Comparative Genomics of Pandoraea, a Genus Enriched in Xenobiotic Biodegradation and Metabolism

Abstract

Comparative analysis of partial gyrB, recA, and gltB gene sequences of 84 Pandoraea reference strains and field isolates revealed several clusters that included no taxonomic reference strains. The gyrB, recA, and gltB phylogenetic trees were used to select 27 strains for whole-genome sequence analysis and for a comparative genomics study that also included 41 publicly available Pandoraea genome sequences. The phylogenomic analyses included a Genome BLAST Distance Phylogeny approach to calculate pairwise digital DNA–DNA hybridization values and their confidence intervals, average nucleotide identity analyses using the OrthoANIu algorithm, and a whole-genome phylogeny reconstruction based on 107 single-copy core genes using bcgTree. These analyses, along with subsequent chemotaxonomic and traditional phenotypic analyses, revealed the presence of 17 novel Pandoraea species among the strains analyzed, and allowed the identification of several unclassified Pandoraea strains reported in the literature. The genus Pandoraea has an open pan genome that includes many orthogroups in the ‘Xenobiotics biodegradation and metabolism’ KEGG pathway, which likely explains the enrichment of these species in polluted soils and participation in the biodegradation of complex organic substances. We propose to formally classify the 17 novel Pandoraea species as P. anapnoica sp. nov. (type strain LMG 31117^T = CCUG 73385^T), P. anhela sp. nov. (type strain LMG 31108^T = CCUG 73386^T), P. aquatica sp. nov. (type strain LMG 31011^T = CCUG 73384^T), P. bronchicola sp. nov. (type strain LMG 20603^T = ATCC BAA-110^T), P. capi sp. nov. (type strain LMG 20602^T = ATCC BAA-109^T), P. captiosa sp. nov. (type strain LMG 31118^T = CCUG 73387^T), P. cepalis sp. nov. (type strain LMG 31106^T = CCUG 39680^T), P. commovens sp. nov. (type strain LMG 31010^T = CCUG 73378^T), P. communis sp. nov. (type strain LMG 31110^T = CCUG 73383^T), P. eparura sp. nov. (type strain LMG 31012^T = CCUG 73380^T), P. horticolens sp. nov. (type strain LMG 31112^T = CCUG 73379^T), P. iniqua sp. nov. (type strain LMG 31009^T = CCUG 73377^T), P. morbifera sp. nov. (type strain LMG 31116^T = CCUG 73389^T), P. nosoerga sp. nov. (type strain LMG 31109^T = CCUG 73390^T), P. pneumonica sp. nov. (type strain LMG 31114^T = CCUG 73388^T), P. soli sp. nov. (type strain LMG 31014^T = CCUG 73382^T), and P. terrigena sp. nov. (type strain LMG 31013^T = CCUG 73381^T).

Introduction

Members of the genus Pandoraea have emerged as rare opportunistic pathogens in persons with cystic fibrosis (Jørgensen et al., 2003; Johnson et al., 2004; Pimentel and MacLeod, 2008; Kokcha et al., 2013; Ambrose et al., 2016; Martina et al., 2017; See-Too et al., 2019) and several cases of chronic colonization and patient-to-patient transfer in this patient group have been reported (Jørgensen et al., 2003; Atkinson et al., 2006; Degand et al., 2015; Pugès et al., 2015; Ambrose et al., 2016; Dupont et al., 2017; Greninger et al., 2017). In addition to causing infection in cystic fibrosis patients, Pandoraea isolates have been recovered from blood and from samples from patients with chronic obstructive pulmonary disease or chronic granulomatous disease (Coenye et al., 2000; Daneshvar et al., 2001). Although the small number of patients involved and underlying diseases make it difficult to identify these bacteria as the cause of clinical deterioration (Martina et al., 2017; Green and Jones, 2018), one report described sepsis, multiple organ failure and death in a non-cystic fibrosis patient who underwent lung transplantation for sarcoidosis (Stryjewski et al., 2003).

Of the 11 validly named Pandoraea species, six (i.e., Pandoraea apista, Pandoraea norimbergensis, Pandoraea pulmonicola, Pandoraea pnomenusa, Pandoraea sputorum, and Pandoraea fibrosis) have been recovered from human clinical specimens (Coenye et al., 2000; See-Too et al., 2019), while Pandoraea faecigallinarum, Pandoraea oxalativorans, Pandoraea terrae, Pandoraea thiooxydans, and Pandoraea vervacti have been isolated from environmental samples (Anandham et al., 2010; Sahin et al., 2011; Jeong et al., 2016). An uncultivated endosymbiont of the trypanosomatid Novymonas esmeraldas represents an additional Pandoraea species which was provisionally named Candidatus Pandoraea novymonadis (Kostygov et al., 2017).

A growing number of reports demonstrate that soil and water represent the natural habitats of Pandoraea bacteria where they can be part of rhizosphere communities (Anandham et al., 2010; Jurelevicius et al., 2010; Peeters et al., 2016; Dong et al., 2018) and be involved in oxalate degradation (Jin et al., 2007; Sahin et al., 2011). The latter suggests they may be important contributors to soil formation, soil fertility and retention, and cycling of elements necessary for plant growth (Sahin, 2003). These free-living Pandoraea bacteria are often enriched in polluted soils and participate in the biodegradation of complex organic substances including lignin (Shi et al., 2013; Kumar et al., 2018b; Liu et al., 2019), biodiesel and petroleum by-products (de Paula et al., 2017; Sarkar et al., 2017; Tirado-Torres et al., 2017), p-xylene (Wang et al., 2015), δ-hexachlorocyclohexane (Pushiri et al., 2013), di-n-butyl phthalate (Yang et al., 2018), biphenyl, benzoate and naphthalene (Uhlik et al., 2012), and tetracycline (Wu et al., 2019) and β-lactam antibiotics (Crofts et al., 2017). A particularly well-documented Pandoraea strain, i.e., JB1^T (LMG 31106^T), was isolated in the 1980s from garden soil (Parsons et al., 1988) and was able to use biphenyl, 2-, 3- and 4-chloro-biphenyl, m-toluate, p-toluate naphthalene, m-hydroxybenzoate and diphenylmethane (Springael et al., 1996). Although this strain also represented a separate novel Pandoraea species, it was not formally classified (Coenye et al., 2000) pending the availability of more than one strain representing the same novel species, a taxonomic practice that has been largely abandoned today.

The genome sequences of several strains with bioremediation potential have been reported, but a growing number of studies fail to provide species level identification of such strains (Pushiri et al., 2013; Chan et al., 2015; Kumar M. et al., 2016; Crofts et al., 2017; Liu et al., 2018; Wu et al., 2019). In addition, in our studies on the diversity and epidemiology of opportunistic pathogens in persons with cystic fibrosis, we isolated a considerable number of Pandoraea strains that represent novel species (unpublished data). The present study aimed to clarify the taxonomy and formally name these novel Pandoraea species, and to make reference cultures and whole-genome sequences of each of these versatile bacteria publicly available.

Materials and Methods

Bacterial Strains and Growth Conditions

Isolates representing novel Pandoraea species are listed in Table 1, along with their isolation source details. These strains were initially assigned to the genus Pandoraea on the basis of sequence analysis of 16S rRNA, gyrB or recA genes (data not shown). Well-characterized reference strains and recent field isolates identified in the present study as established Pandoraea species are listed in Supplementary Table S1. Strains were grown aerobically on Tryptone Soya Agar (Oxoid) and incubated at 28°C. Cultures were preserved in MicroBank^TM vials at −80°C.

TABLE 1.

Isolates representing novel Pandoraea species.

Strain	Other strains designations	Source	Depositor
Pandoraea anapnoica sp. nov.
LMG 31117T	AU1288T, CCUG 73385T	CF patient (United States, 1999)	Own isolate
Pandoraea anhela sp. nov.
LMG 31108T	AU12140T, CCUG 73386T	CF patient (United States, 2006)	Own isolate
Pandoraea aquatica sp. nov.
LMG 31011T	CCUG 73384T	Pond water in greenhouse (Belgium, 2013)	Own isolate
Pandoraea bronchicola sp. nov.
LMG 20603T	CDC H652T, ATCC BAA-110T	CF sputum (United States, 1998)	CDC
R-10961	AU1775	CF patient (United States, 2000)	Own isolate
R-14318	AU2478	CF patient (United States, 2000)	Own isolate
R-52718	AU17726	CF patient (United States, 2009)	Own isolate
Pandoraea capi sp. nov.
LMG 20602T	CDC G9805T, ATCC BAA-109T	Non-CF sputum (United States, 1996)	CDC
R-15265	AU2777	CF patient (United States, 2001)	Own isolate
R-52714	AU12983	CF patient (United States, 2007)	Own isolate
Pandoraea captiosa sp. nov.
LMG 31118T	AU16660T, CCUG 73387T	CF patient (United States, 2008)	Own isolate
Pandoraea cepalis sp. nov.
LMG 31106T	JB1T, CCUG 39680T	Garden soil (Netherlands)	M. Mergeay
LMG 31107		Soil of house plant (Belgium, 2003)	Own isolate
R-51030		Pond water in greenhouse (Belgium, 2013)	Own isolate
Pandoraea commovens sp. nov.
LMG 31010T	CCUG 73378T	CF patient (Belgium, 2002)	C. De Boeck
LMG 24770	AI-S128	Plant root surface (India, 2002)	M. Madhaiyan
R-15662	AU3099	CF patient (United States)	Own isolate
Pandoraea communis sp. nov.
LMG 31110T	CCUG 73383T	CF patient (Belgium, 2012)	D. Pierard
LMG 31111		River water (Belgium, 2002)	Own isolate
R-17388		Maize rhizosphere soil (Belgium, 2002)	Own isolate
R-20591		River water (Belgium, 2002)	Own isolate
Pandoraea eparura sp. nov.
LMG 31012T	CCUG 73380T	Soil of house plant (Belgium, 2003)	Own isolate
Pandoraea horticolens sp. nov.
LMG 31112T	CCUG 73379T	Garden soil (Belgium, 2003)	Own isolate
Pandoraea iniqua sp. nov.
LMG 31009T	CCUG 73377T	Maize rhizosphere soil (Belgium, 2002)	Own isolate
LMG 31115	AU1290	CF patient (United States, 1999)	Own isolate
Pandoraea morbifera sp. nov.
LMG 31116T	AU12324T, CCUG 73389T	CF patient (United States, 2006)	Own isolate
R-54947	AU23671	CF patient (United States, 2011)	Own isolate
Pandoraea nosoerga sp. nov.
LMG 31109T	AU17017T, CCUG 73390T	CF patient (United States, 2008)	Own isolate
R-12863	AU2028	CF patient (United States, 2000)	Own isolate
R-13299	00BC460		P. Evans
R-15344	AU2347	CF patient (United States, 2000)	Own isolate
R-34565		CF patient (Australia, 2006)	M. Aravena-Roman
R-46874		CF patient (Belgium, 2011)	G. Ieven
R-47614		CF patient (Belgium, 2011)	H. Franckx
R-50065		CF patient (Belgium, 2012)	G. Ieven
R-50587		CF patient (Belgium, 2013)	C. De Boeck
R-52720	AU14034	CF patient (United States, 2007)	Own isolate
R-52722	AU18716	CF patient (United States, 2009)	Own isolate
Pandoraea pneumonica sp. nov.
LMG 31114T	AU18032T, CCUG 73388T	CF patient (United States, 2009)	Own isolate
Pandoraea soli sp. nov.
LMG 31014T	CCUG 73382T	Soil of house plant (Belgium, 2003)	Own isolate
Pandoraea terrigena sp. nov.
LMG 31013T	CCUG 73381T	Soil of house plant (Belgium, 2003)	Own isolate

CF, cystic fibrosis. LMG, BCCM/LMG Bacteria Collection, Laboratory of Microbiology, Ghent University, Ghent, Belgium; CCUG, Culture Collection University of Gothenburg, Department of Clinical Bacteriology, Sahlgrenska University Hospital, Gothenburg, Sweden; CDC, Centers for Disease Control, United States Public Health Service, Atlanta, GA, United States.

DNA Preparation

DNA was extracted using an automated Maxwell^® DNA preparation instrument (Promega, United States). The final extract was treated with RNAse (2 mg/ml, 5 μL per 100 μL extract) and incubated at 37°C for 1 h. DNA quality was checked using 1% agarose gel electrophoresis and DNA quantification was performed using the QuantiFluor ONE dsDNA system and the Quantus fluorometer (Promega, United States). DNA was stored at −20°C prior to further analysis.

Single Locus Sequence Analyses

Nearly complete 16S rRNA sequences were obtained as described previously (Peeters et al., 2013).

Partial recA gene sequences (663 bp) were amplified by PCR using forward primer 5′-AGG ACG ATT CAT GGA AGA WAG C-3′ and reverse primer 5′-GAC GCA CYG AYG MRT AGA ACT T-3′ (Spilker et al., 2009). Each 25 μl PCR reaction consisted of 1x PCR buffer (Qiagen), 1 U of Taq polymerase (Qiagen), 250 μM of each dNTP (Applied Biosystems), 1 × Q-solution (Qiagen), 1 μM of each primer and 2 μl of DNA (Peeters et al., 2013). PCR was performed using a Veriti 96 Well Thermal Cycler (Applied Biosystems). Initial denaturation for 2 min at 94°C was followed by 30 cycles of 30 s at 94°C, 45 s at 58°C and 1 min at 72°C, and a final elongation for 10 min at 72°C. Amplicons were purified using a NucleoFast 96 PCR clean-up kit (Macherey-Nagel). Sequencing primers (one per sequencing reaction) were the same as the amplification primers. Sequence analysis was performed with an Applied Biosystems 3130xl Genetic Analyzer and protocols of the manufacturer using the BigDye Terminator Cycle Sequencing Ready kit. Sequence assembly was performed using BioNumerics v7.6 (Applied Maths, Belgium).

Partial gyrB sequences (573 bp) were amplified by PCR using forward primer 5′-GAC AAY GGB CGY GGV RTB CC-3′ (this study) and reverse primer 5′-YTC GTT GWA RCT GTC GTT CCA CTG C-3′ (Spilker et al., 2009). The PCR protocol was the same as for recA, except that 2 μM of primer was used and an annealing temperature of 60°C. Sequencing primers (one per sequencing reaction) were 5′-ACG ACA AGC ACG ARC CSA AGC G-3′ (this study) and the same reverse primer as for amplification. Sequence analysis and assembly were performed as described above for the recA gene.

Partial gltB sequences were amplified by PCR using forward primer 5′-CTG CAT CAT GAT GCG CAA GTG-3′ (Spilker et al., 2009) and reverse primer 5′-GTT GCC ACG GAA RTC GTT GG-3′ (this study). The PCR protocol was the same as for recA, except that 0.4 μM of primer was used. Sequencing primers (one per sequencing reaction) were the same as the amplification primers. Sequence analysis and assembly were performed as described above for the recA gene.

Gene sequences of recA, gyrB, and gltB were aligned based on their amino acid sequences using Muscle (Edgar, 2004) in MEGA7 (Kumar S. et al., 2016). Phylogenetic trees were constructed using RAxML v8.2.11 (Stamatakis, 2014) with the GTRCAT substitution model and 1000 bootstrap analyses. Visualization and annotation of the phylogenetic trees was performed using iTOL (Letunic and Bork, 2016).

Whole-Genome Sequencing

The genome sequences of 27 strains (Table 2 and Supplementary Table S2) were determined using the Illumina HiSeq4000 platform (PE150) at the Oxford Genomics Centre. Quality reports were created by FastQC. Reads were trimmed using Trimmomatic (Bolger et al., 2014) with the MAXINFO:50:0.8 and MINLEN:50 options. Genome size was estimated using kmc (Kokot et al., 2017) and reads were subsampled with seqtk¹ to 80x coverage depth for assembly. Assembly was performed using SPAdes v3.12.0 (Bankevich et al., 2012) with error correction, default k-mer sizes (21, 33, 55, 77) and mismatch correction. Contigs were filtered on length (minimum 500 bp) and coverage (minimum 0.5x and maximum 8x overall coverage). Raw reads were mapped against the assemblies using bwa mem (Li, 2013) and contigs were polished using Pilon 1.22 (Walker et al., 2014) with default parameters. Quast (Gurevich et al., 2013) was used to create quality reports of the resulting assemblies. Annotation was performed using Prokka 1.12 (Seemann, 2014) with a genus-specific database based on publicly available genomes.

TABLE 2.

Genomes included in the present study.

Strain	Project	Contigs	Size(bp)	%GC	CDS	References
P. apista DSM 16535T	PRJNA305052	2a	5,571,260	62.6	4,871
P. apista LMG 18089	PRJEB30685	27	5,815,466	62.7	5,279	This study
P. apista AU2161	PRJNA284212	1a	5,574,863	62.7	4,655	Greninger et al., 2017
P. apista TF80G25	PRJNA281013	1a	5,609,637	62.6	4,691	Greninger et al., 2017
P. apista TF81F4	PRJNA271830	1a	5,582,097	62.6	4,676	Greninger et al., 2017
P. apista FDAARGOS_126	PRJNA231221	1a	5,326,503	62.7	4,621
P. apista PA_200	PRJNA497126	82	5,677,857	62.5	4,969
P. apista PA_201	PRJNA497126	69	5,680,846	62.5	4,957
P. apista Pa13324	PRJNA507897	132	5,656,881	62.7	4,987
P. apista Pa14367	PRJNA441551	97	5,596,190	62.8	4,932
P. apista Pa14697	PRJNA507897	107	5,680,889	62.7	4,989
P. apista Pa15518	PRJNA507897	106	5,631,909	62.7	4,924
P. apista Pa15674	PRJNA507897	208	5,497,617	62.8	4,839
P. apista Pa16226	PRJNA381452	23	5,724,490	62.7	4,998
P. apista Pa16800	PRJNA507897	171	5,729,367	62.7	5,028
P. apista Pa17292	PRJNA507897	113	5,621,546	62.7	4,909
P. apista Pa18364	PRJNA507241	130	5,457,038	62.7	4,787
P. apista Pa18384	PRJNA507897	136	5,678,267	62.7	4,914
P. apista Pa18495	PRJNA507558	124	5,442,190	62.7	4,766
P. apista Pa18531	PRJNA507897	143	5,525,740	62.7	4,817
P. faecigallinarum DSM 23572T	PRJNA286722	3a	5,732,664	63.5	4,858
P. fibrosis 6399T	PRJNA266749	70	5,574,251	62.9	4,356	Ee et al., 2015
P. fibrosis 7641	PRJNA266765	66	5,574,850	62.9	4,363	Ee et al., 2015
P. fibrosis LMG 31113	PRJEB30745	43	5,605,513	62.8	4,943	This study
P. norimbergensis DSM 11628T	PRJNA305058	1a	6,167,370	63.1	5,237
P. oxalativorans DSM 23570T	PRJNA262701	5a	6,500,731	63.1	5,400
P. pnomenusa DSM 16536T	PRJNA261997	1a	5,389,285	64.9	4,586
P. pnomenusa LMG 31119	PRJEB30696	46	5,305,298	64.9	4,699	This study
P. pnomenusa 3kgm	PRJNA226227	1a	5,429,298	64.7	4,251
P. pnomenusa E26	PRJNA229202	96	5,476,952	64.7	4,869	Chan et al., 2015
P. pnomenusa MCB032	PRJNA319140	4a	5,819,834	64.6	4,872
P. pnomenusa RB38	PRJNA242373	1a	5,378,916	64.8	4,562
P. pnomenusa RB-44	PRJNA230133	1a	5,385,946	64.9	4,646
P. pulmonicola DSM 16583T	PRJNA270151	1a	5,867,621	64.3	4,868
P. sputorum DSM 21091T	PRJNA262705	1a	5,742,997	62.8	4,884
P. sputorum LMG 20601	PRJEB30706	62	6,264,179	62.7	5,539	This study
P. sputorum LMG 31120	PRJEB30707	17	5,956,418	62.7	5,301	This study
P. sputorum LMG 31121	PRJEB30708	113	6,453,978	62.8	5,652	This study
P. terrae LMG 30175T	PRJEB30813	81	6,176,823	62.8	5,575	This study
P. thiooxydans DSM 25325T	PRJNA285516	1a	4,464,186	63.2	3,999
P. thiooxydans ATSB16	PRJNA309453	1a	4,464,185	63.2	4,388
P. vervacti NS15T	PRJNA275368	2a	5,736,282	63.5	4,811
P. anapnoica sp. nov. LMG 31117T	PRJEB30755	48	6,126,688	62.4	5,364	This study
P. anhela sp. nov. LMG 31108T	PRJEB30724	61	6,046,012	63.4	5,188	This study
P. aquatica sp. nov. LMG 31011T	PRJEB30756	17	5,958,127	62.9	5,238	This study
P. bronchicola sp. nov. LMG 20603T	PRJEB30725	34	5,351,123	63.0	4,753	This study
P. capi sp. nov. LMG 20602T	PRJEB30721	31	5,852,144	63.4	5,056	This study
P. capi sp. nov. ISTKB	PRJNA325244	115	6,367,971	63.2	5,356	Kumar M. et al., 2016
P. captiosa sp. nov. LMG 31118T	PRJEB30757	36	6,139,582	63.3	5,340	This study
P. cepalis sp. nov. LMG 31106T	PRJEB30715	56	5,274,229	63.7	4,730	This study
P. cepalis sp. nov. LMG 31107	PRJEB30716	32	5,159,566	63.5	4,626	This study
P. cepalis sp. nov. B-6	PRJNA169519	148	5,035,498	63.6	4,570	Liu et al., 2018
P. commovens sp. nov. LMG 31010T	PRJEB30753	26	6,036,949	62.6	5,308	This study
P. communis sp. nov. LMG 31110T	PRJEB30740	17	5,708,603	62.6	5,067	This study
P. communis sp. nov. LMG 31111	PRJEB30741	55	5,566,071	62.5	5,064	This study
P. communis sp. nov. SD6-2	PRJNA174277	37	5,772,015	62.5	5,148	Pushiri et al., 2013
P. eparura sp. nov. LMG 31012T	PRJEB30718	35	5,205,577	63.7	4,621	This study
P. horticolens sp. nov. LMG 31112T	PRJEB30744	68	6,008,490	62.3	5,378	This study
P. iniqua sp. nov. LMG 31009T	PRJEB30748	17	6,339,129	63.1	5,521	This study
P. iniqua sp. nov. LMG 31115	PRJEB30749	14	6,296,634	63.1	5,445	This study
P. morbifera sp. nov. LMG 31116T	PRJEB30750	47	5,233,298	64.7	4,676	This study
P. nosoerga sp. nov. LMG 31109T	PRJEB30729	41	4,862,114	66.1	4,266	This study
P. pneumonica sp. nov. LMG 31114T	PRJEB30747	12	5,845,078	62.5	5,202	This study
P. soli sp. nov. LMG 31014T	PRJEB30720	51	4,961,982	63.6	4,395	This study
P. terrigena sp. nov. LMG 31013T	PRJEB30719	35	5,356,606	63.5	4,878	This study
Pandoraea sp. PE-S2R-1	PRJNA385617	189	6,227,302	63.1	5,387	Crofts et al., 2017
Pandoraea sp. PE-S2T-3	PRJNA385617	37	6,176,158	63.2	5,310	Crofts et al., 2017
Ca. Pandoraea novymonadis E262	PRJNA369045	6	1,157,259	43.8	968	Kostygov et al., 2017

^aStatus complete.

Publicly Available Genomes

All 41 publicly available (January 29, 2019) whole-genome sequences of Pandoraea bacteria were downloaded from the NCBI database (Table 2). Burkholderia cenocepacia J2315^T was used as an outgroup in the phylogenomic analyses. For strains B-6 (Liu et al., 2018), E26 (Chan et al., 2015), PE-S2R-1 and PE-S2T-3 (Crofts et al., 2017) no annotation was available and therefore annotation was performed using Prokka as described above.

Phylogenomic Analyses

The GBDP approach was used to calculate pairwise digital DNA–DNA hybridization (dDDH) values and their confidence intervals (formula 2) using the Genome-to-Genome Distance Calculator (GGDC 2.1²) under recommended settings (Meier-Kolthoff et al., 2013). ANI values were calculated with the OrthoANIu algorithm (Yoon et al., 2017). Whole-genome phylogeny was assessed based on 107 single-copy core genes found in a majority of bacteria (Dupont et al., 2012) using bcgTree (Ankenbrand and Keller, 2016). Visualization and annotation of the phylogenetic tree was performed using iTOL (Letunic and Bork, 2016).

Functional Genome Analyses

To enable a comparative genomic study, each protein-coding gene (CDS) in the 68 Pandoraea genomes (n = 331,123) was functionally classified using the COG (Galperin et al., 2015) and KEGG orthologies (Kanehisa and Goto, 2000; Kanehisa et al., 2017). COGs were assigned by a reversed position-specific BLAST (RPSBLAST v2.6.0+) with an e-value cut-off of 1E-3 against the NCBI conserved domain database (CDD v3.16) (Tange, 2011). KEGG orthology was inferred using the KEGG automated annotation server (KAAS) (Moriya et al., 2007). Based on COG and K numbers, each CDS was assigned to the respective COG category and KEGG hierarchy. In case multiple COG categories were defined for the same COG, the first category was considered as the primary category. Protein orthologous groups (orthogroups) were inferred using OrthoFinder v2.2.7 (Emms and Kelly, 2015) with default parameters. For each orthogroup, we mapped the genomes and species in which it was present, the specificity (core, multiple species, single species or single isolate), and COG and KEGG functional classification.

Data mapping, visualization and statistical analyses were performed using RStudio with R v3.5.2. Pearson’s chi-square analyses were used to test the association between different sets of categorical variables. When a significant relationship was found between two variables, we further examined the standardized Pearson residuals. Standardized Pearson residuals with high absolute values indicate a lack of fit of the null hypothesis of independence in each cell (Agresti, 2002) and thus indicate observed cell frequencies in the contingency table that are significantly higher or lower than expected based on coincidence.

DNA Base Composition

The G + C content of all strains was calculated from their genome sequences using Quast (Gurevich et al., 2013).

Biochemical Characterization

Biochemical characterization was performed as described previously (Draghi et al., 2014).

Fatty Acid Methyl Ester Analysis

After a 24 h incubation period at 28°C on Tryptone Soya Agar (BD), a loopful of well-grown cells was harvested and fatty acid methyl esters were prepared, separated and identified using the Microbial Identification System (Microbial ID) as described previously (Vandamme et al., 1992).

Results and Discussion

Single Locus Sequence Analyses

The 16S rRNA gene sequences determined in the present study are publicly available through the GenBank/EMBL/DDBJ accession numbers listed in the species descriptions. Because the 16S rRNA sequences of Pandoraea species show high levels of similarity (Coenye et al., 2000; Daneshvar et al., 2001), gyrB gene sequence analysis has been introduced for species level identification of Pandoraea isolates (Coenye and LiPuma, 2002). To provide more robust phylogenetic analysis, partial sequences of the gyrB gene, and also of the recA and gltB genes were generated for a total of 84 Pandoraea reference strains and field isolates, and were used to construct phylogenetic trees (Figure 1 and Supplementary Figures S1, S2). The gltB, gyrB and gltB sequences determined in the present study are publicly available through the GenBank/EMBL/DDBJ accession numbers listed in Figure 1 and Supplementary Figures S1, S2 and in the species descriptions.

FIGURE 1.

Phylogenetic tree based on partial gyrB sequences of all Pandoraea strains examined. Sequences (495–573 bp) were aligned based on their amino acid sequences and phylogeny was inferred using the Maximum Likelihood method and GTRCAT substitution model in RAxML. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches if greater than 50%. Burkholderia cenocepacia J2315^T was used as outgroup. The scale bar indicates the number of substitutions per site. Isolates selected for whole-genome sequencing are shown in bold character type.

Overall, the three phylogenetic trees had comparable topologies, but while taxonomic reference strains of established Pandoraea species (Supplementary Table S1) and several groups of field isolates formed well-delineated clusters, others did not (Figure 1 and Supplementary Figures S1, S2). Each of these phylogenetic trees was therefore used to select a total of 27 isolates (shown in bold character type in Figure 1 and Supplementary Figures S1, S2) for whole-genome sequence analysis. These included 6 isolates that were tentatively assigned to established Pandoraea species using single locus sequence analyses, 20 isolates that clustered separately or whose assignment was equivocal, and P. terrae LMG 30175^T, the sole Pandoraea type strain for which there was no publicly available whole-genome sequence at the time of writing.

Genome Characteristics

The assembly of the Illumina HiSeq 150 bp paired end reads resulted in assemblies with 12–113 contigs and a total of 4.86–6.45 Mbp (Table 2 and Supplementary Table S2). The number of predicted CDS in the newly sequenced genomes ranged from 4,266 to 5,652 (Table 2). No clustered regularly interspaced short palindromic repeats (CRISPRs) were identified. The annotated assemblies of these 27 genomes were submitted to the European Nucleotide Archive and are publicly available through the GenBank/EMBL/DDBJ accession numbers listed in Table 2 and in the species descriptions. The G + C content of the newly sequenced strains, as calculated from their genome sequences, ranged from 62.3 to 66.1 mol% (Table 2). These values are similar those of other Pandoraea genomes, except for Ca. Pandoraea novymonadis that has a G + C content of 43.8% (Kostygov et al., 2017).

Phylogenomic Analyses

The 27 genomes from the present study were compared to all 41 publicly available Pandoraea genomes (GenBank database, January 29, 2019), which included 6 unclassified Pandoraea strains (Pushiri et al., 2013; Chan et al., 2015; Crofts et al., 2017; Kumar et al., 2018a; Liu et al., 2018). Pairwise dDDH and ANI values among the 68 genome sequences were calculated and are listed in Supplementary Tables S3, S4, respectively. Species delineation based on the 70% dDDH (Meier-Kolthoff et al., 2013) and 95–96% ANI thresholds (Yoon et al., 2017) yielded 30 species, which included the 11 validly named species, Ca. Pandoraea novymonadis, a total of 17 novel species for which we propose the names shown in Table 1, and a novel species represented by strains PE-S2R-1 and PE-S2T-3 (Crofts et al., 2017) (see below). One of these novel species, i.e., Pandoraea cepalis, corresponds with Pandoraea genomospecies 1, which we reported earlier (Coenye et al., 2000). Two novel species, i.e., Pandoraea capi and Pandoraea bronchicola, correspond with Pandoraea genomospecies 3 and 4, respectively, reported by Daneshvar et al. (2001). Finally, the phylogenomic data (Figure 2 and Supplementary Tables S3, S4), but also each of the single locus sequence analyses, showed that Pandoraea genomospecies 2 LMG 20602 should be classified as P. sputorum, which contradicts earlier wet-lab DNA-DNA hybridization results (Daneshvar et al., 2001).

FIGURE 2.

Phylogenetic tree based on 107 single-copy core genes. BcgTree was used to extract the nucleotide sequence of 107 single-copy core genes and to construct their phylogeny by partitioned maximum-likelihood analysis. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Burkholderia cenocepacia J2315^T was used as outgroup. Bar, 0.01 changes per nucleotide position.

The use of dDDH and ANI threshold levels was generally straightforward, yet some pairs of strains showed values close to the generally applied taxonomic threshold levels (Supplementary Tables S3, S4) (Meier-Kolthoff et al., 2013; Yoon et al., 2017). The two strains classified as P. capi showed 96.4% ANI and 69.6% dDDH with a dDDH confidence interval of 66.6–72.5%, and these strains were therefore classified as the same species. Similarly, the three strains classified as P. cepalis showed 96.2–98.4% ANI, 68.4–86.0% dDDH, and the 70% dDDH threshold level was in the confidence interval; these strains were therefore classified as one species. P. soli LMG 31014^T showed 95.0–95.8% ANI and 60.7–65.0% dDDH toward P. cepalis strains, and the 70% dDDH threshold level was not part of the confidence interval, so this strain was classified as a separate species. Similarly, P. horticolens LMG 31112^T showed 95.0-95.3% ANI and 60.0-62.2% dDDH toward P. communis, and the 70% dDDH threshold level was not part of the confidence interval so this strain was also classified as a separate species.

The phylogenomic analyses also allowed us to identify 4 out of 6 unclassified Pandoraea strains for which genome sequences are publicly available: strain ISTKB (Kumar M. et al., 2016) was assigned to P. capi, strain B-6 (Liu et al., 2018) to P. cepalis, strain SD6-2 (Pushiri et al., 2013) to P. communis, and strain E26 (Chan et al., 2015) to P. pnomenusa (Figure 2 and Table 2). Finally, strains PE-S2R-1 and PE-S2T-3 (Crofts et al., 2017) formed a separate cluster, which represented yet another novel Pandoraea species that remains to be formally classified (Supplementary Tables S3, S4).

The phylogenomic tree based on 107 single-copy marker genes was well resolved and the clusters delineated by dDDH and ANI formed monophyletic groups with a high bootstrap support (Figure 2). The clades in the phylogenomic tree of the present study showed a branching order similar to a previously published tree based on 119 conserved proteins (Kostygov et al., 2017). The results of the phylogenomic analyses along with the clustering in the individual recA, gyrB, and gltB single locus sequence analyses (Figure 1 and Supplementary Figures S1, S2) were used to identify each of the 84 isolates included in the present study. P. sputorum strain LMG 31121 clustered with the remaining P. sputorum strains in the gyrB and gltB trees but grouped aberrantly in the recA tree. In addition, P. cepalis proved particularly difficult to identify through single locus sequence analysis as it exhibited more variation in each of the sequences examined (Figure 1 and Supplementary Figures S1, S2) than any other Pandoraea species.

Phenotypic Characterization

The type strains of each of 11 established Pandoraea species and of 17 novel Pandoraea species reported in the present study were included in an extensive phenotypic characterization. Among Pandoraea species, P. thiooxydans not only occupies a separate phylogenetic position (Figures 1, 2 and Supplementary Figures S1, S2) but also has a distinctive phenotype (Table 3). While all other Pandoraea species show normal growth on general microbiological growth media (i.e., they generate colonies of 1–4 mm in diameter after 2 days of incubation at 37°C), P. thiooxydans LMG 24779^T requires prolonged incubation up to 7 days before the same colony size was obtained.

TABLE 3.

Differential biochemical characteristics of all strains examined.

Characteristic	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	25	26	27	28
Growth at 45°Ca	−	−	+	−	−	+	−	−	−	−	−	+	−	−	−	+	−	+	−	−	−	−	−	−	−	−	−	+
Growth at 5% NaCl	+	+	−	+	+	+	+	+	−	−	−	−	+	−	+	+	+	+	−	+	+	+	−	+	−	−	−	+
Growth at 6% NaCl	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	+	−	−	−	−	−	−	−	−	−	−	−
Growth on MacConkey agar	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	−	+
Catalase activity	+	+	+	+	+	+	+	−	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
Hydrolysis of tween 20	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	−	+	+	+
Hydrolysis of tween 80	−	−	−	+	−	−	−	−	−	−	−	−	−	−	+	−	−	−	−	−	−	−	+	−	−	−	−	−
Alkaline phosphatase	−	w	w	−	w	w	−	−	−	w	−	w	−	−	−	−	w	−	−	−	−	+	−	−	−	−	−	−
Acid phosphatase	+	+	+	+	w	w	+	w	+	+	w	+	+	w	+	+	+	w	w	+	w	+	−	−	−	w	+	+
C4 esterase activity	w	w	w	−	+	+	w	w	+	−	w	+	w	w	+	w	+	+	w	w	−	−	−	−	w	w	+	w
Naphtol-AS-BI-β-D-glucuronide	w	w	w	−	w	w	w	w	−	w	−	+	−	w	−	w	+	w	w	w	w	+	−	w	−	w	+	w
Nitrate reduction	−	−	−	−	−	−	−	+	+	−	+	−	−	−	−	+	−	−	−	−	+	−	+	−	−	+	+	−
Assimilation of D-glucose	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	+	−	−	−	−	−	−	−	−	−	w	−
Assimilation of D-gluconate	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	w	−	+	+	+	+	+	+	+	+	+
Assimilation of caprate	+	+	−	+	+	+	−	−	+	+	−	−	+	+	w	−	−	−	−	+	−	+	−	−	+	−	−	−
Assimilation of citrate	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	−	+	+	+	+	+	+	+	+	+	+
Assimilation of phenylacetate	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	−	+	+	+	+	+	+	+	+	+	+

Species: 1, P. anapnoica LMG 31117^T; 2, P. anhela LMG 31108^T; 3, P. apista LMG 16407^T; 4, P. aquatica LMG 31011^T; 5, P. bronchicola LMG 20603^T; 6, P. capi LMG 20602^T; 7, P. captiosa LMG 31118^T; 8, P. cepalis LMG 31106^T; 9, P. commovens LMG 31010^T; 10, P. communis LMG 31110^T; 11, P. eparura LMG 31012^T; 12, P. faecigallinarum LMG 28171^T; 13, P. fibrosis LMG 29626^T; 14, P. horticolens LMG 31112^T; 15, P. iniqua LMG 31009^T; 16, P. morbifera LMG 31116^T; 17, P. norimbergensis LMG 18379^T; 18, P. nosoerga LMG 31109^T; 19, P. oxalativorans LMG 28169^T; 20, P. pneumonica LMG 31114^T; 21, P. pnomenusa LMG 18087^T; 22, P. pulmonicola LMG 18106^T; 23, P. soli LMG 31014^T; 24, P. sputorum LMG 18819^T; 25, P. terrae LMG 30175^T; 26, P. terrigena LMG 31013^T; 27, P. thiooxydans LMG 24779^T; 28, P. vervacti LMG 28170^T. +, present; −, absent; w, weak reaction. ^aGrowth characteristics were recorded after 4 days of incubation, except for P. thiooxydans LMG 24779^T for test results were recorded after 7 days of incubation.

The following biochemical characteristics were shared by all Pandoraea strains investigated: growth at 15, 28, and 37°C, but not at 4°C; growth in the presence of 0–4% NaCl, but not in the presence of 6–10% NaCl; growth at pH 6, 7, and 8, but not at pH 4, 5, or 9. No anaerobic growth. Oxidase activity is present. No hydrolysis of starch or casein. No DNase activity. No denitrification. Assimilation of L-malate, but not L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, maltose or adipate. No fermentation of glucose. No indole production, esculin hydrolysis, arginine dihydrolase, urease or PNP-β-galactosidase activity, or liquefaction of gelatin. Leucine arylamidase activity is present, but no C₈-ester-lipase, C₁₄-lipase, valine or cystine arylamidase, trypsin, chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase or α-fucosidase activity.

An overview of biochemical characteristics useful for distinguishing the type strains of Pandoraea species is shown in Table 3.

The fatty acid profiles of all type strains are shown in Table 4. Both quantitative and qualitative differences were present. The predominant fatty acids in all strains investigated were C_16:0, C_17:0 cyclo, C_16:0 3-OH, C_18:1 ω7c, C_19:0 cyclo ω8c, summed feature 2 (comprising C_14:0 3-OH, C_16:1 iso I, an unidentified fatty acid with equivalent chain length of 10.928, or C_12:0 ALDE, or any combination of these fatty acids), or summed feature 3 (comprising C_16:1 ω7c or C_15:0 iso 2-OH or both).

TABLE 4.

Fatty acid composition of all strains examined.

Fatty acid	1	2	3	4	5	6	7	8	9	10	11	12	13	14
C12:0	3.40	1.38	3.48	9.30	2.22	3.16	3.48	4.54	9.69	4.42	4.30	2.34	9.07	4.19
C12:0 2-OH	Tr	1.09	1.99	1.09	1.90	1.30	1.24	ND	Tr	Tr	ND	Tr	1.61	Tr
C14:0	Tr	1.77	1.06	Tr	Tr	Tr	Tr	Tr	Tr	Tr	Tr	1.88	ND	Tr
C14:0 2-OH	ND	1.01	ND	ND	ND	ND	ND	ND	ND	ND	ND	Tr	ND	ND
C16:0	18.95	20.54	20.21	18.38	23.45	16.09	15.29	15.29	17.76	18.82	15.47	15.77	19.98	17.45
C16:0 2-OH	1.01	1.13	Tr	Tr	1.01	1.28	1.78	1.21	Tr	1.67	1.22	Tr	ND	1.56
C16:0 3-OH	6.32	8.46	8.65	7.18	6.98	7.31	8.57	6.39	6.48	9.50	6.82	8.83	5.83	10.96
C16:1 2-OH	1.29	Tr	4.27	1.08	1.45	2.04	1.99	3.99	Tr	1.76	3.47	5.00	ND	2.76
C17:0 cyclo	19.83	18.78	20.57	13.72	26.44	19.46	20.35	14.29	9.14	18.10	9.52	8.30	2.47	20.72
C18:0	Tr	ND	ND	Tr	ND	Tr	Tr	ND	Tr	Tr	Tr	Tr	Tr	ND
C18:1 2-OH	3.39	3.35	3.91	3.07	2.49	5.18	5.94	2.71	2.17	3.15	3.54	5.86	ND	3.84
C18:1 ω7c	18.07	13.55	8.90	19.62	10.03	16.95	4.80	17.36	25.76	11.86	25.08	21.23	28.21	4.38
C19:0 cyclo ω8c	13.93	14.04	11.06	8.41	11.84	13.54	23.14	13.82	3.97	13.79	5.93	3.40	Tr	17.86
Summed feature 2∗	7.02	8.64	10.79	9.12	7.70	7.87	9.95	8.22	8.98	9.89	7.87	10.13	7.72	11.65
Summed feature 3∗	3.52	4.12	3.38	8.16	3.05	3.55	1.20	11.01	12.47	4.51	14.42	13.42	22.01	2.22
Fatty acid	15	16	17	18	19	20	21	22	23	24	25	26	27	28
C12:0	8.47	5.15	4.54	ND	3.93	4.18	2.03	3.02	4.31	2.86	5.59	4.35	3.97	4.18
C12:0 2-OH	1.01	1.51	Tr	ND	Tr	Tr	1.72	1.98	ND	2.23	Tr	ND	ND	Tr
C14:0	Tr	1.19	Tr	1.71	Tr	Tr	Tr	Tr	Tr	Tr	Tr	Tr	Tr	Tr
C14:0 2-OH	ND	ND	ND	8.27	ND	ND	Tr	ND	ND	ND	2.02	ND	ND	ND
C16:0	18.03	15.83	15.09	14.82	15.40	21.94	18.34	18.19	12.28	17.13	22.91	13.49	26.61	15.72
C16:0 2-OH	ND	ND	1.06	3.66	1.26	1.61	1.33	1.79	1.67	1.14	ND	1.48	ND	1.16
C16:0 3-OH	6.82	9.40	7.53	14.18	7.03	7.88	7.90	9.48	3.14	8.64	7.06	6.56	1.84	9.05
C16:1 2-OH	Tr	1.07	2.41	2.20	4.93	1.35	2.78	2.19	5.96	Tr	2.00	4.84	Tr	3.51
C17:0 cyclo	14.43	4.84	22.11	15.45	11.45	22.81	16.32	16.55	8.49	14.59	8.24	11.33	19.60	19.36
C18:0	Tr	1.41	Tr	ND	Tr	Tr	Tr	Tr	Tr	ND	Tr	Tr	Tr	ND
C18:1 2-OH	1.61	3.77	3.09	5.69	4.56	2.49	5.19	3.29	3.66	3.99	ND	3.85	Tr	6.58
C18:1 ω7c	21.55	30.31	12.94	1.25	20.47	8.85	17.84	7.05	16.75	21.18	18.83	23.91	12.42	9.54
C19:0 cyclo ω8c	7.75	5.58	14.19	13.03	8.57	14.85	10.16	20.19	3.33	11.56	5.31	5.65	3.12	17.50
Summed feature 2∗	8.63	9.75	10.50	18.27	8.30	9.07	8.49	11.07	12.00	9.87	9.45	8.15	12.29	9.59
Summed feature 3∗	9.10	9.26	3.58	ND	11.51	2.76	5.10	2.19	16.19	4.49	15.15	13.39	15.25	1.94

Species: 1, P. anapnoica LMG 31117^T; 2, P. anhela LMG 31108^T; 3, P. apista LMG 16407^T; 4, P. aquatica LMG 31011^T; 5, P. bronchicola LMG 20603^T; 6, P. capi LMG 20602^T; 7, P. captiosa LMG 31118^T; 8, P. cepalis LMG 31106^T; 9, P. commovens LMG 31010^T; 10, P. communis LMG 31110^T; 11, P. eparura LMG 31012^T; 12, P. faecigallinarum LMG 28171^T; 13, P. fibrosis LMG 29626^T; 14, P. horticolens LMG 31112^T; 15, P. iniqua LMG 31009^T; 16, P. morbifera LMG 31116^T; 17, P. norimbergensis LMG 18379^T; 18, P. nosoerga LMG 31109^T; 19, P. oxalativorans LMG 28169^T; 20, P. pneumonica LMG 31114^T; 21, P. pnomenusa LMG 18087^T; 22, P. pulmonicola LMG 18106^T; 23, P. soli LMG 31014^T; 24, P. sputorum LMG 18819^T; 25, P. terrae LMG 30175^T; 26, P. terrigena LMG 31013^T; 27, P. thiooxydans LMG 24779^T; 28, P. vervacti LMG 28170^T. Those fatty acids for which the amount for all taxa was <1% are not included, therefore, the percentages may not add up to 100%. Tr, trace amount (<1%); ND, not detected. ^∗Summed feature 2 comprises iso-C_16:1I and/or C_14:03-OH; summed feature 3 comprises iso-C_15:02-OH and/or C_16:1ω7c.

Functional Genome Analyses

The 68 Pandoraea genomes in the present study comprised 331,123 CDS, of which 273,692 (83%) and 128,054 (39%) could be assigned to the COG and KEGG orthologies, respectively (Supplementary Table S5). Orthologous genes were identified to determine the conserved genome content of the genus Pandoraea. Ortholog analysis revealed 10,783 orthogroups (325,879 CDS) in total, of which 738 (51,633 CDS) were present in all genomes, 897 (62,692 CDS) were present in all genomes except Ca. Pandoraea novymonadis, 8,003 (207,937 CDS) were present in multiple species, 1,130 (3,581 CDS) were species-specific and 15 (36 CDS) were isolate-specific (Figure 3). For further analyses, the core orthogroups were defined as those present in all genomes or all genomes except Ca. Pandoraea novymonadis (n = 1,635). COG and KEGG could be assigned to 7,243 (67%) and 3,655 (34%) of a total of 10,783 orthogroups (Supplementary Table S6). A previous pan genome analysis of 36 Pandoraea genomes by Wu et al. (2019) revealed a core genome of 1,903 CDS. As shown by these authors, the pan genome of Pandoraea is open (Wu et al., 2019) and the number of core genes decreases with an increasing number of genomes analyzed.

FIGURE 3.

Genomes per orthogroup. Core, present in all genomes or all genomes except Ca. Pandoraea novymonadis.

The frequency of orthologous versus non-orthologous CDS varied significantly per isolate [X²(67) = 7423, p < 0.001] and species [X²(29) = 5863, p < 0.001]. The number of non-orthologous CDS per genome ranged from 0 to 632, with P. terrae LMG 30175^T showing the highest percentage of non-orthologous CDS (Figure 4 and Supplementary Table S7). To identify biological functions that were over- or underrepresented in the core genome, we looked at the COG and KEGG functional classification of the orthogroups versus their specificity (core, multiple species, single species or single isolate). The specificity of the orthogroups varied significantly among the COG categories [X²(66) = 522, p < 0.001] and highest levels of the KEGG pathways [X²(10) = 130, p < 0.001]. The core orthogroups were significantly enriched in the COG categories Translation, ribosomal structure and biogenesis (J), Posttranslational modification, protein turnover, chaperones (O), Nucleotide transport and metabolism (F) and Coenzyme transport and metabolism (H) (Figure 5 and Supplementary Table S8) and in the KEGG pathway Genetic Information Processing (09120) (Figure 6 and Supplementary Table S9).

FIGURE 4.

The frequency of orthologous versus non-orthologous CDS varies among species. Bar plots show the number of orthologous and non-orthologous CDS per species [X²(29) = 5863, p < 0.001].

FIGURE 5.

Orthogroup specificity varies among COG categories. Bar plot shows the number of orthogroups and their specificity per COG category [X²(66) = 522, p < 0.001]. J, translation, ribosomal structure and biogenesis; K, transcription; L, replication, recombination and repair; B, chromatin structure and dynamics; D, cell cycle control, cell division, chromosome partitioning; V, defense mechanisms; T, signal transduction mechanisms; M, cell wall/membrane/envelope biogenesis; N, cell motility; W, extracellular structures; U, intracellular trafficking, secretion, and vesicular transport; O, posttranslational modification, protein turnover, chaperones; X, mobilome: prophages, transposons; C, energy production and conversion; G, carbohydrate transport and metabolism; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; R, general function prediction only; S, function unknown.

FIGURE 6.

Orthogroup specificity varies among KEGG categories. Bar plot shows the number of orthogroups and their specificity per KEGG category [X²(10) = 130, p < 0.001].

Because many Pandoraea strains participate in the biodegradation of recalcitrant xenobiotics (Uhlik et al., 2012; Pushiri et al., 2013; Shi et al., 2013; Wang et al., 2015; Crofts et al., 2017; de Paula et al., 2017; Sarkar et al., 2017; Tirado-Torres et al., 2017; Kumar et al., 2018b; Yang et al., 2018; Liu et al., 2019; Wu et al., 2019), we specifically looked at the orthogroups in the KEGG pathway Xenobiotics biodegradation and metabolism (Figure 7). Most orthogroups in this pathway were present in multiple species (n = 28) and some were even present in the core Pandoraea genome (n = 6). This confirmed the potential of Pandoraea for degrading xenobiotics. In particular, the widespread capacity to utilize benzoate derivatives (Figure 7, pathways 362, 364, 627, and 633) explains why several strains have the potential to degrade lignin (Shi et al., 2013; Kumar et al., 2018a; Liu et al., 2019) and other aromatic compounds (Springael et al., 1996; Uhlik et al., 2012; Wang et al., 2015). Finally, P. fibrosis and P. thiooxydans showed a unique capacity to degrade specific compounds (Figure 7). P. fibrosis was only recently described and named after its origin from a cystic fibrosis patient (See-Too et al., 2019) but its unique capacity to degrade nitrotoluene derivatives is yet another example of the versatility in one Pandoraea species.

FIGURE 7.

Orthogroup specificity in KEGG pathway Xenobiotics biodegradation and metabolism. Bar plot shows the number of orthogroups and their specificity.

Conclusion

The present study extends the number of formally named Pandoraea species considerably and makes reference cultures and their whole-genome sequences publicly available. The genus Pandoraea further emerges as a group of environmental bacteria with strong biodegradation capacities and as opportunistic human pathogens, especially in persons with cystic fibrosis. Within this genus, P. thiooxydans and P. terrae and Candidatus P. novymonadis cluster outside the main Pandoraea lineage. The aberrant phylogenomic position of the former is further supported by a distinctive phenotype. The classification of these bacteria within this monophyletic genus could therefore be questioned.

Taking into account the source and identification of strains ISTKB (a rhizospheric soil isolate, Kumar M. et al., 2016) and B-6 (an eroded bamboo slip isolate, Liu et al., 2018), and, to be as comprehensive as possible, also some additional unpublished own data (JL and PV), the novel species P. aquatica, P. capi, P. cepalis, P. commovens, P. communis, and P. iniqua, but also the established species P. faecigallinarum, P. norimbergensis, P. pnomenusa, and P. fibrosis, have all been isolated from both human clinical and environmental sources. Thus far, the novel species P. anapnoica, P. anhela, P. bronchicola, P. captiosa, P. morbifera, P. nosoerga, and P. pneumonica, but also the established species P. apista, P. pulmonicola, and P. sputorum, have all been isolated from human clinical sources only; while the novel species P. eparura, P. horticolens, P. soli and P. terrigena, and the established species P. oxalativorans, P. terrae, P. thiooxydans, and P. vervacti have thus far been isolated from environmental samples only.

The present study provides genomic, chemotaxonomic and phenotypic data that enable a formal proposal of 17 novel Pandoraea species as outlined below. By making reference cultures and whole-genome sequences of each of these versatile bacteria publicly available, we aim to contribute to future knowledge about the metabolic versatility and pathogenicity of these organisms.

Description of Pandoraea anapnoica sp. nov.

Pandoraea anapnoica sp. nov. (a.na.pnoi’ca. Gr. masc. adj. anapnoikos, affecting respiration; N.L. fem. adj. anapnoica affecting respiration).

The phenotypic description is as presented above and in Table 3.

Isolated from human clinical samples in the United States.

The type strain is LMG 31117^T (=CCUG 73385^T) and was isolated from a cystic fibrosis specimen in the United States in 1999. Its G + C content is 62.4 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 31117^T are publicly available through the accession numbers LR536847, LR536866–LR536868, and CABPSP010000000, respectively.

Description of Pandoraea anhela sp. nov.

Pandoraea anhela sp. nov. (an.he’la. L. fem. adj. anhela breath-taking).

The phenotypic description is as presented above and in Table 3.

Isolated from human clinical samples in the United States.

The type strain is LMG 31108^T (=CCUG 73386^T) and was isolated from a cystic fibrosis specimen in the United States in 2006. Its G + C content is 63.4 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 31108^T are publicly available through the accession numbers LR536848, LR536863-LR536865 and CABPSB010000000, respectively.

Description of Pandoraea aquatica sp. nov.

Pandoraea aquatica sp. nov. (a.qua’ti.ca. L. fem. adj. aquatica aquatic).

The phenotypic description is as presented above and in Table 3.

Isolated from human clinical samples in the United States and from pond water in Belgium.

The type strain is LMG 31011^T (=CCUG 73384^T) and was isolated from pond water in a greenhouse in Belgium in 2013. Its G + C content is 62.9 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 31011^T are publicly available through the accession numbers LR536849, LR536869–LR536871, and CABPSN010000000, respectively.

Description of Pandoraea bronchicola sp. nov.

Pandoraea bronchicola sp. nov. (bron.chi’co.la. L. neut. pl. n. bronchia, the bronchial tubes; L. suff. -cola [from L. n. incola] a dweller, inhabitant; N.L. fem. n. bronchicola a dweller of bronchi, coming from the bronchi).

The phenotypic description is as presented above and in Table 3.

Isolated from human clinical samples in the United States.

The type strain is LMG 20603^T (= ATCC BAA-110^T = CDC H652^T) and was isolated from cystic fibrosis sputum in the United States in 1998. Its G + C content is 63.0 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 20603^T are publicly available through the accession numbers LR536994, LR536872–LR536874, and CABPST010000000, respectively.

Description of Pandoraea capi sp. nov.

Pandoraea capi sp. nov. (ca’pi. Gr. masc. n. kapos, breath; N.L. gen. n. capi, referring to the lung as niche of these bacteria).

The phenotypic description is as presented above and in Table 3.

Isolated from human clinical samples in the United States and from rhizospheric soil in India.

The type strain is LMG 20602^T (=ATCC BAA-109^T = CDC G9805^T) and was isolated from sputum of a non-cystic fibrosis patient in the United States in 1996. Its G + C content is 63.4 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 20602^T are publicly available through the accession numbers LR536850, LR536884–LR536886, and CABPRV010000000, respectively.

Description of Pandoraea captiosa sp. nov.

Pandoraea captiosa sp. nov. (cap.ti.o’sa. L. fem. adj. captiosa, harmful, disadvantageous).

The phenotypic description is as presented above and in Table 3.

Isolated from human clinical samples in the United States.

The type strain is LMG 31118^T (=CCUG 73387^T) and was isolated from a cystic fibrosis specimen in the United States in 2008. Its G + C content is 63.3 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 31118^T are publicly available through the accession numbers LR536851, LR536893–LR536895, and CABPSQ010000000, respectively.

Description of Pandoraea cepalis sp. nov.

Pandoraea cepalis sp. nov. [ce.pa’lis. Gr. n. kepos, garden; -alis L. adjective forming suffix, pertaining to; N.L. fem. adj. cepalis pertaining to garden (soil)].

The phenotypic description is as presented above and in Table 3.

Isolated from soil and water samples in Belgium and the Netherlands, from human clinical samples in the United States, and from historical bamboo slips in China.

The type strain is LMG 31106^T (=CCUG 39680^T) and was isolated from garden soil in The Netherlands. Its G + C content is 63.7 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 31106^T are publicly available through the accession numbers LR536852, LR536896–LR536898, and CABPSL010000000, respectively.

Description of Pandoraea commovens sp. nov.

Pandoraea commovens sp. nov. (com.mo’vens. L. v. commovere, to trouble, upset; L. pres. part. commovens troubling).

The phenotypic description is as presented above and in Table 3.

Isolated from human clinical samples in Belgium and the United States, from soil samples in Belgium, and from plant roots in India.

The type strain is LMG 31010^T (=CCUG 73378^T) and was isolated from sputum of a cystic fibrosis patient in Belgium in 2002. Its G + C content is 62.6 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 31010^T are publicly available through the accession numbers LR536853, LR536902–LR536904, and CABPSA010000000, respectively.

Description of Pandoraea communis sp. nov.

Pandoraea communis sp. nov. (com.mu’nis. L. fem. adj. communis common, widespread).

The phenotypic description is as presented above and in Table 3.

Isolated from human clinical, soil and water samples in Belgium, and from soil in Australia.

The type strain is LMG 31110^T (=CCUG 73383^T) and was isolated from sputum of a cystic fibrosis patient in Belgium in 2012. Its G + C content is 62.6 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 31110^T are publicly available through the accession numbers LR536854, LR536911-LR536913 and CABPSJ010000000, respectively.

Description of Pandoraea eparura sp. nov.

Pandoraea eparura sp. nov. (ep.a.ru’ra. Gr. masc. adj. eparouros, attached to the soil; N.L. fem. adj. eparura attached to the soil).

The phenotypic description is as presented above and in Table 3.

The type (and thus far only) strain is LMG 31012^T (=CCUG 73380^T) and was isolated from soil of a house plant in Belgium in 2003. Its G + C content is 63.7 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 31012^T are publicly available through the accession numbers LR536855, LR536923–LR536925, and CABPSH010000000, respectively.

Description of Pandoraea horticolens sp. nov.

Pandoraea horticolens sp. nov. (hor.ti’co.lens. L. n. hortus garden; L. v. colere to dwell; L. pres. part. colens dwelling; N.L. part. adj. horticolens because the type strain was isolated from garden [soil]).

The phenotypic description is as presented above and in Table 3.

The type (and thus far only) strain is LMG 31112^T (=CCUG 73379^T) and was isolated from garden soil in Belgium in 2003. Its G + C content is 62.3 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 31112^T are publicly available through the accession numbers LR536857, LR536926-LR536928 and CABPSM010000000, respectively.

Description of Pandoraea iniqua sp. nov.

Pandoraea iniqua sp. nov. (in.i’qua. L. fem. adj. iniqua disadvantageous, hostile).

The phenotypic description is as presented above and in Table 3.

Isolated from soil samples in Belgium and human clinical samples in the United States.

The type strain is LMG 31009^T (=CCUG 73377^T) and was isolated from maize rhizosphere soil in Belgium in 2002. Its G + C content is 63.1 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 31009^T are publicly available through the accession numbers LR536856, LR536929–LR536931, and CABPSF010000000, respectively.

Description of Pandoraea morbifera sp. nov.

Pandoraea morbifera sp. nov. (mor.bi’fe.ra, L. fem. adj. morbifera that brings disease).

The phenotypic description is as presented above and in Table 3.

Isolated from human clinical samples in the United States.

The type strain is LMG 31116^T (=CCUG 73389^T) and was isolated from a cystic fibrosis specimen in the United States in 2006. Its G + C content is 64.7 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 31116^T are publicly available through the accession numbers LR536858, LR536935–LR536937, and CABPSD010000000, respectively.

Description of Pandoraea nosoerga sp. nov.

Pandoraea nosoerga sp. nov. (no.so.er’ga, Gr. masc. adj. nosoergos, causing sickness; N.L. fem. adj. nosoerga).

The phenotypic description is as presented above and in Table 3.

Isolated from human clinical samples in Australia, Belgium, Germany, United Kingdom and the United States.

The type strain is LMG 31109^T (=CCUG 73390^T) and was isolated from a cystic fibrosis specimen in the United States in 2008. Its G + C content is 66.1 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 31109^T are publicly available through the accession numbers LR536859, LR536941-LR536943 and CABPSC010000000, respectively.

Description of Pandoraea pneumonica sp. nov.

Pandoraea pneumonica sp. nov. (pneu.mo’ni.ca, Gr. masc. adj. pneumonikos, of the lungs; N.L. fem. adj. pneumonica).

The phenotypic description is as presented above and in Table 3.

The type (and thus far only) strain is LMG 31114^T (=CCUG 73388^T) and was isolated from a cystic fibrosis specimen in the United States in 2009. Its G + C content is 62.5 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 31114^T are publicly available through the accession numbers LR536861, LR536974–LR536976, and CABPSK010000000, respectively.

Description of Pandoraea soli sp. nov.

Pandoraea soli sp. nov. (so’li. L. gen. n. soli of soil, the source of the type strain).

The phenotypic description is as presented above and in Table 3.

The type (and thus far only) strain is LMG 31014^T (=CCUG 73382^T) and was isolated from soil of a house plant in Belgium in 2003. Its G + C content is 63.6 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 31014^T are publicly available through the accession numbers LR536860, LR536980–LR536982, and CABPSG010000000, respectively.

Description of Pandoraea terrigena sp. nov.

Pandoraea terrigena sp. nov. (ter.ri’ge.na. L. fem. n. terra soil; L. v. gignere to bear; L. fem. n. terrigena [nominative in apposition] born of, or from, soil, soil-born).

The phenotypic description is as presented above and in Table 3.

The type (and thus far only) strain is LMG 31013^T (=CCUG 73381^T) and was isolated from soil of a house plant in Belgium in 2003. Its G + C content is 63.5 mol% (calculated based on its genome sequence). The 16S rRNA, gltB, gyrB, recA and whole-genome sequence of LMG 31013^T are publicly available through the accession numbers LR536862, LR536977–LR536979, and CABPRU010000000, respectively.

Data Availability Statement

The datasets generated for this study can be found in the European Nucleotide Archive PRJEB30806, PRJEB30685, PRJEB30686, PRJEB30687, PRJEB30688, PRJEB30689, PRJEB30690, PRJEB30807, PRJEB30745, PRJEB30746, PRJEB30808, PRJEB30691, PRJEB30692, PRJEB30809, PRJEB30810, PRJEB30693, PRJEB30694, PRJEB30695, PRJEB30696, PRJEB30697, PRJEB30699, PRJEB30700, PRJEB30701, PRJEB30698, PRJEB30811, PRJEB30702, PRJEB30703, PRJEB30812, PRJEB30704, PRJEB30705, PRJEB30706, PRJEB30707, PRJEB30708, PRJEB30714, PRJEB30709, PRJEB30710, PRJEB30711, PRJEB30712, PRJEB30713, PRJEB30813, PRJEB30814, PRJEB30815, PRJEB30755, PRJEB30724, PRJEB30756, PRJEB30725, PRJEB30726, PRJEB30727, PRJEB30728, PRJEB30721, PRJEB30722, PRJEB30723, PRJEB30757, PRJEB30715, PRJEB30716, PRJEB30717, PRJEB30753, PRJEB30752, PRJEB30754, PRJEB30740, PRJEB30741, PRJEB30742, PRJEB30743, PRJEB30718, PRJEB30744, PRJEB30748, PRJEB30749, PRJEB30750, PRJEB30751, PRJEB30729, PRJEB30730, PRJEB30731, PRJEB30732, PRJEB30733, PRJEB30734, PRJEB30735, PRJEB30736, PRJEB30737, PRJEB30738, PRJEB30739, PRJEB30747, PRJEB30720, and PRJEB30719.

Author Contributions

PV, JL, and CP conceived the study. PV and CP wrote the manuscript. EDB, EDC, TS, and CP performed single locus sequence analyses. CP performed phylogenetic analyses. CP, ED, and BV carried out the genomic data analyses. EDC, MC, EDB, and CS carried out wet-lab phenotypical analyses. PV and JL generated the required funding. All authors read and approved the final manuscript.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

ANI

average nucleotide identity

COG

cluster of orthologous groups

dDDH

digital DNA–DNA hybridization

GBDP

genome blast distance phylogeny

GGDC

genome-to-genome distance calculator

KEGG

kyoto encyclopedia of genes and genomes.

Funding

Part of this work was performed in the framework of the Belgian National Reference Centre for Burkholderia, supported by the Ministry of Social Affairs through a fund within the National Health Insurance System. This funding agency had no role in study design, data collection and interpretation, or the decision to submit the work for publication. JL and TS receive support from the Cystic Fibrosis Foundation (United States).

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2019.02556/full#supplementary-material