiMttapnPtganerssWelseyernas(scp-,etsHsPaotoureBnaMsngdpPrgeWpoeaerseagon-sautpePftdlnstBtwissiedPanephsssetgepr)aiscecoo.echiigfrfinSaeuts(ctwrleKaraehewelSptsymegiEttazcaod_uelhsgml4lsaset.6yysrthsolonc1eyHemt9oshichsg0feecrphao)osavd-wisnbcsemisfeete-foaolrdeinifeninlrnieegpnskcsnpeaiehhuntpooliingtrgaaHwieuhrtdosMimnloo.fy5wngWb3pthl.eoeye5-oiTc4rPpgm,habBth5oinPee5t--f 019
whiH whiI sigF sigN crgA¼whiP() TheamihownwbldBgen ThisORFmitteran oSgCrlooOgw3ot1uhs56aton)dSk.nscpooowerlnuicloalttooironAb.3eO(2en)xpgtrheenesseesodt(hSedCruOr3hin7agn7d1a,eatrnhiadel
a sb c
402 Complete Genome Sequence of Kitasatospora setae [Vol. 17,
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Figure4. AminoacidphylogenetictreesofDapF(A)andHMW-PBPs(B).Brancheswithlessthan70%bootstrapsupportarerepresentedin
dottedline.Thedistributionofpeptidoglycanaminoacidcomponentisalsopresentedin(A).StreptomycescoelicolorA3(2)PBPsand
theirorthologsweregroupedandexpressionpatternswereassignedaccordingtoNoensetal.55in(B);thoseknowntobeexpressed
in aerial mycelium and vegetative mycelium are shown by AM and VM, respectively. Amino acid sequences were derived from the
HAMAPdatabase(MF_00197).