BACKGROUND
bacteria of the actinomycetales order represent an amazing source of biologically active compounds which are produced as secondary metabolites  <cit> .
the broad spectrum of structural features and wide array of activities of these metabolites attract attention as a limitless source of novel chemical scaffolds as well as new drugs for human and veterinary medicine, and agriculture.
with the recent advances in sequencing technologies significant progress in sequencing and analysis of actinobacterial genomes was achieved  <cit> .
not only genomes of biotechnologically important and typical representatives of the taxonomical order were sequenced, but also strains with interesting and unusual features, leading to deeper insights into phylogeny, genetics, physiology, ecology, and the secondary metabolism biosynthetic potential of these bacteria.
one of the major discoveries of the genomic era in actinomycetes research is the presence of multiple gene clusters dedicated to secondary metabolism in one genome.
this observation caused an increase of interest in diverse groups of actinomycetes  as a possible source of new biologically active metabolites.
one of such groups includes species belonging to the pseudonocardiaceae family.
multiple genome projects dedicated to this group of bacteria are in progress, with seven being completed and published  <cit> .
here we report the sequencing and analysis of the complete genome of kutzneria albida dsm 43870t   <cit> .
the genus kutzneria is a minor branch of the pseudonocardiaceae family currently containing only  <dig> species .
to our knowledge, this is the first member of the kutzneria genus and only the eighth representative of the family pseudonocardiaceae for which a complete genome sequence is available.
k albida was isolated from soil samples collected in the rocky regions of gunma prefecture of japan  <cit> .
the strain is known as a producer of aculeximycin, a macrolide antibiotic with an interesting chemical structure and activity against gram-positive bacteria, fungi, and mosquito larvae  <cit> .
being generally toxic, aculeximycin cannot be used in medicine  <cit> .
however, some structural features, especially the unusual glycoside chain, make it an attractive source of building blocks for use in synthetic biotechnology and combinatorial biosynthesis of secondary metabolites  <cit> .
sequencing k. albida genome allowed not only to identify genes responsible for aculeximycin biosynthesis, but also to discover an unusual abundance and diversity of secondary metabolites that could potentially be produced by this species.
to the best of our knowledge, this genome is the most enriched with clusters involved in secondary metabolites among actinobacterial genomes.
RESULTS
general features of the genome
after gaps closure, a single contig with the size of  <dig> ,926 bp and a  <dig> % g + c content was obtained.
general features of k. albida genome are summarized in table  <dig>  the genome of k. albida consists of a single circular replicon; no extrachromosomal replicon was detected.
the origin of replication  was identified as a  <dig>  nt non-coding region between the two genes – dnaa, encoding the chromosomal replication initiation factor, and dnan, coding for the β-subunit of the dna polymerase iii .
interestingly, the oric has no classic dnaa boxes , and shows almost no similarity to the oric of streptomyces species  <cit>
.
on the opposite side of the chromosome from oric, a putative dif  locus was identified.
its sequence is in good accordance with actinobacterial dif sites  <cit> .
additionally, the obvious cumulative gc skew inversion in the regions of oric and dif supports our oric assignment and suggests the existence of two replichores composing the circular k. albida chromosome .table  <dig>
general features of the
k. albida
chromosome
topology
number of replicons
total size
g + c content
protein coding genes
rrna genes
rrna operons
transfer rnas
average gene length
coding density
secondary metabolite clustersschematic representation of the
kutzneria albida
genome, created with the help of circos ; megabases are labeled; smaller ticks correspond to  <dig> kbp segments.
from outside: genes on the forward and the reverse strands ;  <dig> secondary metabolite clusters coloured by type ; genomic islands; g + c content, 10kbp window ; g + c content,  <dig> kbp window ; g + c skew ; cumulative g + c skew.
the oric is placed at coordinate zero.
unlike the linear topology of streptomyces genomes, the chromosome of k. albida is circular   <cit> .
this appears to be a common feature of pseudonocardiaceae representatives genomes, since the chromosomes of other representatives of this family sequenced so far also have a circular topology  <cit> .
with a total length of  <dig> ,926 bp, the k. albida chromosome is larger than that of streptomyces coelicolor a <dig>   <cit>  and streptomyces avermitilis ma- <dig>   <cit> , as well as genomes of pseudonocardiaceae saccharomonospora viridis p101t   <cit> , pseudonocardia dioxanivorans cb <dig>   <cit> , saccharothrix espanaensis dsm 44229t   <cit> , and saccharopolyspora erythraea nrrl  <dig>   <cit> , but slightly smaller than the genome of another pseudonocardiaceae rifamycin producer amycolatopsis mediterranei s <dig>   <cit> .
the k. albida genome is among the largest actinobacterial genomes sequenced so far.
k. albida chromosome contains  <dig>  predicted protein-coding sequences  .
three ribosomal rna  operons were identified in the genome along with  <dig> trna genes   <cit> .
the rrn operons are located on the leading strand of the chromosome proximally to oric and have remarkably low g + c content in comparison to the genome average.
functional annotation of k. albida genes within the actnog  subset of the eggnog database   <cit> , showed that  <dig>   out of  <dig>  genes had at least one biological function assigned , with some of the genes assigned to more than one category.
of the remainder,  <dig> cdss  had no hits against actnogs, and  <dig>  genes  had hits but were not assigned to functional categories; of the latter,  <dig> are highly conserved.
among the genes with functional assignment,  <dig>   are implicated in metabolism, including  <dig>  in amino acid metabolism,  <dig>  in carbohydrate metabolism, and  <dig>  participating in secondary metabolism .
at the same time, 10% of the annotated genes  code for proteins involved in transcriptional processes, including  <dig> sigma factors,  <dig> anti-sigma factors,  <dig> anti-anti-sigma factors, and  <dig>  putative dna binding regulators.
the large number of proteins implicated in transcriptional processes suggests a high complexity of k. albida gene expression control.
the high proportion of proteins involved in transcriptional regulation is accompanied by a similarly high percentage of proteins involved in signal transduction pathways , which suggests a close connection between extracellular nutrient sensing and transcriptional regulation of uptake and degradation pathways.
this feature of the k. albida genome is typical for the majority of known soil living actinobacteria and reflects their life style and ecological niche, providing higher flexibility of the strain adaptation to changes in growth conditions  <cit> .
in addition to this, a large portion of the genome encodes extracellular polymer-degrading enzymes.
in total,  <dig> putative secretory hydrolase encoding genes were found in the k. albida genome , including  <dig> glycoside hydrolases and  <dig> extracellular proteases.table  <dig>
assignment of  <dig>
k. albida
genes to the functional groups of the actnog subset of the eggnog database
information storage and processing
<dig>
translation, ribosomal structure and biogenesis
rna processing and modification
transcription
replication, recombination and repair
chromatin structure and dynamics
cellular processes and signaling
cell cycle control, cell division, chromosome partitioning
nuclear structure
defense mechanisms
signal transduction mechanisms
cell wall/membrane/envelope biogenesis
cell motility
cytoskeleton
extracellular structures
intracellular trafficking, secretion, and vesicular transport
posttranslational modification, protein turnover, chaperones
metabolism
energy production and conversion
carbohydrate transport and metabolism
amino acid transport and metabolism
nucleotide transport and metabolism
coenzyme transport and metabolism
lipid transport and metabolism
inorganic ion transport and metabolism
secondary metabolites biosynthesis, transport and catabolism
poorly characterized
general function prediction only
function unknown
a region of the chromosome  extending either side of oric appears to contain the majority of the genes predicted to be essential with the highest density in close proximity to oric .
this distribution of housekeeping genes with the notable core region is typical for actinobacterial genomes  <cit> .
outside this region, the chromosome has apparently undergone major expansion by acquiring novel genetic elements as a result of horizontal gene transfer.
this oric-distal region is enriched with the genetic loci encoding secondary metabolism, many of which have little similarity to other pseudonocardiaceae secondary metabolism genes.
after manual correction of antismash results  <cit> , approximately 14% of the k. albida chromosome  was found to be involved in secondary metabolites biosynthesis.
this includes  <dig> orfs, or  <dig> % of all genes annotated in the genome.
thus, secondary metabolism genes occupy a significantly bigger proportion of k. albida genome when compared to other actinomycetes, including sequenced genomes of pseudonocardiaceae.
for example, s. coelicolor a <dig> secondary metabolism genes cover only 5% of the chromosome, whereas in s. avermitilis ma- <dig> secondary metabolism genes represent  <dig> % of the genome length  <cit> .
another feature of the k. albida chromosome is the presence of numerous genomic islands outside the ancestral core genome.
these regions comprise the youngest part of the chromosome acquired as a result of horizontal gene transfer events.
they are characterized by lower than average genomic g + c content, biased codon usage, as well as presence of numerous insertion elements and transposon relicts.
among k. albida genomic islands, the most interesting are the two large regions with a length of  <dig> kbp  and  <dig> kbp , respectively .
these regions are significantly distinct from the rest of the genome.
additional synteny analysis between k. albida and draft genome sequence of k. spp.
<dig>  clearly showed that the longer genomic island is acquired  <cit> .
we were unable to determine the origin of the  <dig> kbp genomic island, but in the case of the  <dig> kbp region there were clearly no counterparts identified in sequence of k. spp.
<dig> genome, even though the regions around this portion of the k. albida chromosome are similar in both species.
both regions designated as genomic island are surrounded by the long inverted repeats  that might point on their origin from the large plasmids that were inserted into the chromosome.
however, other possibilities cannot be excluded.
interestingly, the location of the predicted genomic islands corresponds to the locations of the regions with high density of secondary metabolism gene clusters indicating their possible origin .
phylogenetic and orthologous analysis
k. albida was originally classified as “streptosporangium albidum” in  <dig>  <cit> .
however, later it was renamed to kutzneria albida due to the primary structures of 5s rrna and 16 s rrna, as well as to chemotaxonomic properties of the strain and the electrophoretic mobility of its ribosomal proteins  <cit> .
consequently, the genus was transferred to the family pseudonocardiaceae, due to the range of taxonomic features that were found to be more similar to the typical strain of the family, saccharothrix australiensis atcc 31497t
<cit> .
an unsupervised nucleotide blast analysis of the dna sequence corresponding to the 16s rrna gene from k. albida with the 16s rrna of different actinobacteria and e. coli as out-group was performed to determine phylogenetic relationships of the strain within the taxon .
this analysis clearly showed that k. albida is distinct from the genus streptosporangium and streptomyces, and closer related to representatives of pseudonocardiaceae.
the highest similarity was observed between 16s rrna of k. albida and lentzea albida, actinosynnema mirum, saccharothrix algeriensis, streptoalloteichus tenebrarius, saccharopolyspora erythraea and amycolatopsys alba, all of which belong to the pseudonocardiacea family.
at the same time, it is clear that the closest relatives of k. albida inside pseudonocardiaceae are streptoalloteichus tenebrarius, lentzea albida, actinosynnema mirum, and saccharothrix algeriensis .
these species are forming a branch distinct from other representatives of the family.figure  <dig>
the 16s rrna phylogram of representative
actinobacteria
strains, with an outgroup containing e. coli as non-related strains.
ribosomal rna sequences for all strains but s. albus were obtained from the silva rrna database  <cit> .
bootstrap value  <dig>
the molecular phylogeny fully corresponds to and supports the data obtained during comparison of 16s rrna.
as expected, k. albida shares the highest number of orthologous genes  with the pseudonocardiaceae and less with the streptosporangiaceae .
ten genomes of actinobacteria were used in this analysis: kutzneria albida dsm 43870t, actinosynnema mirum dsm 43827t, amycolatopsis mediterranei s <dig>  kitasatospora setae nbrc 14216t, saccharopolyspora erythrea nrrl  <dig>  saccharotrix espanaensis dsm 44229t, streptomyces avermitilis ma- <dig>  streptomyces coelicolor a <dig>  streptomyces griseus subsp.
griseus nbrc  <dig>  streptosporangium roseum dsm 43021t.
as expected, k. albida shares the highest number of orthologs with a. mediterranei , followed by saccharotrix espanaensis , actinosynnema mirum, and s. erythraea .
in comparison, the number of genes shared with streptosporangium roseum is  <dig> .
in contrast, the k. albida genome shares less orthologous genes with all the species from the genus streptomyces tested: s. coelicolor –  <dig> , s. avermitilis –  <dig> , s. griseus –  <dig> .
furthermore, when comparing all analyzed genomes, the number of orthologs drops to  <dig>  genes, defining the non-strict minimal core genome shared between the analyzed species.
this number is bigger than the previously predicted core genome for the family  <cit>  because of a less strict method of calculating orthologs used in this study.
our result should not be regarded as a disagreement with earlier results, because our goal is only to provide additional support for the proper placement of kutzneria albida among other pseudonocardiaceae – we are not aiming at the identification of the 10-species core genome.
most of these  <dig>  genes are located around oric, while genes unique to k. albida or conserved in only one species are located further away from the oric.table  <dig>
number of orthologous genes shared by
k. albida
and each one of the other 9
actinomycetales
genomes
<dig>  saccharotrix espanaensis
<dig>  actinosynnema mirum
<dig>  amycolatopis mediterranei
<dig>  streptosporangium roseum
<dig>  saccharopolispora erythraea
<dig>  kutzneria albida
<dig>  streptomyces coelicolor
<dig>  streptomyces avermitilis
<dig>  streptomyces griseus
<dig>  kitasatospora setae
last row shows orthologs common to all  <dig> genomes.
comparison of the secondary metabolome of different actinobacteria
s. espanaensis
dsm 44229
t
a. mirum
dsm 43827
t
a. mediterranei
s699
s. roseum dsm 43021
t
s. erythraea
nrrl 2338
k. albida
dsm 43870
t
<dig>  <dig> 926
5
8
16
7
10
46
s. coelicolor
a3
s. avermitilis
ma-4680
s. griseus
nbrc 13350
k. setae
nbrc 14216
t
s. viridis
p101
t
p. dioxanivorans
cb1190
aculeximycin biosynthesis gene cluster
the only characterized secondary metabolite produced by k. albida is aculeximycin   <cit> .
this compound is particularly interesting due to its activity against a broad range of gram-positive bacteria, as well as against fungi and mosquitoes  <cit> .
aculeximycin, like similar metabolite streptoviridin from kutzneria viridogrisea, exerts strong general toxicity caused by uncoupling of oxidative phosphorylation in mitochondria  <cit> .
on the other hand, this compound has an intriguing chemical structure with five sugars attached to the macrolactone  <cit> .
analysis of the k. albida genome sequence revealed the entire set of genes required for the aculeximycin molecule assembly clustered in a region of the chromosome approximately  <dig>  mbp away from the oric.
the acu cluster is  <dig>  kbp long and contains  <dig> orfs .
the core of the cluster comprises eight genes encoding type i polyketide synthases  with  <dig> modules in total, each containing different sets of reductase and acyltransferase domains as predicted by antismash and searchpks   <cit> .
since biosynthesis of the aculeximycin polyketide scaffold is predicted to require  <dig> condensation steps including loading it makes us believe that one module is skipped during elongation process or alternate utilization of two modules takes place  <cit> .
the first module encoded by acuai , a three-modular synthase, contains a full set of ketoreduction domains that corresponds to the first condensation step of biosynthesis.
however, neither the loading module nor the other four first elongation steps could be predicted based on a collinear logic of polyketide assembly, since the sets of ketoreduction domains in the second and third modules of acuai  and three modules of acuaii  did not correspond to the primary structure of the aculeximycin polyketide  <cit> .
the last module encoded by acuaviii , contains a thioesterase domain  similar to te domains of erythromycin and pikromycin synthases that are catalysing lactonization of the matured chain  <cit> .
malonate, methylmalonate, and ethylmalonate are used as precursors in polyketide chain extension.
the hydroxyl group at c <dig> position is most probably incorporated as post-pks oxygenation, and the gene encoding a hydroxylase, acuo <dig> , is present within the cluster .figure  <dig>
chemical structure of aculeximycin and genetic organization of the
acu
biosynthesis gene cluster.
pks domain prediction was performed with antismash and searchpks tools  <cit> .
domain abbreviations: dd – docking domain, ks – ketosynthase, at – acyltransferase, dh – dehydratase, er – enoylreductase, kr – ketoreductase, acp – acyl carrying protein, te – thioesterase.
aculeximycin is a highly glycosylated compound .
two sugars – d-mannose and l-vancosamine – are attached at positions c <dig> and c <dig>  respectively  <cit> .
a short oligosaccharide chain named aculexitriose -o--6-deoxy-d-glucopyranose) is attached at position c <dig>  d-mannose is supplied from the primary metabolism, as in the case of biosynthesis of other antibiotics containing this moiety  <cit> .
deoxysugar biosynthesis genes are present within the cluster: the genes acus <dig>  and acus <dig>  encode glucose-1-phosphate thymidylyltransferase and dtdp-glucose  <dig> -dehydratase, respectively – enzymes participating in the initial common steps in deoxysugars biosynthesis  <cit> .
genes for downstream enzymes leading to the production of each individual sugar from ndp-4-keto-6-deoxy-d-glucose are also present within the cluster.
l-vancosamine is built by the sequential action of acus <dig> , acus <dig> , acus <dig> , acus <dig> , and acun <dig>  or acun <dig>  aminotransferases, similar to what had been described for the glycopeptide antibiotic chloroeremomycin  <cit> ; 6-deoxy-glucopyranose – acus5; 3-amino- <dig> ,6-trideoxy-beta-d-arabino-hexopyranose – acus <dig>  acus <dig>  acun <dig> or acun <dig> .
eight putative glycosyltransferase genes  were found within the aculeximycin biosynthesis gene cluster.
the redundant number of gts can be explained by the fact that some of them might participate in self-resistance mechanism.
acugt <dig>  has a high degree of similarity to numerous glycosyltransferases involved in resistance to macrolides, including one involved in self-resistance of oleandomycin producer  <cit> .
another component of this resistance complex might be the gene product of acuh  encoding a β-hexosaminidase homologue.
this family of enzymes is involved in the cleavage of galnac residues from oligosaccharides.
the acu cluster also contains acuw  gene encoding type iii abc transporter protein containing both transmembrane and atp binding domains within one polypeptide  <cit> .
in addition,  <dig> putative regulatory genes were found within the acu cluster.
one of them, acur <dig> , belongs to the tetr family of transcription regulators and is located next to the acuw gene.
four other regulators  belong to the luxr family and might participate in control and fine tuning of the aculeximycin production   <cit> .
secondary metabolites production potential
besides aculeximycin, no other secondary metabolites are known to be produced by k. albida.
however, genome analysis using antismash revealed  <dig> gene clusters potentially involved in secondary metabolism, including the acu cluster  <cit> .
manual correction of the obtained data resulted in  <dig> gene clusters related to secondary metabolism .
this makes us think that the k. albida genome is one of the richest in terms of secondary metabolism genes reported till now.
general features of the secondary metabolism gene clusters of k. albida are summarized in additional file 1: table s <dig>
the most represented type of secondary metabolite biosynthesis genes within the k. albida genome are non-ribosomal peptide synthases  .
core genes in clusters kal  <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> encode nrps proteins with only three domains: adenylation , peptidyl-carrier protein , and an approximately  <dig> aa long n-terminal domain that is proposed to act as condensation domain   <cit> .
nrps genes in these clusters are accompanied by genes usually found in the biosynthesis of siderophores .
several other nrps clusters in the genome could be designated as siderophores producing due to the presence of genes encoding ornithine n-monooxygenase  or isochorismate  synthase  known to be involved in biosynthesis of precursors , or conserved fe3+-siderophore abc transport system genes   <cit> .
besides nrps produced siderophores, kal <dig> cluster is predicted to be responsible for biosynthesis of the aerobactin-like fe3+-chelating compound found in different species of corynebacterium and bacillus
<cit> .
the second most abundant k. albida secondary metabolism genes are encoding polyketide biosynthesis.
analysis of the genome revealed  <dig> type i pks  and  <dig> type ii pks  gene clusters .
the kal <dig> cluster consists of one gene encoding single module pks  with a full set of ketoreductase domains.
the closest homologues of this enzyme encode mycocerosic acid synthase in stigmatella aurantiaca and in several mycobacterium species involved in the production of the multimethyl-branched fatty acids by the elongation of fatty acids with  <dig> units of methyl-malonate  <cit> .
the core genes in the cluster kal <dig>  are similar to genes encoding omega-3-polyunsaturated fatty acid synthases.
these unusual fatty acids are known to accumulate in the membranes of cold- and high pressure-resistant marine bacteria  <cit> .
other type i pks gene clusters  have no homologues in another sequenced bacterial genomes and their products could be just partially predicted from the genes organization.
two other pks clusters encode type ii pks systems that show some similarity to whie clusters involved in spore pigment production in different streptomycetes   <cit>  and to genes involved in biosynthesis of the aromatic polyketides mithramycin  <cit>  and nogalamycin  <cit>  .
seven k. albida gene clusters combine type i pks and nrps biosynthetic pathways   <cit> .
gene clusters kal <dig> and kal <dig> encode type i pkss and a nrpss with a single condensation domain.
the pks portion of kal <dig> resembles the ks involved in production of phenolic glycolipids in mycobacteria, another fe3+-chelating compound  <cit> .
the nrps part might act at the initiation step by direct loading of the starting unit into the acp of pks, similar to the mechanism proposed for biosynthesis of mycobactins, mycobacterial siderophores of the phenolic glycolipids family  <cit> .
<dig> gene clusters for terpenoid biosynthesis could be found in the k. albida genome .
the c <dig> precursors for these secondary metabolites are provided by the non-mevalonate  pathway, genes for which are present in the genome  <cit> .
at the same time we were unable to identify any genes encoding enzymes of the mevalonate pathway.
the kal <dig> and kal <dig> gene clusters a predicted to be involved in the production of the earthy flavored sesquiterpene geosmin and moldy-smelling monoterpene 2-methylisoborneol  respectively  <cit> .
core genes of both of them are highly conserved among different actinomycetes.
interestingly, there is one more germacradienol synthase gene  located upstream from geo <dig>  in kal <dig> cluster.
its function is not clear.
kal <dig> cluster is predicted to be involved in the biosynthesis of carotenoids based on the key gene ctrb <dig>  similarity to the known phytoene synthases  <cit> .
similar, kal <dig> gene cluster is suggested to be involved in the biosynthesis of hopanoids, bacterial pentacyclic triterpenoids  <cit> .
several other types of secondary metabolites could be potentially produced by k. albida .
the gene cluster kal <dig> encodes  <dig> enzymes involved in the biosynthesis of compatible solutes ectoine and 5-hydroxyectoine  <cit> .
the kal <dig> cluster is similar to genes involved in biosynthesis of indolocarbazole group of secondary metabolites  <cit> .
several lantibiotics biosynthesis gene clusters could be found in the genome of k. albida as well .
interestingly, orfs  <dig> and  <dig> from kal <dig> are coding for ycao domain-containing proteins involved in the formation of thiazole/oxazole, another post-translational modifications observed in ribosomally synthesized natural products  <cit> .
the cluster kal <dig> contains only one gene , which encodes a 13 kda protein with the high degrees of similarity to bacteriocins of the linocin m <dig> family  <cit> .
linocin m <dig> was first isolated from brevibacterium linens due to its ability to inhibit the growth of several listeria species, and similar genes were later discovered in other bacterial genomes.
recent findings indicate that linocins might play a role in the compartmentalization of oxidative-stress response processes in bacterial cells  <cit> .
at the same time, the product of kalb_ <dig> is two times shorter than typical linocin m <dig>
testing secondary metabolism potential
the genome sequence of k. albida unveiled enormous secondary metabolites biosynthetic potential of this bacterium .
however, so far only aculeximycin is known to be produced by this strain  <cit> .
to further elucidate the biosynthetic potential of k. albida the strain was grown in different media and extra- and intra-cellular accumulated metabolites were tested using high resolution lc-ms .
after 7 days of growth aculeximycin and its aglycone production was observed only in two out of six used media.
at the same time, multiple secondary metabolites accumulation was observed in all cases.
the dnp analysis  <cit>  of obtained data led to idea that majority of compounds accumulated by k. albida are not described yet.
furthermore, extracts were found to be active against bacillus subtilis, even those that did not contain aculeximycin.
these facts are making further analysis of secondary metabolites produced by k. albida especially interesting.
as expected from the secondary metabolism genes analysis many of the metabolites produced by k. albida are acting as siderophores .
a siderophores accumulation test using a modified cas assay clearly showed that k. albida produced significant amounts of fe3+-chelating compounds during growth on different media when compared to s. coelicolor and s. albus   <cit> .
additionally, extracts from cultures grown on tested media including liquid and solid cas media were separated by tlc and overlaid with cas agarose for siderophores detection .
as a result, k. albida was found to accumulate from one to at least  <dig> fe3+-chelating compounds with different physicochemical properties when growing at different conditions.
any of them could be predicted based on information available in dnp  <cit> .
this finding supports our prediction of evolving of the k. albida genome in directions leading to adaptation to iron deficient environment, where the strain was isolated from  <cit> .
one of the previously described compounds that were identified within the extracts from k. albida is a cyclic leucilphenylalanine  .
it was produced in  <dig> out of  <dig> tested media.
this compound accumulation could be directly linked to the cyclodipeptide synthase gene  found in the kal <dig> cluster.
these proteins comprise an interesting class of enzymes utilizing amino-acyl trna as a substrate to produce diketopiperazine containing cyclic dipeptides .
in many cases the cyclodipeptide synthase products are further modified by the decorating enzymes.
no genes possibly involved in post-processing of cyclic peptides were found in close proximity to the kalb_ <dig>  the number of recently discovered cyclodipeptide synthases is expanding.
in many cases these enzymes are not strictly specific for some particular substrates and can produce several types of cyclic peptides  <cit> .
however, we were not able to identify any other possible products of kalb_ <dig> in extracts of k. albida.
in order to test the enzyme specificity the gene was cloned and expressed in e. coli.
comparison of extracts from e. coli containing kalb_ <dig> expression construct and empty vector control led to identification of four new compounds .
one of them, as was predicted from analysis of k. albida extracts, was cfl.
three other compounds were identified as cfm, cfy and cff based on exact mass and fragmentation patterns .
this finding led us to the conclusion that the k. albida enzyme as the first substrate prefers phenylalanine, but can also utilize other amino acids as a second substrate.
re-examination of k. albida extracts led to identification of cff and cfy, however not cfm.figure  <dig>
hplc-ms analysis of extracts from
e. coli
expressing kalb_ <dig>  a. uv–vis traces that correspond to the kalb_ <dig> expressing strain  and control strain  are shown.
cyclic dipeptides are marked and their masses are indicated.
b. ms2-fragmentation spectra of kalb_ <dig> products.
the characteristic neutral losses of  <dig> and 45 da, resulting in the detection of the respective ammonium ions are shown.
CONCLUSIONS
the complete genome of kutzneria albida, the first representative of kutzneria genus was sequenced, annotated and analyzed.
the genome of this strain is one of the biggest circular actinobacterial genomes sequenced thus far.
the phylogenetic and orthology analyses clearly distinguish kutzneria albida from streptosporangiaceae thereby providing the first genomic evidence for transferring the genus into the pseudonocardiaceae family.
two large genomic islands are present in the k. albida genome.
localization of these islands corresponds to regions of a high density of genes involved in secondary metabolism providing clues into the origin of a large part of the strain’s auxiliary metabolism.
in general, about 14% of the chromosome is occupied with the secondary metabolism gene clusters, including cluster predicted to be involved in aculeximycin biosynthesis.
aculeximycin and its aglycone accumulation were observed during growth of the strain in several media.
however, a vast majority of compounds produced by the strain were not found in available secondary metabolites databases.
as predicted from the genome analysis and confirmed experimentally, a large proportion of secondary metabolism of k. albida is devoted to siderophores production.
on the other hand, cyclic dipeptides were found in the extract of the strain.
in summary, sequencing of the k. albida genome provides new insights into understanding the evolution of minor groups of actinobacteria and will attract more attention to these fascinating bacteria as an inexhaustible source of novel biologically active secondary metabolites.
the large diversity of secondary metabolism gene clusters in the genome of k. albida is reflected in metabolites produced.
furthermore, isolation, structural and biological characterization of secondary metabolites produced by this strain might lead to discovery of new interesting biological activities as well as new chemical scaffolds thus proving the concept of genome mining of minor groups of actinobacteria for new secondary metabolites discovery.

 bacteria of the actinomycetales order represent an amazing source of biologically active compounds which are produced as secondary metabolites. the broad spectrum of structural features and wide array of activities of these metabolites attract attention as a limitless source of novel chemical scaffolds as well as new drugs for human and veterinary medicine, and agriculture. with the recent advances in sequencing technologies significant progress in sequencing and analysis of actinobacterial genomes was achieved. not only genomes of biotechnologically important and typical representatives of the taxonomical order were sequenced, but also strains with interesting and unusual features, leading to deeper insights into phylogeny, genetics, physiology, ecology, and the secondary metabolism biosynthetic potential of these bacteria. one of the major discoveries of the genomic era in actinomycetes research is the presence of multiple gene clusters dedicated to secondary metabolism in one genome. this observation caused an increase of interest in diverse groups of actinomycetes as a possible source of new biologically active metabolites. one of such groups includes species belonging to the pseudonocardiaceae family. multiple genome projects dedicated to this group of bacteria are in progress, with seven being completed and published. here we report the sequencing and analysis of the complete genome of kutzneria albida dsm 43870t. the genus kutzneria is a minor branch of the pseudonocardiaceae family currently containing only species. to our knowledge, this is the first member of the kutzneria genus and only the eighth representative of the family pseudonocardiaceae for which a complete genome sequence is available. k albida was isolated from soil samples collected in the rocky regions of gunma prefecture of japan. the strain is known as a producer of aculeximycin, a macrolide antibiotic with an interesting chemical structure and activity against gram-positive bacteria, fungi, and mosquito larvae. being generally toxic, aculeximycin cannot be used in medicine. however, some structural features, especially the unusual glycoside chain, make it an attractive source of building blocks for use in synthetic biotechnology and combinatorial biosynthesis of secondary metabolites. sequencing k. albida genome allowed not only to identify genes responsible for aculeximycin biosynthesis, but also to discover an unusual abundance and diversity of secondary metabolites that could potentially be produced by this species. to the best of our knowledge, this genome is the most enriched with clusters involved in secondary metabolites among actinobacterial genomes.  general features of the genome after gaps closure, a single contig with the size of,926 bp and a % g + c content was obtained. general features of k. albida genome are summarized in table  the genome of k. albida consists of a single circular replicon; no extrachromosomal replicon was detected. the origin of replication was identified as a  nt non-coding region between the two genes – dnaa, encoding the chromosomal replication initiation factor, and dnan, coding for the β-subunit of the dna polymerase iii. interestingly, the oric has no classic dnaa boxes, and shows almost no similarity to the oric of streptomyces species. on the opposite side of the chromosome from oric, a putative dif locus was identified. its sequence is in good accordance with actinobacterial dif sites. additionally, the obvious cumulative gc skew inversion in the regions of oric and dif supports our oric assignment and suggests the existence of two replichores composing the circular k. albida chromosome.table general features of the k. albida chromosome topology number of replicons total size g + c content protein coding genes rrna genes rrna operons transfer rnas average gene length coding density secondary metabolite clustersschematic representation of the kutzneria albida genome, created with the help of circos; megabases are labeled; smaller ticks correspond to kbp segments. from outside: genes on the forward and the reverse strands; secondary metabolite clusters coloured by type; genomic islands; g + c content, 10kbp window; g + c content, kbp window; g + c skew; cumulative g + c skew. the oric is placed at coordinate zero. unlike the linear topology of streptomyces genomes, the chromosome of k. albida is circular. this appears to be a common feature of pseudonocardiaceae representatives genomes, since the chromosomes of other representatives of this family sequenced so far also have a circular topology. with a total length of,926 bp, the k. albida chromosome is larger than that of streptomyces coelicolor a and streptomyces avermitilis ma-, as well as genomes of pseudonocardiaceae saccharomonospora viridis p101t, pseudonocardia dioxanivorans cb, saccharothrix espanaensis dsm 44229t, and saccharopolyspora erythraea nrrl, but slightly smaller than the genome of another pseudonocardiaceae rifamycin producer amycolatopsis mediterranei s. the k. albida genome is among the largest actinobacterial genomes sequenced so far. k. albida chromosome contains predicted protein-coding sequences. three ribosomal rna operons were identified in the genome along with trna genes. the rrn operons are located on the leading strand of the chromosome proximally to oric and have remarkably low g + c content in comparison to the genome average. functional annotation of k. albida genes within the actnog subset of the eggnog database, showed that out of genes had at least one biological function assigned, with some of the genes assigned to more than one category. of the remainder, cdss had no hits against actnogs, and genes had hits but were not assigned to functional categories; of the latter, are highly conserved. among the genes with functional assignment, are implicated in metabolism, including in amino acid metabolism, in carbohydrate metabolism, and participating in secondary metabolism. at the same time, 10% of the annotated genes code for proteins involved in transcriptional processes, including sigma factors, anti-sigma factors, anti-anti-sigma factors, and putative dna binding regulators. the large number of proteins implicated in transcriptional processes suggests a high complexity of k. albida gene expression control. the high proportion of proteins involved in transcriptional regulation is accompanied by a similarly high percentage of proteins involved in signal transduction pathways, which suggests a close connection between extracellular nutrient sensing and transcriptional regulation of uptake and degradation pathways. this feature of the k. albida genome is typical for the majority of known soil living actinobacteria and reflects their life style and ecological niche, providing higher flexibility of the strain adaptation to changes in growth conditions. in addition to this, a large portion of the genome encodes extracellular polymer-degrading enzymes. in total, putative secretory hydrolase encoding genes were found in the k. albida genome, including glycoside hydrolases and extracellular proteases.table assignment of k. albida genes to the functional groups of the actnog subset of the eggnog database information storage and processing translation, ribosomal structure and biogenesis rna processing and modification transcription replication, recombination and repair chromatin structure and dynamics cellular processes and signaling cell cycle control, cell division, chromosome partitioning nuclear structure defense mechanisms signal transduction mechanisms cell wall/membrane/envelope biogenesis cell motility cytoskeleton extracellular structures intracellular trafficking, secretion, and vesicular transport posttranslational modification, protein turnover, chaperones metabolism energy production and conversion carbohydrate transport and metabolism amino acid transport and metabolism nucleotide transport and metabolism coenzyme transport and metabolism lipid transport and metabolism inorganic ion transport and metabolism secondary metabolites biosynthesis, transport and catabolism poorly characterized general function prediction only function unknown a region of the chromosome extending either side of oric appears to contain the majority of the genes predicted to be essential with the highest density in close proximity to oric. this distribution of housekeeping genes with the notable core region is typical for actinobacterial genomes. outside this region, the chromosome has apparently undergone major expansion by acquiring novel genetic elements as a result of horizontal gene transfer. this oric-distal region is enriched with the genetic loci encoding secondary metabolism, many of which have little similarity to other pseudonocardiaceae secondary metabolism genes. after manual correction of antismash results, approximately 14% of the k. albida chromosome was found to be involved in secondary metabolites biosynthesis. this includes orfs, or % of all genes annotated in the genome. thus, secondary metabolism genes occupy a significantly bigger proportion of k. albida genome when compared to other actinomycetes, including sequenced genomes of pseudonocardiaceae. for example, s. coelicolor a secondary metabolism genes cover only 5% of the chromosome, whereas in s. avermitilis ma- secondary metabolism genes represent % of the genome length. another feature of the k. albida chromosome is the presence of numerous genomic islands outside the ancestral core genome. these regions comprise the youngest part of the chromosome acquired as a result of horizontal gene transfer events. they are characterized by lower than average genomic g + c content, biased codon usage, as well as presence of numerous insertion elements and transposon relicts. among k. albida genomic islands, the most interesting are the two large regions with a length of kbp and kbp, respectively. these regions are significantly distinct from the rest of the genome. additional synteny analysis between k. albida and draft genome sequence of k. spp. clearly showed that the longer genomic island is acquired. we were unable to determine the origin of the kbp genomic island, but in the case of the kbp region there were clearly no counterparts identified in sequence of k. spp. genome, even though the regions around this portion of the k. albida chromosome are similar in both species. both regions designated as genomic island are surrounded by the long inverted repeats that might point on their origin from the large plasmids that were inserted into the chromosome. however, other possibilities cannot be excluded. interestingly, the location of the predicted genomic islands corresponds to the locations of the regions with high density of secondary metabolism gene clusters indicating their possible origin. phylogenetic and orthologous analysis k. albida was originally classified as “streptosporangium albidum” in. however, later it was renamed to kutzneria albida due to the primary structures of 5s rrna and 16 s rrna, as well as to chemotaxonomic properties of the strain and the electrophoretic mobility of its ribosomal proteins. consequently, the genus was transferred to the family pseudonocardiaceae, due to the range of taxonomic features that were found to be more similar to the typical strain of the family, saccharothrix australiensis atcc 31497t. an unsupervised nucleotide blast analysis of the dna sequence corresponding to the 16s rrna gene from k. albida with the 16s rrna of different actinobacteria and e. coli as out-group was performed to determine phylogenetic relationships of the strain within the taxon. this analysis clearly showed that k. albida is distinct from the genus streptosporangium and streptomyces, and closer related to representatives of pseudonocardiaceae. the highest similarity was observed between 16s rrna of k. albida and lentzea albida, actinosynnema mirum, saccharothrix algeriensis, streptoalloteichus tenebrarius, saccharopolyspora erythraea and amycolatopsys alba, all of which belong to the pseudonocardiacea family. at the same time, it is clear that the closest relatives of k. albida inside pseudonocardiaceae are streptoalloteichus tenebrarius, lentzea albida, actinosynnema mirum, and saccharothrix algeriensis. these species are forming a branch distinct from other representatives of the family.figure the 16s rrna phylogram of representative actinobacteria strains, with an outgroup containing e. coli as non-related strains. ribosomal rna sequences for all strains but s. albus were obtained from the silva rrna database. bootstrap value the molecular phylogeny fully corresponds to and supports the data obtained during comparison of 16s rrna. as expected, k. albida shares the highest number of orthologous genes with the pseudonocardiaceae and less with the streptosporangiaceae. ten genomes of actinobacteria were used in this analysis: kutzneria albida dsm 43870t, actinosynnema mirum dsm 43827t, amycolatopsis mediterranei s kitasatospora setae nbrc 14216t, saccharopolyspora erythrea nrrl saccharotrix espanaensis dsm 44229t, streptomyces avermitilis ma- streptomyces coelicolor a streptomyces griseus subsp. griseus nbrc streptosporangium roseum dsm 43021t. as expected, k. albida shares the highest number of orthologs with a. mediterranei, followed by saccharotrix espanaensis, actinosynnema mirum, and s. erythraea. in comparison, the number of genes shared with streptosporangium roseum is. in contrast, the k. albida genome shares less orthologous genes with all the species from the genus streptomyces tested: s. coelicolor –, s. avermitilis –, s. griseus –. furthermore, when comparing all analyzed genomes, the number of orthologs drops to genes, defining the non-strict minimal core genome shared between the analyzed species. this number is bigger than the previously predicted core genome for the family because of a less strict method of calculating orthologs used in this study. our result should not be regarded as a disagreement with earlier results, because our goal is only to provide additional support for the proper placement of kutzneria albida among other pseudonocardiaceae – we are not aiming at the identification of the 10-species core genome. most of these genes are located around oric, while genes unique to k. albida or conserved in only one species are located further away from the oric.table number of orthologous genes shared by k. albida and each one of the other 9 actinomycetales genomes saccharotrix espanaensis actinosynnema mirum amycolatopis mediterranei streptosporangium roseum saccharopolispora erythraea kutzneria albida streptomyces coelicolor streptomyces avermitilis streptomyces griseus kitasatospora setae last row shows orthologs common to all genomes. comparison of the secondary metabolome of different actinobacteria s. espanaensis dsm 44229 t a. mirum dsm 43827 t a. mediterranei s699 s. roseum dsm 43021 t s. erythraea nrrl 2338 k. albida dsm 43870 t 926 5 8 16 7 10 46 s. coelicolor a3 s. avermitilis ma-4680 s. griseus nbrc 13350 k. setae nbrc 14216 t s. viridis p101 t p. dioxanivorans cb1190 aculeximycin biosynthesis gene cluster the only characterized secondary metabolite produced by k. albida is aculeximycin. this compound is particularly interesting due to its activity against a broad range of gram-positive bacteria, as well as against fungi and mosquitoes. aculeximycin, like similar metabolite streptoviridin from kutzneria viridogrisea, exerts strong general toxicity caused by uncoupling of oxidative phosphorylation in mitochondria. on the other hand, this compound has an intriguing chemical structure with five sugars attached to the macrolactone. analysis of the k. albida genome sequence revealed the entire set of genes required for the aculeximycin molecule assembly clustered in a region of the chromosome approximately mbp away from the oric. the acu cluster is kbp long and contains orfs. the core of the cluster comprises eight genes encoding type i polyketide synthases with modules in total, each containing different sets of reductase and acyltransferase domains as predicted by antismash and searchpks. since biosynthesis of the aculeximycin polyketide scaffold is predicted to require condensation steps including loading it makes us believe that one module is skipped during elongation process or alternate utilization of two modules takes place. the first module encoded by acuai, a three-modular synthase, contains a full set of ketoreduction domains that corresponds to the first condensation step of biosynthesis. however, neither the loading module nor the other four first elongation steps could be predicted based on a collinear logic of polyketide assembly, since the sets of ketoreduction domains in the second and third modules of acuai and three modules of acuaii did not correspond to the primary structure of the aculeximycin polyketide. the last module encoded by acuaviii, contains a thioesterase domain similar to te domains of erythromycin and pikromycin synthases that are catalysing lactonization of the matured chain. malonate, methylmalonate, and ethylmalonate are used as precursors in polyketide chain extension. the hydroxyl group at c position is most probably incorporated as post-pks oxygenation, and the gene encoding a hydroxylase, acuo, is present within the cluster.figure chemical structure of aculeximycin and genetic organization of the acu biosynthesis gene cluster. pks domain prediction was performed with antismash and searchpks tools. domain abbreviations: dd – docking domain, ks – ketosynthase, at – acyltransferase, dh – dehydratase, er – enoylreductase, kr – ketoreductase, acp – acyl carrying protein, te – thioesterase. aculeximycin is a highly glycosylated compound. two sugars – d-mannose and l-vancosamine – are attached at positions c and c respectively. a short oligosaccharide chain named aculexitriose -o--6-deoxy-d-glucopyranose) is attached at position c d-mannose is supplied from the primary metabolism, as in the case of biosynthesis of other antibiotics containing this moiety. deoxysugar biosynthesis genes are present within the cluster: the genes acus and acus encode glucose-1-phosphate thymidylyltransferase and dtdp-glucose -dehydratase, respectively – enzymes participating in the initial common steps in deoxysugars biosynthesis. genes for downstream enzymes leading to the production of each individual sugar from ndp-4-keto-6-deoxy-d-glucose are also present within the cluster. l-vancosamine is built by the sequential action of acus, acus, acus, acus, and acun or acun aminotransferases, similar to what had been described for the glycopeptide antibiotic chloroeremomycin; 6-deoxy-glucopyranose – acus5; 3-amino-,6-trideoxy-beta-d-arabino-hexopyranose – acus acus acun or acun. eight putative glycosyltransferase genes were found within the aculeximycin biosynthesis gene cluster. the redundant number of gts can be explained by the fact that some of them might participate in self-resistance mechanism. acugt has a high degree of similarity to numerous glycosyltransferases involved in resistance to macrolides, including one involved in self-resistance of oleandomycin producer. another component of this resistance complex might be the gene product of acuh encoding a β-hexosaminidase homologue. this family of enzymes is involved in the cleavage of galnac residues from oligosaccharides. the acu cluster also contains acuw gene encoding type iii abc transporter protein containing both transmembrane and atp binding domains within one polypeptide. in addition, putative regulatory genes were found within the acu cluster. one of them, acur, belongs to the tetr family of transcription regulators and is located next to the acuw gene. four other regulators belong to the luxr family and might participate in control and fine tuning of the aculeximycin production. secondary metabolites production potential besides aculeximycin, no other secondary metabolites are known to be produced by k. albida. however, genome analysis using antismash revealed gene clusters potentially involved in secondary metabolism, including the acu cluster. manual correction of the obtained data resulted in gene clusters related to secondary metabolism. this makes us think that the k. albida genome is one of the richest in terms of secondary metabolism genes reported till now. general features of the secondary metabolism gene clusters of k. albida are summarized in additional file 1: table s the most represented type of secondary metabolite biosynthesis genes within the k. albida genome are non-ribosomal peptide synthases. core genes in clusters kal and encode nrps proteins with only three domains: adenylation, peptidyl-carrier protein, and an approximately aa long n-terminal domain that is proposed to act as condensation domain. nrps genes in these clusters are accompanied by genes usually found in the biosynthesis of siderophores. several other nrps clusters in the genome could be designated as siderophores producing due to the presence of genes encoding ornithine n-monooxygenase or isochorismate synthase known to be involved in biosynthesis of precursors, or conserved fe3+-siderophore abc transport system genes. besides nrps produced siderophores, kal cluster is predicted to be responsible for biosynthesis of the aerobactin-like fe3+-chelating compound found in different species of corynebacterium and bacillus. the second most abundant k. albida secondary metabolism genes are encoding polyketide biosynthesis. analysis of the genome revealed type i pks and type ii pks gene clusters. the kal cluster consists of one gene encoding single module pks with a full set of ketoreductase domains. the closest homologues of this enzyme encode mycocerosic acid synthase in stigmatella aurantiaca and in several mycobacterium species involved in the production of the multimethyl-branched fatty acids by the elongation of fatty acids with units of methyl-malonate. the core genes in the cluster kal are similar to genes encoding omega-3-polyunsaturated fatty acid synthases. these unusual fatty acids are known to accumulate in the membranes of cold- and high pressure-resistant marine bacteria. other type i pks gene clusters have no homologues in another sequenced bacterial genomes and their products could be just partially predicted from the genes organization. two other pks clusters encode type ii pks systems that show some similarity to whie clusters involved in spore pigment production in different streptomycetes and to genes involved in biosynthesis of the aromatic polyketides mithramycin and nogalamycin. seven k. albida gene clusters combine type i pks and nrps biosynthetic pathways. gene clusters kal and kal encode type i pkss and a nrpss with a single condensation domain. the pks portion of kal resembles the ks involved in production of phenolic glycolipids in mycobacteria, another fe3+-chelating compound. the nrps part might act at the initiation step by direct loading of the starting unit into the acp of pks, similar to the mechanism proposed for biosynthesis of mycobactins, mycobacterial siderophores of the phenolic glycolipids family. gene clusters for terpenoid biosynthesis could be found in the k. albida genome. the c precursors for these secondary metabolites are provided by the non-mevalonate pathway, genes for which are present in the genome. at the same time we were unable to identify any genes encoding enzymes of the mevalonate pathway. the kal and kal gene clusters a predicted to be involved in the production of the earthy flavored sesquiterpene geosmin and moldy-smelling monoterpene 2-methylisoborneol respectively. core genes of both of them are highly conserved among different actinomycetes. interestingly, there is one more germacradienol synthase gene located upstream from geo in kal cluster. its function is not clear. kal cluster is predicted to be involved in the biosynthesis of carotenoids based on the key gene ctrb similarity to the known phytoene synthases. similar, kal gene cluster is suggested to be involved in the biosynthesis of hopanoids, bacterial pentacyclic triterpenoids. several other types of secondary metabolites could be potentially produced by k. albida. the gene cluster kal encodes enzymes involved in the biosynthesis of compatible solutes ectoine and 5-hydroxyectoine. the kal cluster is similar to genes involved in biosynthesis of indolocarbazole group of secondary metabolites. several lantibiotics biosynthesis gene clusters could be found in the genome of k. albida as well. interestingly, orfs and from kal are coding for ycao domain-containing proteins involved in the formation of thiazole/oxazole, another post-translational modifications observed in ribosomally synthesized natural products. the cluster kal contains only one gene, which encodes a 13 kda protein with the high degrees of similarity to bacteriocins of the linocin m family. linocin m was first isolated from brevibacterium linens due to its ability to inhibit the growth of several listeria species, and similar genes were later discovered in other bacterial genomes. recent findings indicate that linocins might play a role in the compartmentalization of oxidative-stress response processes in bacterial cells. at the same time, the product of kalb_ is two times shorter than typical linocin m testing secondary metabolism potential the genome sequence of k. albida unveiled enormous secondary metabolites biosynthetic potential of this bacterium. however, so far only aculeximycin is known to be produced by this strain. to further elucidate the biosynthetic potential of k. albida the strain was grown in different media and extra- and intra-cellular accumulated metabolites were tested using high resolution lc-ms. after 7 days of growth aculeximycin and its aglycone production was observed only in two out of six used media. at the same time, multiple secondary metabolites accumulation was observed in all cases. the dnp analysis of obtained data led to idea that majority of compounds accumulated by k. albida are not described yet. furthermore, extracts were found to be active against bacillus subtilis, even those that did not contain aculeximycin. these facts are making further analysis of secondary metabolites produced by k. albida especially interesting. as expected from the secondary metabolism genes analysis many of the metabolites produced by k. albida are acting as siderophores. a siderophores accumulation test using a modified cas assay clearly showed that k. albida produced significant amounts of fe3+-chelating compounds during growth on different media when compared to s. coelicolor and s. albus. additionally, extracts from cultures grown on tested media including liquid and solid cas media were separated by tlc and overlaid with cas agarose for siderophores detection. as a result, k. albida was found to accumulate from one to at least fe3+-chelating compounds with different physicochemical properties when growing at different conditions. any of them could be predicted based on information available in dnp. this finding supports our prediction of evolving of the k. albida genome in directions leading to adaptation to iron deficient environment, where the strain was isolated from. one of the previously described compounds that were identified within the extracts from k. albida is a cyclic leucilphenylalanine. it was produced in out of tested media. this compound accumulation could be directly linked to the cyclodipeptide synthase gene found in the kal cluster. these proteins comprise an interesting class of enzymes utilizing amino-acyl trna as a substrate to produce diketopiperazine containing cyclic dipeptides. in many cases the cyclodipeptide synthase products are further modified by the decorating enzymes. no genes possibly involved in post-processing of cyclic peptides were found in close proximity to the kalb_ the number of recently discovered cyclodipeptide synthases is expanding. in many cases these enzymes are not strictly specific for some particular substrates and can produce several types of cyclic peptides. however, we were not able to identify any other possible products of kalb_ in extracts of k. albida. in order to test the enzyme specificity the gene was cloned and expressed in e. coli. comparison of extracts from e. coli containing kalb_ expression construct and empty vector control led to identification of four new compounds. one of them, as was predicted from analysis of k. albida extracts, was cfl. three other compounds were identified as cfm, cfy and cff based on exact mass and fragmentation patterns. this finding led us to the conclusion that the k. albida enzyme as the first substrate prefers phenylalanine, but can also utilize other amino acids as a second substrate. re-examination of k. albida extracts led to identification of cff and cfy, however not cfm.figure hplc-ms analysis of extracts from e. coli expressing kalb_ a. uv–vis traces that correspond to the kalb_ expressing strain and control strain are shown. cyclic dipeptides are marked and their masses are indicated. b. ms2-fragmentation spectra of kalb_ products. the characteristic neutral losses of and 45 da, resulting in the detection of the respective ammonium ions are shown.  the complete genome of kutzneria albida, the first representative of kutzneria genus was sequenced, annotated and analyzed. the genome of this strain is one of the biggest circular actinobacterial genomes sequenced thus far. the phylogenetic and orthology analyses clearly distinguish kutzneria albida from streptosporangiaceae thereby providing the first genomic evidence for transferring the genus into the pseudonocardiaceae family. two large genomic islands are present in the k. albida genome. localization of these islands corresponds to regions of a high density of genes involved in secondary metabolism providing clues into the origin of a large part of the strain’s auxiliary metabolism. in general, about 14% of the chromosome is occupied with the secondary metabolism gene clusters, including cluster predicted to be involved in aculeximycin biosynthesis. aculeximycin and its aglycone accumulation were observed during growth of the strain in several media. however, a vast majority of compounds produced by the strain were not found in available secondary metabolites databases. as predicted from the genome analysis and confirmed experimentally, a large proportion of secondary metabolism of k. albida is devoted to siderophores production. on the other hand, cyclic dipeptides were found in the extract of the strain. in summary, sequencing of the k. albida genome provides new insights into understanding the evolution of minor groups of actinobacteria and will attract more attention to these fascinating bacteria as an inexhaustible source of novel biologically active secondary metabolites. the large diversity of secondary metabolism gene clusters in the genome of k. albida is reflected in metabolites produced. furthermore, isolation, structural and biological characterization of secondary metabolites produced by this strain might lead to discovery of new interesting biological activities as well as new chemical scaffolds thus proving the concept of genome mining of minor groups of actinobacteria for new secondary metabolites discovery. 
kutzneria albida is one of the eight known members of the genus. kutzneria albida genome sequencing and analysis allow a deeper understanding of evolution of this genus of pseudonocardiaceae, provide new insight in the phylogeny of the genus, as well as decipher the hidden secondary metabolic potential of these rare actinobacteria. to explore the biosynthetic potential of kutzneria albida to its full extent, the complete genome was sequenced. kutzneria albida is the first species of the genus kutzneria with a completely sequenced genome. moreover, the genome revealed additional putative secondary metabolite gene clusters, suggesting a huge biosynthetic potential, which makes kutzneria albida a very rich source of natural products. comparison of the kutzneria albida genome to genomes of other actinobacteria clearly shows its close relations with pseudonocardiaceae in line with the taxonomic position of the genus. genome analysis revealed gene clusters potentially encoding secondary metabolite biosynthesis pathways. two large genomic islands were identified, containing regions most enriched with secondary metabolism gene clusters. kutzneria is a representative of a rarely observed genus of the family pseudonocardiaceae. large parts of this secondary metabolism “clustome” are dedicated to siderophores production.
 kutzneria is a representative of a rarely observed genus of the family pseudonocardiaceae. kutzneria species were initially placed in the streptosporangiaceae genus and later reconsidered to be an independent genus of the pseudonocardiaceae. kutzneria albida is one of the eight known members of the genus. this strain is a unique producer of the glycosylated polyole macrolide aculeximycin which is active against both bacteria and fungi. kutzneria albida genome sequencing and analysis allow a deeper understanding of evolution of this genus of pseudonocardiaceae, provide new insight in the phylogeny of the genus, as well as decipher the hidden secondary metabolic potential of these rare actinobacteria.  to explore the biosynthetic potential of kutzneria albida to its full extent, the complete genome was sequenced. with a size of,926 bp, coding for genes, it stands alongside other pseudonocardiaceae with large circular genomes. genome analysis revealed gene clusters potentially encoding secondary metabolite biosynthesis pathways. two large genomic islands were identified, containing regions most enriched with secondary metabolism gene clusters. large parts of this secondary metabolism “clustome” are dedicated to siderophores production.  kutzneria albida is the first species of the genus kutzneria with a completely sequenced genome. genome sequencing allowed identifying the gene cluster responsible for the biosynthesis of aculeximycin, one of the largest known oligosaccharide-macrolide antibiotics. moreover, the genome revealed additional putative secondary metabolite gene clusters, suggesting a huge biosynthetic potential, which makes kutzneria albida a very rich source of natural products. comparison of the kutzneria albida genome to genomes of other actinobacteria clearly shows its close relations with pseudonocardiaceae in line with the taxonomic position of the genus. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords kutzneriagenomegenomic islandssecondary metabolismaculeximycinissue-copyright-statement© the author 2014 