Complete genome sequence of Jiangella gansuensis strain YIM 002T (DSM 44835T), the type species of the genus Jiangella and source of new antibiotic compounds.

Jiangella gansuensis strain YIM 002T is the type strain of the type species of the genus Jiangella, which is at the present time composed of five species, and was isolated from desert soil sample in Gansu Province (China). The five strains of this genus are clustered in a monophyletic group when closer actinobacterial genera are used to infer a 16S rRNA gene sequence phylogeny. The study of this genome is part of the GenomicEncyclopedia ofBacteria andArchaea project, and here we describe the complete genome sequence and annotation of this taxon. The genome of J. gansuensis strain YIM 002T contains a single scaffold of size 5,585,780 bp, which involves 149 pseudogenes, 4905 protein-coding genes and 50 RNA genes, including 2520 hypothetical proteins and 4 rRNA genes. From the investigation of genome sizes of Jiangella species, J. gansuensis shows a smaller size, which indicates this strain might have discarded too much genetic information to adapt to desert environment. Seven new compounds from this bacterium have recently been described; however, its potential should be higher, as secondary metabolite gene cluster analysis predicted 60 gene clusters, including the potential to produce the pristinamycin.

Introduction

strain YIM 002 T (=DSM 44835 T =CCTCC AA 204001 T =KCTC 19044 T) is the type strain of . This organism is an aerobic, Gram-positive, haloduric filamentous actinomycete, placed within the genus [1].

The genus was first identified by Song et al. in 2005, including five halotolerant species listed at present by LPSN [2]. Members of this taxon isolated from different habitats, respectively, are rarely described except for their polyphasic approach based on combination of phenotypic and genotypic characteristics [1, 3–6]. The was originally identified as a new genus of the family within the suborder [1] based on phenotypic and genotypic criteria. However, the reconstruction of the phylogenetic relationships of at higher taxa was done later based on using the 16S rRNA genes and other related evidences, such as taxon-specific 16S rRNA gene signature nucleotides [7, 8]. After the genus was described by Tang et al., the genus together with the genus were placed in a novel family belong to subord. nov., mainly because of theirs signature nucleotide patterns, 16S rRNA gene similarity and phylogenetic criteria [9]. Presently, the is placed in the family of the order within the class [10].

The capacity of YIM 002 T to produce seven new compounds (five pyrrol-2-aldehyde compounds, jiangrines A-E; one indolizine derivative, jiangrine F; one glycolipid, jiangolide) has previously been shown [11], highlighting the importance of this bacterium and its analysis as a novel source of secondary metabolites. As part of the GEBA project and considering its phylogenetic position and biological significance, we finally decided to sequence the genome of the type strain of . Here we present a summary classification and a set of features for YIM 002 T, together with the description of genomic sequencing and annotation. At the same time, we will provide a brief introduction of its genome in this article.

Organism information

Classification and features

Strain YIM 002 T is a free-living isolate collected from a desert soil sample of Gansu Province during an investigation into microbial diversity of extreme environments. This actinobacterium forms well-differentiated non-sporulating aerial and substrate mycelia. Its aerial hypha was observed to have yellow-white color at the earliest and finally turns to orange-yellow after few days on NA medium, and its substrate mycelia fragmented into short or elongated rods in the early phase of the growth (Fig. 1). Growth was observed on ISP 2, ISP 3, ISP 4, ISP 5, nutrient agar and Czapek’s agar [1, 12]. The type strain of this taxon is able to tolerate a pH range between 5.0 and 10.0, and able to growth at the salinity between 0 and 10% (w/v NaCl), with no growth observed at 12.5%. Optimal growth of strain YIM 002 T occurs at pH 7.0–8.0, 1–5% (w/v) NaCl and 28 °C. The diamino acid in the peptidoglycan is LL-2,6-diaminopimelate. MK-9(H4) is the predominant menaquinone. The primary phospholipids profile of strain DSM 44835 T was found to consist of phosphatidylinositol mannosides, phosphatidylinositol and diphosphatidylglycerol. Its major cellular fatty acids (>10%) are anteiso-C15:0, anteiso-C17:0 and iso-C15:0. Whole cell sugar composition includes glucose and ribose, whereas the amino acids in the peptidoglycan layer were LL-A2pm, alanine, glycine and glutamic acid [1]. The DNA G + C content of the type strain was previously determined as 70% while genome analysis showed a higher value of 70.91%.

Fig. 1

Scanning electron micrograph of Jiangella gansuensis strain YIM 002T grown on ISP medium 2 for 14d at 28 °C. Bar size: 2 μm

The draft genome of YIM 002 T has one almost full-length 16S rRNA gene sequence, which correspond perfectly with the original sequence from the species description (AY631071). The comparison of this 16S rRNA sequence of YIM 002 T using the EzTaxon-e server [13], showed highest similarity to YIM 61503 T (98.93%), with close relationships to other species within the genus, 15-Je-017T (98.88%), 3SM4-07T (98.49%) and D8-87T (98.10%). Closest other genera are [9] and [14]. The strains of the genus have many 16S rRNA gene signature nucleotides compared with most of other described actinomycetes. This allows for distinguished them easily from other actinobacteria, especially in 11 unique positions, including 127:234 (G-C), 598:640 (C-G), 672:734 (G–C), 831:855 (U–A), 833:853 (G–C), 840:846 (A–U), 950:1231 (G–C), 952:1229 (G–C), 955:1225 (G–U), 986:1219 (U–G) and 987:1218 (C–G) [9].

Phylogenetic analyses were performed using both neighbor-joining (NJ) and maximum-likehood (ML) algorithms. The NJ phylogenetic tree of the genus based on 16S rRNA genes provide an evidence of its independent taxon (Figs. 2 and Additional file 1: Figure S1), together with the genera and , which arouse ours reflection on the relationship of three families among , and . The ML tree (Additional file 1: Figure S1) demonstrates the same positions in compared with the NJ tree. Minimum Information about the Genome Sequence is provided in Table 1.

Fig. 2

Phylogenetic tree showing the relationship of J. gansuensis YIM 002T with some other actinobacteria based on 16S rRNA gene sequences. The Neighbour-joining tree was built using MEGA 5 [39] with the Kimura 2-parameter model. Bootstrap values (percentages of 1000 replicates) are shown at branch points. Asterisks denote nodes that were also recovered using the Maximum Likelihood method in the branch of Jiangellaceae. The Haloglycomyces albus act as the outgroup

Table 1

Classification and general features of Jiangella gansuensis strain YIM 002T in accordance with the MIGS recommendations [20], List of Prokaryotic names with Standing in Nomenclature [40] and the Names for Life database [41]

MIGS ID	Property	Term	Evidence codea
	Current classification	Domain Bacteria	TAS [42]
		Phylum Actinobacteria	TAS [43]
		Class Actinobacteria	TAS [7]
		Order Jiangellales	TAS [44]
		Family Jiangellaceae	TAS [9]
		Genus Jiangella	TAS [1]
		Species Jiangella gansuensis	TAS [1]
		Type strain YIM 002T (=DSM 44835T)	TAS [1]
	Gram stain	Positive	IDA
	Cell shape	Filamentous	IDA
	Motility	Non motile	IDA
	Sporulation	Non-sporulating	IDA
	Temperature range	10–45 °C	IDA
	Optimum temperature	28 °C	IDA
	pH range; Optimum	7.0–8.0	TAS [1]
	Carbon source	Various	IDA
	Energy source	Chemoorganotroph	IDA
MIGS-6	Habitat	Desert soil	IDA
MIGS-6.3	Salinity	Halotolerant	IDA
MIGS-22	Oxygen requirement	Aerobic	IDA
MIGS-15	Biotic relationship	Free living	IDA
MIGS-14	Pathogenicity	None	IDA
MIGS-4	Geographic location	Gansu Province, China	IDA
MIGS-5	Sample collection time	2005 or before	NAS
MIGS-4.1	Latitude	Not reported	NAS
MIGS-4.2	Longitude	Not reported
MIGS-4.4	Altitude	Not reported

a Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from of the Gene Ontology project [45]

Genome sequencing information

Genome project history

This organism was selected for sequencing on the basis of its important phylogenetic position and biological significance [15, 16], and for a better understanding of the school of ‘evolutionary taxonomy’ [17]. Sequencing of YIM 002 T is part of Genomic Encyclopedia of Bacteria and Archaea pilot project [18], which aims for generating high quality draft genomes for bacterial and archaeal strains. The genome project is deposited in the Genomes OnLine Database (GOLD) [19], and the finished genome sequence was deposited in GenBank. Genome sequencing, finishing and annotation were performed by the Department of Energy, Joint Genome Institute (JGI) using state of the art genome sequencing technology [20]. A summary of project information is shown in Table 2, compliance with MIGS version 2.0 [21].

Table 2

Genome sequencing project information

MIGS ID	Property	Term
MIGS 31	Finishing quality	Non-contiguous Finished
MIGS-28	Libraries used	Illumina Std shotgun library
MIGS 29	Sequencing platforms	454-GS-FLX-Titanium Illumina GAii
MIGS 31.2	Fold coverage	Unknown
MIGS 30	Assemblers	ALLPATHS v. R37654
MIGS 32	Gene calling method	Prodigal 1.4, GenePRIMP
	Locus Tag	JIAGA
	GenBank ID	AZXT00000000
	GenBank Date of Release	15-08-2013
	GOLD ID	Gp0001209
	BIOPROJECT	PRJNA224116, PRJNA63165
MIGS 13	Source Material Identifier	YIM 002, DSM 44835
	Project relevance	Tree of Life, GEBA

Growth conditions and genomic DNA preparation

strain YIM 002 T (=DSM 44835 T) was grown in DSMZ medium 65 (GYM medium) at 28 °C. Genomic DNA was isolated using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) following the standard protocol provided by the manufacturer. Some modifications were included for cell lysis, first freezing for 20 min (−70 °C), then heating 5 min (98 °C), and cooling 15 min to 37 °C; adding 1.5 ml lysozyme (standard: 0.3 ml, only), 1.0 ml achromopeptidase, 0.12 ml lysostaphine, 0.12 ml mutanolysine, 1.5 ml proteinase K (standard: 0.5 ml, only), followed by overnight incubation at 35 °C.

Genome sequencing and assembly

All general aspect of library construction and sequencing performed can be found at the JGI website. The complete sequence in one scaffold was obtained from 9 contigs with the assembly method ALLPATHS v. R37654, obtaining a total size of 5.5 Mbp from a total volume data of 4 Gbases (Fig. 3).

Fig. 3

Graphical map of the J. gansuensis strain YIM 002T chromosome. The genome circular map was set up by the CGView Server [46]. From the outside to the center: Genes on forward strand (colored by COG categories), Genes on reverse strand (colored by COG categories), GC content, GC skew, where green indicates positive values and magenta indicates negative values

Genome annotation

Prodigal [22] was used to identify genes as part of the JGI genome annotation pipeline [23, 24] followed by a round of manual curation using the JGI GenePRIMP pipeline [25]. The National Center for Biotechnology Information non-redundant database, UniProt, TIGR/Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases were used to analyse the predicted CDSs after translation. RNA genes identification was done using HMMER 3.0 [26] (rRNAs) and tRNAscan-SE 21.23 [27] (tRNAs). INFERNAL 1.0.2 [28] was used for prediction of other non-coding genes. Integrated Microbial Genomes Expert Review platform [29] permitted the additional gene prediction analysis and functional annotation. CRISPR elements were detected with CRT [30] and PILER-CR [31]. General statistics are shown in Table 3.

Table 3

Genome Statistics

Attribute	Value	% of total a
Genome size (bp)	5,585,780	100.00
DNA coding (bp)	4,761,339	85.24
DNA G + C (bp)	3,960,974	70.91
DNA scaffolds	1	-
Total genes	5,104	-
Protein-coding genes	4,905	98.03
RNA genes	50	0.98
Pseudo genes	149	2.98
Genes in internal clusters	1763	34.54
Genes with function prediction	2,504	48.86
Genes assigned to COGs	2,156	42.07
Genes with Pfam domains	1,734	33.97
Genes with signal peptides	456	8.69
Genes with trandmembrane helices	1230	23.43
CPISPR repeats	0	-

a The total is based on either the size of genome in base pairs or the total number of genes in the predicted genome

Genome properties

The assembly of the draft genome sequence consists of one scaffold for the strain YIM 002 T (Fig. 1), with 70.9% GC content (Table 3) in 5,585,780 nucleotides. From a total of 5104 genes, there were 4905 protein-coding genes, 149 pseudogens and 50 RNA genes. Numbers of the genes were assigned a putative function (48.86%), while the remaining protein-coding genes were annotated as hypothetical proteins. COGs categories distributions for the genes are presented in Table 4.

Table 4

Number of genes associated with the general COG functional categories

Code	Value	% age a	Description
J	160	3.18	Translation, ribosomal structure and biogenesis
A	1	0.02	RNA processing and modification
K	230	4.58	Transcription
L	116	2.31	Replication, recombination and repair
B	1	0.02	Chromatin structure and dynamics
D	21	0.42	Cell cycle control, cell division, chromosome partitioning
V	60	1.19	Defence mechanisms
T	75	1.49	Signal transduction mechanisms
M	96	1.91	Cell wall/membrane biogenesis
N	0	0.00	Cell motility
U	18	0.36	Intracellular trafficking, secretion, and vesicular transport
O	69	1.37	Posttranslational modification, protein turnover, chaperones
C	160	3.18	Energy production and conversion
G	223	4.44	Carbohydrate transport and metabolism
E	298	5.93	Amino acid transport and metabolism
F	56	1.11	Nucleotide transport and metabolism
H	114	2.27	Coenzyme transport and metabolism
I	111	2.21	Lipid transport and metabolism
P	179	3.56	Inorganic ion transport and metabolism
Q	84	1.67	Secondary metabolites biosynthesis, transport and catabolism
R	311	6.19	General function prediction only
S	151	3.00	Function unknown
-	2868	57.09	Not in COGs

aThe total is based on the total number of protein-coding genes in the genome

Insights from the genome sequence

The genome of YIM 002 T with a high G + C content and the smallest size within the genomes (Table 3) may be the result of selection and mutation [32], which could involve several factors, such as environment, aerobiosis and others [33]. Generally speaking, a larger genome size may correlate with more complex habitat, suggesting that the genome encodes a large metabolic and stress-tolerance potential [34]. However, after we investigated the genome size of other type strains of species, we found the size of the other three strains sequenced of this genus, , and greater than 7 Mbp based on the genome data from NCBI. This result could implicate that the tight packing and small size of is likely an adaptation for reproductive efficiency or competitiveness [35]. As a halotolerant actinobacterium, solute and ion transporter were predicted in its genome. At the same time, the genome shows properties related to solution of nitrate and sulfonate transport systems. Moreover, nitrite reductase and nitrogen fixation protein NifU were also detected.

The capacity of this microorganism to produce antibiotics has been recently proved with the description of seven new compounds (five pyrrol-2-aldehyde compounds, jiangrines A-E; one indolizine derivative, jiangrine F; one glycolipid, jiangolide) [11]. However, its potential should be higher, taken account the 45 biosynthetic clusters found within the JGI tool [36] and the 497 genes implicated in these clusters. As most of the clusters appear to be putative genes in this analysis, a second approach was carried out to detect the variety of biosynthetic types and enhance manual genome annotations of secondary metabolite biosynthesis. The software pipeline antiSMASH for secondary metabolite gene cluster identification, annotation and analysis was used [37, 38]. From this analysis, 60 gene clusters were identified, including 20 gene clusters in which the most similar clusters were still unknown (Additional file 2: Table S1). The result of the analysis shown the potential of to produce pristinamycin, an antibiotic derived from pristinaespiralis effective against staphylococcal infections, and other antibiotics.

Conclusions

The genome sequence and annotation of YIM 002 T were presented. This draft genome possess a smaller size (5.59 Mb) compared with other species, and contents 2504 function predicted proteins, indicating that possibly discarded many genes to adapt to the extreme desert conditions during its evolution. Although the processes of nitrous metabolism and secondary metabolism need further investigation to fully understand the related pathways, we believe that participates in nitrogen cycling and has an important ability to produce secondary metabolites. This genome will contribute to further studies on phylogenetics and the mechanisms of environmental adaptation. A combined study together with genomes of other members in the family will help us to better understand the ecological role of this taxon and its relationships to other actinobacteria.