Complete genome sequence of Klebsiella variicola subsp. variicola ML.9ba2, an endophytic strain isolated from aerial roots of Philodendron erubescens

TL;DR

This paper reports the complete genome sequence of a Klebsiella variicola strain isolated from a plant in Hong Kong.

Contribution

The study provides the complete genome sequence of an endophytic Klebsiella variicola strain isolated from Philodendron erubescens.

Findings

The complete genome of Klebsiella variicola subsp. variicola ML.9ba2 is 5,682,083 bp with 57.29% G+C content.

The genome includes a single chromosome and an IncF plasmid.

Abstract

The endophytic strain Klebsiella variicola subsp. variicola ML.9ba2 was isolated from aerial roots of Philodendron erubescens in Hong Kong. Its complete genome of 5,682,083 bp (57.29% G+C), comprising a single chromosome and an IncF plasmid, was established through hybrid assembly.

Tables

TABLE 1. Predicted genesa in ML.9ba2 potentially linked to plant-growth promotion and endophytism

Gene(s)	Protein(s)	Locus	Function
nif cluster	nifJHDKTXENXUSVWZMFLABQ	MKK01_RS08305 to MKK01_RS08400	Nitrogen fixation
Pho regulon	phoPQ, phoH, phoBR, pstSCAB-phoU	MKK01_RS16770 to MKK01_RS16775,MKK01_RS17230,	Phosphate solubilizationand utilization
		MKK01_RS20955 to MKK01_RS20950,
		MKK01_RS26375 to MKK01_RS26395,
aphA	Acid phosphatase	MKK01_RS24820
phoA	Alkaline phosphatase	MKK01_RS21020
phyb	Phytaseb	MKK01_RS19375
bcs operon	bcsOQABCDZ (type Ib),bcsGFERQABZC (type IIa),	MKK01_RS01075 to MKK01_RS01150	Cell-wall modification
chiAb	Chitinase (EC 3.2.1.14)b	MKK01_RS15875	Fungal defense
nhhABG	Nitrile hydratase (EC 4.2.1.84)	MKK01_RS13035 to MKK01_RS13045	Indole-3-acetic acid (IAA) synthesis,auxin transport, and plantontogenesis.
ami	Amidase (EC 3.5.1.4)	MKK01_RS13030
tynA	Monoamine oxidase (EC 1.4.3.4)	MKK01_RS14785
aldBb	Aldehyde dehydrogenase(EC 1.2.1.3)b	MKK01_RS05580, MKK01_RS19890
aec	Auxin efflux carrier (AEC) family transporter	MKK01_RS05085, MKK01_RS09265,MKK01_RS14270
ent-fep gene cluster	entHABEC-fepB-entS-fepDGC- entF-mbtH-fes-fepA-entD	MKK01_RS19440 to MKK01_RS19510	Enterobactin biosynthesis andtransport, root colonizationthrough ROS defense
cirA, fiu	Ferric catecholate siderophore receptor	MKK01_RS07705, MKK01_RS15625	Iron-catecholate transport, plantcolonization
fhu operon	fhuABCD, fhuE, fhuF, ferric hydroxamatetransport operon	MKK01_RS21875 to MKK01_RS21890,MKK01_RS22900	Ferrichrome transport andutilization, albomycin uptake

Full text

ANNOUNCEMENT

While Klebsiella pneumoniae species complex (KpSC) members are clinically relevant as opportunistic human pathogens (1) with intrinsic resistance to penicillins (2, 3) often extending to cephalosporins (4, 5) and carbapenems (6, 7), they are also found as endophytes (8). Kingdom-crossing is especially pronounced in strains of K. variicola (9), which are not only nitrogen fixers and plant colonizers, isolated from sources such as sugarcane (10), rice and bananas (11), but also found in bloodstream infections (12), cattle urine (13) and termite gut microbiota (14).

Here, ML.9ba2 was isolated from aerial roots of Philodendron erubescens in Hong Kong (22.266384N, 114.191128E; 2021-06-25). 20 mm lengths of three root tips from a single plant were surface-sterilized using 1% wt:vol 8-hydroxyquinoline sulfate before incubation in nitrogen-free Jensen’s broth M710 (15, 16) for 7 days at 32°C. Following subculture to fresh broth and incubation under the same conditions, a 100 µL aliquot was spread on M710 agar and the resultant colonies tested for phosphate solubilization on Pikovskaya’s agar (17). A single colony of ML.9ba2, which grows well on M710 and gives clear zones on Pikovskaya’s agar, was passaged to purity on M710 agar before streaking a single colony onto Luria agar (18) and harvesting for DNA extraction following overnight growth (DNeasy PowerSoil Pro kit, Qiagen GmbH, Hilden, Germany). All agar incubations conducted at 27°C.

Paired-end short-read sequencing libraries were prepared using a NexteraXT DNA Library Preparation Kit (Illumina, Inc., USA) and sequenced via the Illumina MiSeq platform using v3 chemistry (2 × 300 bp). 798,646 raw read pairs were quality-filtered and trimmed using TrimGalore! v0.6.7 (https://github.com/FelixKrueger/TrimGalore) (stringency:3; -e:0.2), producing 790,949 read pairs (median length 170 bp) totaling ~279 Mbp (SRX21353898). Long-read libraries, prepared from the same extracted DNA using the Rapid Barcoding Kit SQK-RBK004, were sequenced using a Spot-ON Flow Cell (vR9) and MinION sequencer, with MinKNOW v3.1.8 software and base-calling by Guppy v2.1.3 high-accuracy mode (all from Oxford Nanopore Technologies plc, UK). The final long-read data set of 429,202 reads was trimmed by Filtlong v0.2.1 (https://github.com/rrwick/Filtlong) (min_length:2000; target_bases:350Mbp; length_weight:10) to give 56,702 reads (350 Mbp) (SRX21353899) with a median length of 4,712 bp (N50 7,681). Default parameters were used for all software unless otherwise specified.

Hybrid assembly of MiSeq and MinION filtered data sets, overlap removal, and sequence rotation were performed using Unicycler v0.4.3 (19) to yield a circular chromosome of 5,515,756 bp (57.48% G+C) and a circular plasmid (166,327 bp, 51.1% G+C), rotated to begin DNAA_KLEP3 (dnaA) and Q6U5J6_KLEPN (repA), respectively. The genome was judged 100% complete (0.36% contamination) by CheckM v.1.1.6 (20) and submitted to NCBI PGAP v6.4 (21) for annotation. Mash/MinHash at BV-BRC 3.34.11 (22) found ML.9ba2 closest to Klebsiella variicola subsp. variicola strain F2R9T (CP084765) with an average nucleotide identity of 99.09% (23).

PGAP predicted numerous genes associated with plant-growth promotion and endophytism (Table 1) (24–28) as well as a chromosomally encoded LEN-19 beta-lactamase (MKK01_RS13570) (29). Plasmid pML9ba2 was classified as type IncFIB(K)_5(cladeI) by the KpVR web-based tool (https://bioinfo-mml.sjtu.edu.cn/KpVR/index.php), which found no antimicrobial resistance genes.