Genome insights from the identification of a novel Pandoraea sputorum isolate and its characteristics.

In this study, we sequenced a bacteria isolate Pandoraea sp. 892iso isolated from a Phytophthora rubi strain which is an important plant pathogenic oomycete, identified through genome and combined the data with existing genomic data from other 28 the genus of Pandoraea species. Next, we conducted a comparative genomic analysis of the genome structure, evolutionary relationships, and pathogenic characteristics of Pandoraea species. Our results identified Pandoraea sp. 892iso as Pandoraea sputorum at both the genome and gene levels. At the genome level, we carried out phylogenetic analysis of single-copy, gene co-linearity, ANI (average nucleotide identity) and AAI (average amino acid identity) indices, rpoB similarity, MLSA phylogenetic analysis, and genome-to-genome distance calculator calculations to identify the relationship between Pandoraea sp. 892iso and P. sputorum. At the gene level, the quorum sensing genes ppnI and ppnR and the OXA-159 gene were assessed. It is speculated that Pandoraea sp. 892iso is the endosymbiont of the Oomycetes strain of Phytophthora rubi.

Introduction

The genus Pandoraea, originating from the term “Pandora’s box”, refers to the source of all evil in Greek mythology and was established by Coenye et al. in 2000 [1]. The species are characterized as nonspore-forming, catalase-positive, aerobic, gram-negative rods with polar flagella. Some species in this genus were once identified closest to Burkholderia cepacia complex (Bcc), Ralstonia pickettii, or Ralstonia paucula based on phenotype. [1-3]. The genus Pandoraea includes 28 named species (Pandoraea anapnoica, P. anhela, P. apista, P. aquatica, P. bronchicola, P. capi, P. captiosa, P. cepalis, P.commovens, P. communis, P. eparura, P. faecigallinarum, P. fibrosis, P. horticolens, P. iniqua, P. morbifera, P. norimbergensis, P. nosoerga, P. oxalativorans, P. pneumonica, P. pnomenusa, P. pulmonicola, P. soli, P. sputorum, P. terrae, P. terrigena, P. thiooxydans and P. vervacti [3-5]. Pandoraea sp. types have been predominantly isolated from patients with septicemia or respiratory tract infections (mostly cystic fibrosis), as well as from food, water, soil, and food [2, 4, 6–9].

Clinical manifestations of this terrorizing pathogen revolve around nosocomial infections and its ability to deteriorate lung function and even cause multiple organ impairment [10-12]. These organisms appear to be potential pathogens for individuals with cystic fibrosis as well for cross-infection [13]. Further, Pandoraea spp. isolated from environmental samples have considerable potential for biotechnological application given various beneficial degradation abilities, such as removing isomers of 1,2,3,4,5,6-hexachlorocyclohexane (HCH) [13], catalyzing the aerobic transformation of biphenyl and various polychlorinated biphenyls (PCBs) [14, 15], catalyzing the decarboxylation of 2,6-dihydroxybenzoate and regioselective carboxylation of 1,3-dihydroxybenzene to 2,6-dihydroxybenzoate, catalyzing the regioselective carboxylation of phenol and 1,2-dihydroxybenzene [16], degrading kraft lignin without any cosubstrate under high alkaline conditions [17], degrading chlorobenzene [18], biodegrading endosulfan classified as an organochlorine pesticide [19], treating malachite green [20], and metabolizing oxalate [21].

Reflecting on previous research, Pandoraea spp. have frequently been misidentified in many clinical laboratories, leading to a lack of clinical documentation on their virulence potential. Therefore, it is important to accurately identify Pandoraea spp.. Earlier classification of prokaryotes was based solely on phenotypic similarities [22], but modern prokaryote characterization has been strongly influenced by advances in genetic methods. One criterion to be considered a species is to be essentially a collection of types that are characterized by at least one diagnostic phenotypic trait and to have purified DNA molecules that show at least 70% cross-hybridization (DNA-DNA hybridization, DDH) [22-25]. This is pragmatic and universally applicable within the bacterial domain, while the lack of this standard has been increasingly found when it comes to reliable diagnosis of infectious disease agents, international regulations for transport, quarantine, and so on [26-28]. Subsequently, this parameter has been applied most frequently in species identification at the whole genome level [29-32]. Genome Blast Distance Phylogeny (GBDP) [33], the core and pangenome [32], and the genomic-distance index based on DNA maximal unique matches (MUM) [34] are used to identify new species. Unfortunately, our understanding of Pandoraea spp. at the genomic level is relatively superficial, whereby the majority of the literature focuses principally on the usage of genotypic data to facilitate accurate genus- and species-level identification and secondarily on biotechnological potential [1, 2, 18, 21].

In the present study, suspected bacteria isolated from an oomycete strain was identified through whole genome sequencing. The taxonomic status of this isolate was verified at the genome and gene levels, and its phylogenetic relationship with similar species was explored using indices, such as ANI/AAI, MLSA (Multi-locus Sequence Analysis) phylogenetic analysis, genome-to-genome distance calculations, quorum sensing, and oxacillinase gene analysis.

Materials and methods

Strains, cultures, and DNA extraction

When we performed morphological observations on the hyphae of a Phytophthora rubi strain (No. 109892) from Westerdijk Fungal Biodiversity Institute, we inadvertently discovered the structure of suspected bacteria present in the mycelia. The structure still existed after the isolation by monofilament isolation and monospore isolation of the fungus. After isolation and culture, we obtained an analytical strain of bacteria, so that part of the name of which is called “892iso isolate”. Separation, purification, and culture were carried out on beef extract peptone medium plates at 30°C for 48 h. A TIANamp Bacteria DNA Kit (Tiangen, China) was used for genomic DNA.

Sequencing, assembly, and annotation

The whole genome was sequenced and assembled by a strategy that combined paired-end and mate-paired libraries. One targeted insert size of 500 bp was constructed using the TruSeq Nano DNA LT Library Prep Kit (Illumina, USA). One mate-paired library (2 kb) was constructed by the Nextera Mate Pair Sample Prep Kit (Illumina, FC-132–1001, USA) on the Illumina HiSeq 2500 platform. SOAPdenovo (v2.04) was used for de novo assembly. The assembled genome was annotated with a web-based tool called RAST (http://rast.nmpdr.org). RAST can identify repeat sequences in the genome, protein-encoding rRNA and tRNA genes, and assign functions to the genes.

Whole genome alignment and some indices calculation

Mauve (version 2.3.1) was used to align genomes for synteny analysis. The calculation of ANI and AAI was based on BLAST alignment results using a Perl script. The genome-to-genome distance calculator calculations were based on a web server (https://ggdc.dsmz.de/) that uses multi-FASTA files as input. The ppnI/ppnR genes of P. pnomenusa were download from NCBI (accession ID KF887500.1 and KF900148.1), then aligned with all Pandoraea gene sets, all matches with the identity greater than 0.3 and score greater than 100 were retained. The ppnI candidates should contain PF00765 domain and ppnR candidates contain PF03472 domain, and the candidate pairs should be adjacent to each other. An intrinsic Carbapenem-Hydrolyzing Oxacillinases gene of Pandoraea sp. HD7676 was download from NCBI (accession ID: KP771987.1). BLAST was employed to identify homolog genes in the 28 Pandoraea species.

Comparative genome analysis

All protein sequences in reference genomes were downloaded and set as the query for all-vs-all BLASTP. OrthoMCL (version 2.0.8) was used to identify single-copy genes with I (inflation) set at 1.5. Next, MUSCLE (version 3.8.425) was used to align the sequences of the associated proteins. PAL2NAL (version 14.0) was used to convert the protein alignment to codon alignment. Gblock (version 0.91b) was used to remove the alignment results that were deemed unreliable. The phylogenetic tree was built by single-copy genes, with Burkholderia cepacia strain LO6 as the outgroup. MCMCTree software in PAML (version 4.7) was used to estimate the divergence time. CAFÉ (version 4) was used to calculate the expansion and contraction of these gene families.

Results

Genome assembly, annotation, and validation of protein-coding genes

The genome of the Pandoraea sp. 892iso isolate was assembled from sequencing data generated by HiSeq 2000 by SOAPDenovo2 assembler. The total length of the top 48 longest scaffolds was 5.83 Mb, representing approximately 82.6-fold genome sequence coverage. The N50 and maximum lengths of scaffolds was 1.43 kb. Most of the length was concentrated on 12 scaffold sequences over 1,000 bp, of which the longest sequence was 2.06 MB (Fig 1). A total of 5,367 protein-coding genes were predicted from the genome assembly, 5,131 (95.60%) of which were supported by the RNA-seq data (coverage > = 90%). Within these protein-coding genes, 4,274 (79.63%) were assigned a biological function. Among the 1,093 ORFs without known function, 736 showed similarity to other database entries. For Pandoraea sp. 892iso isolate, the coding regions from the predicted genes constituted 88.61% of the genome (total length of all genes divide the genome size) and the average gene density was 919 genes per 1 Mb (total number of all genes divide the genome size, times with 100000bp), which were more or fewer than most of other sequenced Pandoraea species. The GC content of the genome, coding sequences, and repetitive elements were 62.66%, 63.32%, and 57.52%, respectively. A total of 63 tRNA genes were predicted from the assembly. The genome characteristics of Pandoraea sp. 892iso and other Pandoraea species are shown in Table 1.

Fig 1

Structure of the genome assembly.

Table 1

Genome and gene comparison of Pandoraea sp. 892iso and other Pandoraea species.

Content	Pandoraea sp. 892iso	Pandoraea anapnoica	Pandoraea anhela	Pandoraea apista	Pandoraea aquatica	Pandoraea bronchicola	Pandoraea capi	Pandoraea captiosa	Pandoraea cepalis	Pandoraea commovens	Pandoraea communis
Accession number		GCF_902459765.1	GCF_902459655.1	GCF_001465595.2	GCF_902459565.1	GCF_902459805.1	GCF_902459735.1	GCF_902459775.1	GCF_902459625.1	GCF_902459615.1	GCF_902459745.1
Genome
Scaffold Number	48	48	61	2	17	34	31	36	32	26	17
Total Length (Mb)	5.83	6.13	6.05	5.57	5.96	5.35	5.85	6.14	5.16	6.04	5.71
GC Content (%)	62.7	62.4	63.35	62.63	62.89	62.97	63.44	63.3	63.54	62.63	62.57
N50 Length	1,430,084	278,466	256,277	-	442,715	323,330	401,082	280,257	286,967	434,611	434,208
N90 Length	768,040	128,458	71,214	-	244,118	147,078	151,211	137,159	88,384	144,451	240,155
Longest scaffold	2,057,907	677,688	731,537	-	1,296,496	977,860	812,744	1,307,560	1,062,234	1,441,918	1,422,275
Gene
Gene Number	5367	5,348	5,178	4,969	5,197	4,734	5,049	5,328	4,602	5,246	5,051
Gene Length (bp)	5,175,057	5,334,275	5,189,856	4,830,549	5,211,988	4,656,675	5,074,754	5,326,122	4,478,447	5,288,517	4,979,502
GC Content in Gene Region (%)	63.32	63.11	63.92	63.21	63.6	63.52	64.13	63.89	64.03	63.33	63.13
Gene Length/Genome (%)	88.61	87.07	85.84	86.7	87.48	87.02	86.72	86.75	86.8	87.6	87.23
Gene Average Length (bp)	964	997	1,002	972	1,003	984	1,005	1,000	973	1,008	986
Intergenic Region Length (bp)	665,083	792,413	856,156	740,711	746,139	694,448	777,390	813,460	681,119	748,432	729,101
GC Content in Intergenic Region (%)	57.52	57.63	59.87	58.9	57.95	59.27	58.97	59.45	60.3	57.73	58.74
Intergenic Region Length/Genome (%)	11.39	12.93	14.16	13.3	12.52	12.98	13.28	13.25	13.2	12.4	12.77
Content	Pandoraea eparura	Pandoraea faecigallinarum	Pandoraea fibrosis	Pandoraea horticolens	Pandoraea iniqua	Pandoraea morbifera	Pandoraea norimbergensis	Pandoraea nosoerga	Pandoraea oxalativorans	Pandoraea pneumonica	Pandoraea pnomenusa
Accession number	GCF_902459725.1	GCF_001029105.3	GCF_000807775.2	GCF_902459555.1	GCF_902459685.1	GCF_902459575.1	GCF_001465545.3	GCF_902459585.1	GCF_000972785.3	GCF_902459645.1	GCF_000504585.2
Genome
Scaffold Number	35	3	1	68	17	47	1	41	5	12	1
Total Length (Mb)	5.21	5.73	5.59	6.01	6.34	5.23	6.17	4.86	6.5	5.85	5.39
GC Content (%)	63.68	63.45	62.82	62.31	63.06	64.65	63.06	66.13	63.08	62.45	64.89
N50 Length	259,402	-	-	290,798	382,973	316,192	-	229,370	-	265,947	-
N90 Length	102,841	-	-	73,897	241,289	80,719	-	91,075	-	5,636	-
Longest scaffold	893,217	-	-	787,753	1,308,188	801,833	-	664,052	-	2,096,772	-
Gene
Gene Number	4,615	5,027	4,855	5,322	5,499	4,652	5,356	4,297	5,648	5,168	4,759
Gene Length (bp)	4,496,889	4,932,939	4,868,583	5,167,287	5,558,312	4,536,412	5,418,712	4,198,421	5,522,745	5,131,811	4,684,824
GC Content in Gene Region (%)	64.17	63.98	63.39	62.98	63.76	65.1	63.72	66.51	63.59	63.06	65.36
Gene Length/Genome (%)	86.39	86.05	87.06	86	87.68	86.68	87.86	86.35	84.96	87.8	86.98
Gene Average Length (bp)	974	981	1,003	971	1,011	975	1,012	977	978	993	984
Intergenic Region Length (bp)	708,688	799,725	723,482	841,203	780,817	696,886	748,658	663,693	977,986	713,267	701,122
GC Content in Intergenic Region (%)	60.55	60.15	58.94	58.19	58.13	61.77	58.29	63.71	60.23	58.09	61.71
Intergenic Region Length/Genome (%)	13.61	13.95	12.94	14	12.32	13.32	12.14	13.65	15.04	12.2	13.02
Content	Pandoraea pulmonicola	Pandoraea soli	Pandoraea sp. XY-2	Pandoraea sputorum	Pandoraea terrae	Pandoraea thiooxydans	Pandoraea vervacti
Accession number	GCF_000815105.2	GCF_902459595.1	GCF_004193915.1	GCF_900187205.1	GCF_902459695.1	GCF_001017775.3	GCF_000934605.2
Genome
Scaffold Number	1	51	1	1	81	1	2
Total Length (Mb)	5.87	4.96	5.06	5.74	6.18	4.46	5.74
GC Content (%)	64.3	63.62	63.76	62.78	62.79	63.19	63.52
N50 Length	-	370,563	-	-	194,136	-	-
N90 Length	-	61,129	-	-	60,237	-	-
Longest scaffold	-	921,398	-	-	456,896	-	-
Gene
Gene Number	4,996	4,393	4,512	4,994	5,590	4,091	4,889
Gene Length (bp)	5,040,965	4,324,589	4,386,412	5,002,422	5,421,742	3,998,582	4,955,787
GC Content in Gene Region (%)	65	64.13	64.26	63.5	63.31	63.76	64.11
Gene Length/Genome (%)	85.91	87.15	86.75	87.1	87.78	89.57	86.39
Gene Average Length (bp)	1,009	984	972	1,002	970	977	1,014
Intergenic Region Length (bp)	826,656	637,393	669,794	740,701	755,081	465,604	780,495
GC Content in Intergenic Region (%)	59.97	60.12	60.47	57.89	58.98	58.3	59.82
Intergenic Region Length/Genome (%)	14.09	12.85	13.25	12.9	12.22	10.43	13.61

Comparative genomics and identification at the genome level

Comparative genomic analysis

A total of genes in Pandoraea sp. 892iso were classified through cluster analysis. The distribution of best hits within the genus Pandoraea is shown in Fig 2. In total, 3,849 orthologous genes were shared in common between Pandoraea sp. 892iso and the other four Pandoraea species. The cluster analysis of Pandoraea sp. 892iso, 28 Pandoraea species, and Burkholderia cepacia as an outgroup was carried out by orthoMCL to obtain the result of a common single-copy gene family. The phylogenetic relationship in view of these single-copy genes is shown in Fig 3 and S1 Table, which shows the closest phylogenetic relationship to be between Pandoraea sp. 892iso and the P. sputorum strain DSM21091. Meanwhile, eight specific gene families, including 21 genes, were clustered, 17 genes were hypothetical proteins, and the other four are shown in Table 2.

Fig 2

Venn diagram of genes common to Pandoraea sp. 892iso and the four other Pandoraea types.

Fig 3

Phylogenetic analyses of the evolutionary relationships between Pandoraea sp. 892iso and Pandoraea types.

A neighbor-joining phylogenetic tree constructed based on single-copy genes common to these nine bacterial genomes. The neighbor-joining method was used in MEGA6, where a bootstrap test (1,000 replicates) is shown next to the branches.

Table 2

Details of the four respective genes of Pandoraea sp. 892iso.

Gene	Position	direction	Detail
fig|93222.8.peg.1	C163_3_104	-	DNA-cytosine methyltransferase
fig|93222.8.peg.10	C237_1_126	-	DNA-cytosine methyltransferase
fig|93222.8.peg.14	C273_3_104	-	DNA-cytosine methyltransferase
fig|93222.8.peg.2519	scaffold3_1641978_1640653	-	DNA-cytosine methyltransferase
fig|93222.8.peg.4329	scaffold5_320715_322067	+	DNA-cytosine methyltransferase
fig|93222.8.peg.4363	scaffold5_361619_362311	+	Transcriptional regulator, GntR family
fig|93222.8.peg.4364	scaffold5_363002_362316	-	Transcriptional regulator, GntR family

The global genome clustering and alignment of Pandoraea types were complicated by Mummer. The results showed the best gene co-linearity among these Pandoraea types and that rearrangement was almost absent, except for P. thiooxydans and P. sputorum, which were phylogenetically closest to Pandoraea sp. 892iso (S1 Fig). It was speculated that the small external selection pressure of the Pandoraea group and the genome evolution occurred in a similar way. More attention should be given to Pandoraea sp. 892iso and its proximal P. sputorum, both of which rearranged compared to other Pandoraea types. Rearrangements existed in the five largest scaffold alignments, especially in scaffolds 3, 4, and 5, as shown in S2 Fig. A special unique insertion sequence in scaffold3_1802763_1803544 of Pandoraea sp. 892iso contains the gene fig|93222.8.peg.2650 with the function of ubiquitin in the NR database, which may be related to the function of covalent attachment to other cellular proteins associated with stability changing, localization, and activity of the target protein [35]. The ubiquitin gene in Pandoraea sp. 892iso was found to be different from that in human, mouse, zebrafish, rice, Arabidopsis, yeast, or other model organisms by phylogenetic analysis (Fig 4).

Fig 4

Phylogenetic analyses of evolutionary relationships of ubiquitin genes among Pandoraea sp. 892iso and Pandoraea types.

A neighbor-joining phylogenetic tree constructed based on single-copy genes common to these nine bacterial genomes. The neighbor-joining method was used in MEGA6, where a bootstrap test (1,000 replicates) is shown next to the branches.

ANI and AAI

ANI (average nucleotide identity), as the new method for bacterial species definition, provides several benefits, avoids misplacement based on phenotypic similarities or chemical characteristics, provides a scalable and uniform approach that works for both culturable and nonculturable species, is faster and cheaper than traditional taxonomic methods, and, most importantly, falls in line with Darwin’s vision of classification [30]. AAI (average amino acid identity), a method that compares all conserved protein-coding genes present in a given set of genomes, clusters types into groups that share more than 95% AAI [36]. ANI and AAI characteristics have been used to evaluate the accuracy of these genotypic methods in the identification of Pandoraea species. Given the availability of whole genome sequence data and Pandoraea sp. 892iso nucleotide and amino acid data as query, Blastn by CDS sequence coverage was ≥ 50% and tblastn by protein coverage was ≥ 70%. We performed sequence-based genotypic microbial identification analysis using the RefSeq database by genome comparison between Pandoraea sp. 892iso and Pandoraea sputorum and generated an ANI value of 98.81% and an AAI value of 91.18%; genome comparison with other in-house sequenced Pandoraea species provided an ANI value of less than 93.34% and an AAI value of 84.90% (Table 3). Based on previous results using the ANI value for species definition, ANI and AAI values of ≥ 95% corresponded to the traditional 70% DNA-DNA. Using the ANI and AAI values of Pandoraea sp. 892iso, it can be unequivocally stated that Pandoraea sp. 892iso is phylogenetically close to P. sputorum.

Table 3

Average nucleotide identity (ANI) and average amino acid identity (AAI) analyses.

Genome comparisons of Pandoraea sp. 892iso and other Pandoraea-type species.

Species	ID	ANI		AAI
		value	percent	value	percent
Pandoraea anapnoica	GCF_902459765.1	94.10	83.55	93.57	89.88
Pandoraea anhela	GCF_902459655.1	87.56	61.88	85.97	84.01
Pandoraea apista	GCF_001465595.2	86.43	54.74	84.98	83.38
Pandoraea aquatica	GCF_902459565.1	92.99	83.01	93.06	89.25
Pandoraea bronchicola	GCF_902459805.1	86.63	55.10	84.51	81.14
Pandoraea capi	GCF_902459735.1	87.77	67.77	88.02	87.59
Pandoraea captiosa	GCF_902459775.1	87.24	61.15	86.49	85.41
Pandoraea cepalis	GCF_902459625.1	86.48	48.39	82.57	77.98
Pandoraea commovens	GCF_902459615.1	94.51	85.62	94.43	90.55
Pandoraea communis	GCF_902459745.1	86.60	53.85	83.93	82.50
Pandoraea eparura	GCF_902459725.1	86.58	48.85	81.94	77.51
Pandoraea faecigallinarum	GCF_001029105.3	87.43	60.07	85.94	83.10
Pandoraea fibrosis	GCF_000807775.2	86.50	57.54	85.87	83.29
Pandoraea horticolens	GCF_902459555.1	86.53	53.29	83.58	82.45
Pandoraea iniqua	GCF_902459685.1	85.58	54.26	83.99	86.77
Pandoraea morbifera	GCF_902459575.1	86.21	51.70	83.57	82.34
Pandoraea norimbergensis	GCF_001465545.3	85.45	53.68	83.84	86.68
Pandoraea nosoerga	GCF_902459585.1	86.24	50.42	82.72	78.67
Pandoraea oxalativorans	GCF_000972785.3	93.57	77.73	90.99	86.06
Pandoraea pneumonica	GCF_902459645.1	85.60	52.62	83.58	85.52
Pandoraea pnomenusa	GCF_000504585.2	86.27	53.21	83.90	82.50
Pandoraea pulmonicola	GCF_000815105.2	86.28	53.72	83.78	82.39
Pandoraea soli	GCF_902459595.1	86.52	48.07	82.37	77.75
Pandoraea sp. XY-2	GCF_004193915.1	86.49	48.39	80.31	74.75
Pandoraea sputorum	GCF_900187205.1	99.29	88.49	97.03	90.91
Pandoraea terrae	GCF_902459695.1	82.67	25.17	72.62	73.04
Pandoraea terrigena	GCF_902459705.1	86.36	48.80	82.03	79.21
Pandoraea thiooxydans	GCF_001017775.3	80.09	10.79	67.18	62.49
Pandoraea vervacti	GCF_000934605.2	87.33	60.28	86.32	83.73

rpoB similarity and MLSA phylogenetic analysis

The rpoB gene, encoding the β-subunit of RNA polymerase, has emerged as a core gene candidate for phylogenetic analyses and identification of bacteria; it is a single-copy gene, belongs to the common set of genes, and is long enough to contain phylogenetically useful information for some bacterial declination [37-40]. Multilocus sequence analysis (MLSA) is a currently widely used method for prokaryotic taxonomy, which utilizes internal fragments of several protein-coding genes. It was introduced by Gevers et al. and is increasingly being applied to obtain higher resolution power among species within a genus [39, 41]. As a typing technique for type characterization that shows variation in multiple housekeeping genes, a concatenation of five housekeeping genes, shikimate dehydrogenase (aroE), guanylate kinase (gmk), phosphate acetyltransferase (pta), triosephosphate isomerase (tpi), and acetyl coenzyme A acetyltransferase (yqiL), was recommended for our bacterial delineation, as well as for clarifying the taxonomic situation within the Pandoraea family [39, 41]. The phylogenetic tree topologies of Pandoraea sp. 892iso and other Pandoraea spp. by rpoB similarity (Fig 5A) and MLSA analysis (Fig 5B) revealed Pandoraea sp. 892iso to have the closest phylogenetic relationship with Pandoraea sputorum strain DSM21091.

Fig 5

Phylogenetic tree highlighting the position of Pandoraea sp. 892iso relative to the other Pandoraea species.

The tree was aligned with the characteristics of the rpoB gene (a) and MLSA (b) under the maximum likelihood (ML) criterion.

Genome-to-genome distance calculator

In silico genome-to-genome comparison to obtain an estimate of the overall similarity between the genomes of two types has enabled the taxonomist to perform genome-based species delineation and genome-based subspecies delineation. These distance functions can also cope with heavily reduced genomes and repetitive sequence regions. The Genome-to-Genome Distance Calculator (GGDC) calculates the distances by comparing genomes to obtain HSPs (high-scoring segment pairs) and interfering distances from a set of formulas: 1) HSP length/total length; 2) identities/HSP length; and 3) identities/total length [42]. An estimated GGDC of the overall similarity between Pandoraea sp. 892iso and other Pandoraea species is shown in Table 4. In probability DDG ≥70% index analysis, the pairwise comparison of the genome with P. sputorum was found to be 98.49%, 96.97%, and 99.88% for the HSP length/total length, identities/HSP length, and identities/total length ratios, respectively. Thus, the close relationship of Pandoraea sp. 892iso and P. sputorum was verified.

Table 4

Pairwise comparison of Pandorarae sp. 892iso and Pandoraea species using the GGDC.

Query	Reference	ID	HSP length/total length				identities/HSP length				identities/total length				G+C difference
			Distance	DDH estimate (GLM-based)	Prob. DDH>70%	Prob. DDH>79%	Distance	DDH estimate (GLM-based)	Prob. DDH>70%	Prob. DDH>79%	Distance	DDH estimate (GLM-based)	Prob. DDH>70%	Prob. DDH>79%
Pandoraea sp. 892iso	Pandoraea anapnoica	GCF_902459765.1	78.3	[74.3–81.8%]	0.1411	89.13	52.1	[49.5–54.8%]	0.0669	25.38	74.9	[71.4–78.1%]	0.1985	88.31	0.26
Pandoraea sp. 892iso	Pandoraea anhela	GCF_902459655.1	48.1	[44.7–51.5%]	0.3215	8.92	29	[26.6–31.5%]	0.1476	0.07	42.4	[39.4–45.4%]	0.4217	0.31	0.69
Pandoraea sp. 892iso	Pandoraea apista	GCF_001465595.2	48	[44.6–51.4%]	0.322	8.82	26.6	[24.3–29.1%]	0.1626	0.02	41.3	[38.3–44.3%]	0.4323	0.21	0.03
Pandoraea sp. 892iso	Pandoraea aquatica	GCF_902459565.1	80.7	[76.8–84.1%]	0.1286	91.75	46.5	[43.9–49.1%]	0.0803	10.85	74.9	[71.4–78.1%]	0.1985	88.3	0.23
Pandoraea sp. 892iso	Pandoraea bronchicola	GCF_902459805.1	48.6	[45.2–52%]	0.3173	9.79	27	[24.6–29.5%]	0.1603	0.03	41.8	[38.9–44.9%]	0.4268	0.26	0.31
Pandoraea sp. 892iso	Pandoraea capi	GCF_902459735.1	61	[57.3–64.6%]	0.2337	45.76	29.3	[27–31.8%]	0.1458	0.08	51.6	[48.5–54.7%]	0.3455	4.22	0.78
Pandoraea sp. 892iso	Pandoraea captiosa	GCF_902459775.1	48.7	[45.3–52.2%]	0.3164	9.99	28.4	[26–30.9%]	0.1515	0.05	42.6	[39.6–45.6%]	0.42	0.32	0.64
Pandoraea sp. 892iso	Pandoraea cepalis	GCF_902459625.1	38.3	[34.9–41.8%]	0.4098	1.11	26.9	[24.6–29.4%]	0.1605	0.03	34.5	[31.6–37.6%]	0.5045	0.02	0.88
Pandoraea sp. 892iso	Pandoraea commovens	GCF_902459615.1	85.3	[81.6–88.4%]	0.1056	95.14	54.2	[51.5–56.9%]	0.0625	32.2	81.4	[78–84.3%]	0.1615	96.5	0.03
Pandoraea sp. 892iso	Pandoraea communis	GCF_902459745.1	43.9	[40.5–47.4%]	0.3559	4.04	26.8	[24.4–29.3%]	0.1614	0.02	38.5	[35.6–41.6%]	0.4599	0.08	0.09
Pandoraea sp. 892iso	Pandoraea eparura	GCF_902459725.1	36.8	[33.4–40.3%]	0.426	0.75	27.5	[25.1–30%]	0.1571	0.03	33.6	[30.6–36.7%]	0.5161	0.01	1.02
Pandoraea sp. 892iso	Pandoraea faecigallinarum	GCF_001029105.3	49.8	[46.4–53.2%]	0.3082	11.94	28.8	[26.4–31.3%]	0.1492	0.06	43.5	[40.5–46.5%]	0.4115	0.44	0.79
Pandoraea sp. 892iso	Pandoraea fibrosis	GCF_000807775.2	52.1	[48.6–55.5%]	0.2918	16.86	26.7	[24.4–29.2%]	0.1618	0.02	44.1	[41.1–47.1%]	0.4064	0.52	0.16
Pandoraea sp. 892iso	Pandoraea horticolens	GCF_902459555.1	41.6	[38.3–45.1%]	0.3767	2.47	26.8	[24.5–29.3%]	0.1612	0.02	36.9	[33.9–40%]	0.4771	0.04	0.35
Pandoraea sp. 892iso	Pandoraea iniqua	GCF_902459685.1	39.5	[36.2–43%]	0.397	1.51	25.6	[23.3–28.1%]	0.1697	0.01	35	[32–38%]	0.4994	0.02	0.41
Pandoraea sp. 892iso	Pandoraea morbifera	GCF_902459575.1	43.2	[39.9–46.7%]	0.3621	3.49	26.5	[24.1–29%]	0.1636	0.02	37.9	[34.9–40.9%]	0.4665	0.06	2
Pandoraea sp. 892iso	Pandoraea norimbergensis	GCF_001465545.3	39.3	[36–42.8%]	0.3992	1.44	25.5	[23.2–28%]	0.1702	0.01	34.8	[31.8–37.9%]	0.5015	0.02	0.4
Pandoraea sp. 892iso	Pandoraea nosoerga	GCF_902459585.1	40.8	[37.4–44.2%]	0.3849	2.03	26.8	[24.4–29.3%]	0.1615	0.02	36.3	[33.3–39.3%]	0.4842	0.03	3.47
Pandoraea sp. 892iso	Pandoraea oxalativorans	GCF_000972785.3	62.6	[58.9–66.2%]	0.2242	51.57	49.4	[46.8–52%]	0.073	17.48	61	[57.7–64.2%]	0.2809	29.7	0.43
Pandoraea sp. 892iso	Pandoraea pneumonica	GCF_902459645.1	39.9	[36.6–43.4%]	0.393	1.67	25.3	[23–27.8%]	0.1718	0.01	35.1	[32.2–38.2%]	0.4973	0.02	0.21
Pandoraea sp. 892iso	Pandoraea pnomenusa	GCF_000504585.2	43.6	[40.2–47%]	0.3588	3.78	26.6	[24.3–29.1%]	0.1626	0.02	38.2	[35.3–41.3%]	0.463	0.07	2.23
Pandoraea sp. 892iso	Pandoraea pulmonicola	GCF_000815105.2	41.3	[37.9–44.8%]	0.3797	2.29	26.6	[24.2–29.1%]	0.163	0.02	36.6	[33.6–39.6%]	0.4809	0.04	1.64
Pandoraea sp. 892iso	Pandoraea soli	GCF_902459595.1	39.4	[36–42.9%]	0.3985	1.46	27	[24.7–29.5%]	0.16	0.03	35.4	[32.4–38.4%]	0.4948	0.02	0.96
Pandoraea sp. 892iso	Pandoraea sp. XY-2	GCF_004193915.1	39.7	[36.3–43.1%]	0.3957	1.56	27	[24.6–29.5%]	0.1603	0.03	35.5	[32.6–38.6%]	0.4926	0.03	1.1
Pandoraea sp. 892iso	Pandoraea sputorum	GCF_900187205.1	94.2	[91.7–96%]	0.0565	98.49	94	[92.2–95.4%]	0.0077	96.97	96.1	[94.4–97.3%]	0.0638	99.88	0.12
Pandoraea sp. 892iso	Pandoraea terrae	GCF_902459695.1	18.2	[15.1–21.7%]	0.7652	0	22.6	[20.3–25%]	0.194	0	17.9	[15.3–20.9%]	0.8108	0	0.13
Pandoraea sp. 892iso	Pandoraea terrigena	GCF_902459705.1	39	[35.6–42.4%]	0.403	1.31	26.7	[24.4–29.2%]	0.1621	0.02	34.9	[32–38%]	0.4998	0.02	0.82
Pandoraea sp. 892iso	Pandoraea thiooxydans	GCF_001017775.3	14.2	[11.4–17.6%]	0.9145	0	20.2	[18–22.6%]	0.2177	0	14.4	[12–17.2%]	0.9331	0	0.54
Pandoraea sp. 892iso	Pandoraea vervacti	GCF_000934605.2	50.9	[47.5–54.4%]	0.2999	14.26	28.4	[26–30.9%]	0.1512	0.05	44.1	[41.1–47.2%]	0.4058	0.53	0.87

Some special genes among Pandoraea sp

Quorum sensing (QS)

The most studied QS molecule is N-acyl homoserine lactone (AHL), which is secreted by gram-negative proteobacteria. AHLs are secreted by LuxI homologs until a threshold concentration of AHL is attained before they bind to LuxR homologs and subsequently activate a cascade of QS-regulated gene expression [43]. The predicted putative AHL synthase (ppnI) and AHL receptor protein (ppnR) in Pandoraea sp. 892iso and the nine Pandoraea species are shown in Table 5. The phylogenetic trees of putative AHL synthase (ppnI) and AHL receptor protein (ppnR) are shown in Fig 6.

Table 5

The identified ppnI and ppnR genes in Pandoraea sp. 892iso and nine Pandoraea species.

Species	Accession number	scaffold	gene	start	end	strand
Pandoraea sp. 892iso	fig|93222.8.peg.1246	scaffold3	ppnI	215501	216286	+
Pandoraea sp. 892iso	fig|93222.8.peg.1247	scaffold3	ppnR	216253	216966	-
Pandoraea oxalativorans	WP_046292715.1	NZ_CP011253.3	ppnI	4024825	4025493	-
Pandoraea oxalativorans	WP_046293945.1	NZ_CP011253.3	ppnR	4024031	4024732	+
Pandoraea anapnoica	WP_150739377.1	NZ_CABPSP010000011.1	ppnI	57228	57914	-
Pandoraea anapnoica	WP_150739515.1	NZ_CABPSP010000011.1	ppnR	56433	57134	+
Pandoraea pneumonica	WP_150681584.1	NZ_CABPSK010000004.1	ppnI	583193	583867	-
Pandoraea pneumonica	WP_174988328.1	NZ_CABPSK010000004.1	ppnR	582433	583146	+
Pandoraea morbifera	WP_150566717.1	NZ_CABPSD010000005.1	ppnI	208906	209694	-
Pandoraea morbifera	WP_150566716.1	NZ_CABPSD010000005.1	ppnR	208206	208919	+
Pandoraea sputorum	WP_174555901.1	NZ_LT906435.1	ppnI	1348270	1349055	+
Pandoraea sputorum	WP_039402529.1	NZ_LT906435.1	ppnR	1349022	1349723	-
Pandoraea terrae	WP_150700195.1	NZ_CABPRZ010000043.1	ppnI	19106	19732	-
Pandoraea terrae	WP_150700194.1	NZ_CABPRZ010000043.1	ppnR	18360	19076	+
Pandoraea vervacti	WP_044456583.1	NZ_CP010897.2	ppnI	4037152	4037835	-
Pandoraea vervacti	WP_044458339.1	NZ_CP010897.2	ppnR	4036372	4037073	+
Pandoraea captiosa	WP_150627103.1	NZ_CABPSQ010000011.1	ppnI	88267	88950	-
Pandoraea captiosa	WP_150627162.1	NZ_CABPSQ010000011.1	ppnR	87492	88193	+
Pandoraea pnomenusa	WP_023871914.1	NC_023018.2	ppnI	3778787	3779572	-
Pandoraea pnomenusa	WP_080685145.1	NC_023018.2	ppnR	3778087	3778800	+
Pandoraea commovens	WP_174985011.1	NZ_CABPSA010000008.1	ppnI	204518	205333	+
Pandoraea commovens	WP_150666021.1	NZ_CABPSA010000008.1	ppnR	205300	206013	-
Burkholderia cepacia	WP_042976961.1	NZ_CP045236.1	ppnI	471746	472354	-
Burkholderia cepacia	WP_021162347.1	NZ_CP045236.1	ppnR	473082	473801	+
Pandoraea faecigallinarum	WP_167362711.1	NZ_CP011807.3	ppnI	3690884	3691549	-
Pandoraea faecigallinarum	WP_053059408.1	NZ_CP011807.3	ppnR	3690044	3690820	+
Pandoraea capi	WP_150721274.1	NZ_CABPRV010000004.1	ppnI	224554	225237	-
Pandoraea capi	WP_150721396.1	NZ_CABPRV010000004.1	ppnR	223772	224473	+
Pandoraea norimbergensis	WP_157125706.1	NZ_CP013480.3	ppnI	1418662	1419441	+
Pandoraea norimbergensis	WP_064675185.1	NZ_CP013480.3	ppnR	1419408	1420109	-

Fig 6

Phylogenetic tree of ppnI and ppnR.

Intrinsic carbapenem-hydrolyzing oxacillinases

Oxacillinases are serine β-lactamases of molecular class D. Many bacterial species could produce OXA-type enzymes, some of them with carbapenem-hydrolyzing activity. The nine Pandoraea-derived oxacillinase genes, named OXA-159, encode 292 amino acids and were found to be new oxacillinase variants [44]. The predicted genes with the function of OXA-159 in Pandoraea sp. 892iso and the nine Pandoraea species are shown in Table 6. The phylogenetic trees of genes with the putative function of OXA-159 are shown in Fig 7.

Table 6

The identified genes with the function of OXA-159 in Pandoraea sp. 892iso and nine Pandoraea species.

Species	Accession number
Pandoraea sp. 892iso	fig|93222.8.peg.176
Pandoraea oxalativorans	WP_052653498.1
Pandoraea nosoerga	WP_150556387.1
Pandoraea morbifera	WP_150567617.1
Pandoraea sputorum	WP_063861062.1
Pandoraea communis	WP_150690981.1
Pandoraea fibrosis	WP_052240481.1
Pandoraea pnomenusa	WP_023872076.1
Burkholderia cepacia	WP_153490194.1
Pandoraea faecigallinarum	WP_053059421.1
Pandoraea capi	WP_150719552.1
Pandoraea norimbergensis	WP_058375744.1
Pandoraea anapnoica	WP_150740206.1
Pandoraea bronchicola	WP_150559740.1
Pandoraea iniqua	WP_150791439.1
Pandoraea apista	WP_048627819.1
Pandoraea pneumonica	WP_150680540.1
Pandoraea cepalis	WP_150607462.1
Pandoraea sp. XY-2	WP_130026801.1
Pandoraea pulmonicola	WP_052266736.1
Pandoraea soli	WP_150552526.1
Pandoraea vervacti	WP_063389849.1
Pandoraea aquatica	WP_150576315.1
Pandoraea captiosa	WP_150626879.1
Pandoraea commovens	WP_150664304.1
Pandoraea horticolens	WP_150619975.1
Pandoraea anhela	WP_150669648.1

Fig 7

Phylogenetic tree of OXA-159 genes.

The neighbor-joining method was used in MEGA7, where a bootstrap test (1,000 replicates) is shown next to the branch.

Conclusions

We sequenced Pandoraea sp. 892iso from the genome of a Phytophthora rubi strain (numbered 109892) and combined the data with existing genomic data for other Pandoraea species. Next, we conducted a comparative genomic analysis of the genome structure, evolutionary relationships, and pathogenic characteristics of Pandoraea species. Our results identified Pandoraea sp. 892iso as Pandoraea sputorum at both the genome and gene levels. At the genome level, we carried out phylogenetic analysis of single-copy, gene co-linearity, ANI and AAI indices, rpoB similarity, MLSA phylogenetic analysis, and genome-to-genome distance calculator calculations to identify the relationship between Pandoraea sp. 892iso and P. sputorum. At the gene level, the quorum sensing genes ppnI and ppnR and the OXA-159 gene were analyzed. It is speculated that Pandoraea sp. 892iso is the endosymbiont of the Phytophthora rubi strain.

Diagram of linear genomic organization among Pandoraea types.

(DOC)

Click here for additional data file.

Diagram of linear genomic organization between Pandoraea sp. 892iso and Pandoraea sputorum.

Scaffold1, scaffold2, scaffold3, scaffold4, and scaffold5 were the five largest sequences.

(DOC)

Click here for additional data file.

The list of single copy gene in the genome of Pandoraea sp. 892iso.

(XLSX)

Click here for additional data file.

13 Dec 2021

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Reviewer #1: PONE-D-21-31328: Genome insights from the identification of a Pandoraea sputorum strain

The authors describe the relationship between a strain of Pandoraea derived from Phytophthora rubi with some previously described Pandoraea species at the genomic level and conclude that it is likely to belong to the species P. sputorum. They also compare quorum sensing and oxacillinase genes from their strain with those from other species of this genus.

Major comments: While the authors have been quite systematic in their comparison of strain Pandoraea sp. 892iso with other species at the genomic level, they have not included a large number of species which have been added to this genus in the last few years. In addition, while the analysis is thorough, a considerable portion of their manuscript focuses on the description of where this strain sits in the taxonomy of this genus and this seems to be out of proportion with respect to the final conclusion i.e. that this strain belongs to P. sputorum. It would be better if the authors focused less on the where their strain fits in this genus and more on the interesting aspects concerning their quorum sensing and oxacillinase gene comparisons and their MLSA typing approach.

Minor comments:

Please avoid using the word “strain”, unless required, or else preface it with “type” or “reference” where appropriate (for example in the legend for figure 3).

Title:

This should be more informative.

Abstract:

Line 13-would be useful to explain what Phytophthora rubi is.

Line 15: Would be useful to clarify which Pandoraea species you are referring to.

Line 21: Please make it clear which Oomycetes strain you are referring to.

Introduction:

Line 27: Use “genus” rather than “species”

Line 30: Please re-structure this sentence as it is unclear.

Line 33: Many additional species have been added to this genus in the last few years. Please see Peeters et al., (2019) Comparative Genomics of Pandoraea, a Genus Enriched in Xenobiotic Biodegradation and Metabolism.Front. Microbiol. 10:2556 and the following for additional species: Anandham et al., 2010; Sahin et al., 2011; Jeong et al., 2016). Please update your manuscript with this information.

Line 40: “Further”

Line 67: “were identified through whole genome sequencing”

Lines 68 and 75: Please use “isolate” rather than strain

Materials and Methods:

Line 90: Do you mean “calculations” rather than “calculate”?

Line 95 and 100: please clarify what you mean by “11 species”.

Results:

Line 114: Which Pandoraea sputorum isolate are you referring to? If it is Pandoraea sp. 892iso It may be better to use the name of the strain at this stage as the evidence for the WGS-based identification has not yet been presented.

Line 134: It would be helpful if the authors included some details about the types of single copy genes chosen for their generating their phylogenetic tree, with the genes listed in a table, or in supplementary information.

Lines 183-188: The decision to choose these particular house-keeping genes should be explained.

Line 207: A much shorter title to this section should be chosen.

Figures and Tables:

Figure 2: The legend for this figure needs further information about the methods used to generate this data.

Table 1: Please clarify the accession numbers and details for the P. pulmonicola, P. sputorum genomes used, and also for the other species for which this information had not been provided.

Table 2: This table needs some description in the legend, and further details in the main text to explain what it depicts.

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Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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19 Jan 2022

Dear reviewer and editor,

Thank you very much for your help on our manuscript entitled ‘Genome insights from the identification of a novel Pandoraea sputorum isolate and its characteristics’.

According to your suggestion, we have revised one by one, and now we resubmit again.

We hope our revisions will meet with approval.

Best wishes,

Sincerely yours,

Ruifang Gao

Submitted filename: renamed_7c11c.docx

Click here for additional data file.

6 Apr 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Paula V Morais, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments:

1- The number of species of Pandorea is not correct. See https://lpsn.dsmz.de/genus/pandoraea

2- To consider a strain a new species we have 3 criteria and not 2. 16S rRNA gene sequence has to be considered (introduction).

3- What made you suspect that the fungus culture was contaminated? Give some detail (material and methods).

4- You still have the number of Pandorea wrong "...KP771987.1). BLAST was employed to identify homolog genes in the 11 Pandoraea species." Did you repeat the comparison with the 28 Pandorea strains?

5- Please, clarify "...per 1 Mb, which is more/fewer than most of the other sequenced bacteria...."

6- Table 1. What was the criteria to include 30 strains of Pandorea species? What was the criteria for the order they are included? I would like to see them by alphabetic order.

7- "In total, 3,456 849

orthologous genes were shared in common between Pandoraea sp. 892iso and the other four Pandoraea straintypes." Which ones?

8- "fig|93222.8.peg.2650 with the function of ubiquitin in the NR database." Do you mean predicted function?

9- Table 3 and Table 4. List the species by alphabetic order unless you could present a relevant criterium for not doing it.

10- "...AHL receptor protein (ppnR) in Pandoraea sp. 892iso and the nine Pandoraea species are shown..." Why 9 Pandorea species?

11- Table 6. I do not understand. How many genes? How namy species type strains?

12- "...and combined the data with existing genomic data for five other Pandoraea" Is it correct?

13. "It is speculated that Pandoraea sp. 892iso is the endosymbiont of the Phytophthora rubi strain." I do not see in your manuscript any result that supports this sentence. Why the isolate is not a contaminant of your culture? This comment is related with the 3 comment. If you want to include this senetence you have to explain in detail the fungus isolation and culture and the bacteria isolation story.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: There should be unification for the term that used after Pandoraea. Three different terms (types, sp., and previously strains) were employed throughout the manuscript. The term “species” is usually used in the literature.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

12 May 2022

Dear editor,

Thank you very much for your help on our manuscript entitled ‘Genome insights from the identification of a novel Pandoraea sputorum isolate and its characteristics’.

A list of the single copy genes in the genome of Pandoraea sp. 892iso had been shown in Supporting Information files, which was noted in the section Comparative genomic analysis in the manuscript.

"The phylogenetic relationship in view of these single-copy genes is shown in Fig 3 and S1 Table, ....."

The separate caption of S1 Table also has been added at the end of manuscript.

We hope our revisions will meet with approval.

Best wishes,

Sincerely yours,

Ruifang Gao

Submitted filename: renamed_7c11c.docx

Click here for additional data file.

3 Jun 2022

Genome insights from the identification of a novel Pandoraea sputorum isolate and its characteristics

PLOS ONE

Dear Dr. Gao,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Figures 4 and 5 need to be improved so they can be published as supplementary figures. The manuscript has to be revised to be adapted and the figures numbered according.

Please submit your revised manuscript by Jul 18 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

4 Jun 2022

Dear editor,

Thank you very much for your help on our manuscript entitled ‘Genome insights from the identification of a novel Pandoraea sputorum isolate and its characteristics’.

According to your suggestion, we have revised and now we resubmit again.

We hope our revisions will meet with approval.

Best wishes,

Sincerely yours,

Ruifang Gao

Submitted filename: 4th-Response to editor.doc

Click here for additional data file.

20 Jul 2022

Genome insights from the identification of a novel Pandoraea sputorum isolate and its characteristics

PONE-D-21-31328R3

Dear Dr. Gao,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Paula V Morais, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

27 Jul 2022

PONE-D-21-31328R3

Genome insights from the identification of a novel Pandoraea sputorum isolate and its characteristics

Dear Dr. Gao:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Paula V Morais

Academic Editor

PLOS ONE