De novo genome assembly of Bacillus altitudinis 19RS3 and Bacillus altitudinis T5S-T4, two plant growth-promoting bacteria isolated from Ilex paraguariensis St. Hil. (yerba mate)

Iliana Julieta Cortese; María Lorena Castrillo; Andrea Liliana Onetto; Gustavo Ángel Bich; Pedro Darío Zapata; Margarita Ester Laczeski

doi:10.1371/journal.pone.0248274

. 2021 Mar 11;16(3):e0248274. doi: 10.1371/journal.pone.0248274

De novo genome assembly of Bacillus altitudinis 19RS3 and Bacillus altitudinis T5S-T4, two plant growth-promoting bacteria isolated from Ilex paraguariensis St. Hil. (yerba mate)

Iliana Julieta Cortese ^1,^*, María Lorena Castrillo ¹, Andrea Liliana Onetto ¹, Gustavo Ángel Bich ¹, Pedro Darío Zapata ¹, Margarita Ester Laczeski ^1,^2,^*

Editor: Ying Ma³

PMCID: PMC7954119 PMID: 33705487

Abstract

Plant growth-promoting bacteria (PGPB) are a heterogeneous group of bacteria that can exert beneficial effects on plant growth directly or indirectly by different mechanisms. PGPB-based inoculant formulation has been used to replace chemical fertilizers and pesticides. In our previous studies, two endophytic endospore-forming bacteria identified as Bacillus altitudinis were isolated from roots of Ilex paraguariensis St. Hil. seedlings and selected for their plant growth-promoting (PGP) properties shown in vitro and in vivo. The purposes of this work were to assemble the genomes of B. altitudinis 19RS3 and T5S-T4, using different assemblers available for Windows and Linux and to select the best assembly for each strain. Both genomes were also automatically annotated to detect PGP genes and compare sequences with other genomes reported. Library construction and draft genome sequencing were performed by Macrogen services. Raw reads were filtered using the Trimmomatic tool. Genomes were assembled using SPAdes, ABySS, Velvet, and SOAPdenovo2 assemblers for Linux, and Geneious and CLC Genomics Workbench assemblers for Windows. Assembly evaluation was done by the QUAST tool. The parameters evaluated were the number of contigs ≥ 500 bp and ≥ 1000 bp, the length of the longest contig, and the N50 value. For genome annotation PROKKA, RAST, and KAAS tools were used. The best assembly for both genomes was obtained using Velvet. The B. altitudinis 19RS3 genome was assembled into 15 contigs with an N50 value of 1,943,801 bp. The B. altitudinis T5S-T4 genome was assembled into 24 contigs with an N50 of 344,151 bp. Both genomes comprise several genes related to PGP mechanisms, such as those for nitrogen fixation, iron metabolism, phosphate metabolism, and auxin biosynthesis. The results obtained offer the basis for a better understanding of B. altitudinis 19RS3 and T5S-T4 and make them promissory for bioinoculant development.

Introduction

Biological products that enhance plant growth are an alternative for improving crop management and degraded soils. The application of native microorganisms reduces the degradation of the agroecosystem and the loss of nutrients, optimizing the yield of crops. Plant growth-promoting bacteria (PGPB) are a heterogeneous group in the rhizosphere, at root surfaces or in association with plant tissues as endophytes. These bacteria exert beneficial effects on plant growth directly, or indirectly. The mechanisms by which PGPB can influence plant growth may differ from one species to another as well as from strain to strain [1]. PGPB-based inoculant formulation and application have been used in integrated management systems to reduce or replace the use of chemical fertilizers and pesticides [2].

Bacteria genera such as Alcaligenes, Acinetobacter, Arthrobacter, Azoarcus, Azospirillum, Azotobacter, Bacillus, Paenibacillus, Burkholderia, Clostridium, Enterobacter, Gluconacetobacter, Klebsiella, Kosakonia, Pseudomonas, Serratia, and Stenotrophomonas include specific strains that have been reported as PGPB for different plant species [3–9]. Their mechanisms are multiple, diverse, and their effects include the transformation of nutrients into forms available to plants, for example, the capacity to fix atmospheric nitrogen [10], the solubilization of inorganic phosphorus, or the mineralization of organic phosphorus [11, 12]. Some PGPB can also promote plant growth indirectly by controlling the associated pathogens by producing antibiotics and other secondary metabolites [4], or by activating the mechanisms of Induced Systemic Resistance (ISR) [13].

Keeping in mind this context, the genus Bacillus presents a great diversity of species that are distributed widely in the environment. It is one of the most studied and promising genera for achieving sustainable and environmentally safe agricultural practices [14].

They have been shown to enhance plant growth through a combination of mechanisms [15, 16], by activating ISR in the plant against both root and foliar pathogens, by increasing abiotic stress tolerance [17], and by showing biocontrol properties [18]. Elicitation of ISR by Bacillus and its metabolites has been demonstrated on a variety of crops to defend against pathogen attack in both the greenhouse and the field [19, 20]. It can also produce numerous antifungal compounds [21], such as lipopeptides [22], bacillomycin [23], fengycin [24], surfactin [25], bacillibactin [26], and bacteriocin [27].

The PGPB activity of some bacilli strains was studied in the last years. B. subtilis is commercially used as a biofertilizer [28]. It can maintain stable contact with higher plants and promote their growth. B. licheniformis shows beneficial effects when inoculated on tomato and pepper [4]. B. megaterium improves different root parameters in mint [29], while B. mucilaginosus, when inoculated in nutrient-limited soil, can increase mineral availability [30, 31]. B. pumilus is used as a bioinoculant to increase the crop yield of a wheat variety in Mongolia [32]. Genome analysis revealed that B. velezensis can be considered a potential biofertilizer and biopesticide [33]. Likewise, Bacillus spp. consortia present the capability to increase the yield, growth, and nutrition of raspberry [34] and banana plants [35].

Different crops have great economic agro-food importance in the world, and it is of interest to improve their production through the implementation of PGPB as a biofertilizer [36, 37]. Ilex paraguariensis St. Hil., a plant that is also commonly called yerba mate, is one of the most economically important crops in the northeast of Argentina. It is widely marketed in South America, but it is also consumed worldwide. It is emphasized that despite this overall consumption, yerba mate can only grow in certain regions of Argentina, Paraguay, and Brazil due to unique soil characteristics, such as lateritic soils, and warm and moist weather [38, 39].

Currently, there are plantations of good performance in the region, however, there are concerns about the increase of degraded crop sites, as a result of the monoculture system, erosion and compaction of soil, and nutrient loss combined with little or no soil fertilization [40]. This motivates the research and development of a biofertilizer from native bacteria isolated from yerba mate to recover crop performance.

In our previous study, two Gram-positive endophytic endospore-forming bacteria, coded as 19RS3 and T5S-T4, were isolated from roots of I. paraguariensis St. Hil. seedlings. These bacteria were selected for their in vitro PGP properties. Both strains were identified morphologically and molecularly as Bacillus altitudinis. Also, B. altitudinis 19RS3 and B. altitudinis T5S-T4 showed in vivo growth promotion in yerba mate seedlings in greenhouse conditions with promising results [41].

The study of the genome of microorganisms used for biofertilizer production is important to bioinoculant technology because it helps to identify genes that contribute to the beneficial activity and increasing knowledge of the molecular mechanisms related to plant growth potential. In the last decade, the development of new bioinformatics tools and next-generation sequencing technologies has allowed researchers to gain deeper insights into the molecular and genetic mechanisms of plant growth-promoting (PGP) activities such as the study of Pho regulon involved in the inorganic phosphate (Pi) solubilization, the detection of nif gene cluster associated to nitrogen fixation, the study of metabolic pathways related to the siderophore production, and the discovery of antibiotics and volatile compounds production mechanisms implicated in biocontrol properties. These advances were accompanied by an exponential increase in the number of assembler algorithms available to obtain complete prokaryotic genomes [42]. Principio del formulario Final del formulario Currently, there are two widely used classes of algorithms: overlap–layout–consensus (OLC) and de-Bruijn-graph (DBG) [43]. The DBG algorithm is based on the k-mers approach [44]. This value divides the short sequences into smaller fragments of size k, and these k-mers overlap with k-1, which represents the next k-mer. Since Illumina sequencing technology entered the global market, several short-read assembly software based on DBG have been developed, such as Velvet [45], ABySS [46], SPAdes [47], and SOAPdenovo2 [48]. Despite this, the selection of assembly tools, the determination of the parameters to be executed, as well as the evaluation of the assemblies, are still a challenge [49].

In this context, to advance knowledge of PGP mechanisms, the genomes of B. altitudinis 19RS3 and B. altitudinis T5S-T4 were sequenced. The purposes of this work were to assemble both genomes, to compare the results obtained using different de novo assemblers available for Windows and Linux operating systems, and to select the best assembly for each B. altitudinis strain. Finally, both genomes were automatically annotated to detect genes involved in PGP capabilities and compare these sequences with other Bacillus sp. genomes reported.

Materials and methods

Bacteria

B. altitudinis 19RS3 and B. altitudinis T5S-T4 were isolated from roots of I. paraguariensis St. Hil. seedlings [41]. Both strains were identified by analysis of 16S rRNA gene sequencing (accession number MH883312 and MH883235, respectively) and characterized as Gram-positive endospore-forming rod-shaped bacteria. B. altitudinis 19RS3 and B. altitudinis T5S-T4 were deposited into the bacterial collection of the Instituto de Biotecnología Misiones “Dra. María Ebe Reca”, under accession numbers LBM250 and LBM251, respectively. Bacteria were preserved in 50% glycerol stocks at -80°C until the performance of this study.

DNA extraction

The strains were cultivated in nutrient broth (Britania Lab. SA) for 24 h at 30°C. The DNA extraction procedures were done using Sambrook´s work protocol modified [50, 51]. The DNA was resuspended by 20 μL of sterile distilled DNAse-free water (BioPack ®). The extracted DNA was qualitatively evaluated by agarose gel (1% w/v) electrophoresis stained with a solution of GelRed® (Sigma-Aldrich, Germany). The DNA quantification was performed by UV spectrophotometry.

Library preparation and genome sequencing

Genomic TruSeq Nano DNA library (350) construction and draft genome paired-end sequencing were performed by Macrogen Co. (Seoul, Korea) services using Illumina HiSeq technology.

Genome assembly and evaluation

The quality of the FASTQ files was verified with FastQC [52] and reads were trimmed to ensure high quality (Phred score > 30) using Trimmomatic version 0.39 [53].

The genomes were assembled using different de novo assemblers available for Linux and Windows operating systems (Table 1).

Table 1. De novo assemblers used for the genome assemblies of Bacillus altitudinis 19RS3 and Bacillus altitudinis T5S-T4 plant growth-promoting bacteria isolated from Ilex paraguariensis St. Hil.

Assembler	Reference	Operating System	Manual
Velvet (v. 1.2.10)	Zerbino et al. [45]	Linux	https://github.com/dzerbino/velvet/wiki/Manual
ABySS (v. 2.0.2)	Simpson et al. [46]	Linux	ftp://ftp.ccb.jhu.edu/pub/dpuiu/Docs/ABYSS.html
SPAdes (v. 3.12.0)	Bankevich et al. [47]	Linux	http://cab.spbu.ru/files/release3.12.0/manual.html
SOAPdenovo2 (v. 2.40)	Luo et al. [48]	Linux	https://vcru.wisc.edu/simonlab/bioinformatics/programs/soap/SOAPdenovo2MANUAL.txt
Geneious (v. 11.0.1)	Kearse et al. [54]	Windows	https://assets.geneious.com/documentation/geneious/GeneiousManual.pdf
CLC Genomics Workbench (v. 12.0.3)	Knudsen et al. [55]	Windows	http://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/900/index.php?manual=Sequence_alignment.html

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The k-mer values were selected according to the manual user instructions of each assembler. In general terms, the values were odd to avoid palindromes and were strictly inferior to read length.

The assemblies obtained in Linux were evaluated using QUAST (Quality Assessment Tool for Genome Assemblies) [56–58]. The assemblies generated in Windows showed their own statistics tables.

The parameters evaluated were the number of contigs ≥ 500 bp, the number of contigs ≥ 1000 bp, the length of the longest contig, and the value of N50.

Genome annotation

Gene prediction and annotation were performed using The Rapid Prokaryotic Genome Annotation (Prokka) [59]. Putative genes involved in plant growth-promoting mechanisms were determined using the Rapid Annotations using Subsystems Technology (RAST) [60] annotation server and KEGG Automatic Annotation Server (KAAS) [61].

Genome comparison

The genomes of B. altitudinis 19RS3, B. altitudinis T5S-T4, B. altitudinis W3 (accession number: NZ_CP011150.1) an NCBI reference sequence, B. altitudinis GQYP101 (accession number: NZ_CP040514.1) an NCBI reference sequence reported as PGPB, and B. velezensis FZB42 (accession number: CP000560.2) [23] a commercial PGPB strain used as an active principle of Biomex® and Rhizovital®42, were compared to locate genes involved in PGP mechanisms using Geneious 11.0.1 software.

Results

The genome sequencing of B. altitudinis 19RS3 showed 9,938,250 paired-end reads of 101 bp with a GC content of 41.014% and an average coverage of 249. While B. altitudinis T5S-T4 showed 12,397,272 paired-end reads of 101 bp with a GC content of 40.390% and an average coverage of 292.

After the quality filtering by Trimmomatic, B. altitudinis 19RS3 and B. altitudinis T5S-T4 resulted in 9,329,838 and 10,923,782 paired-end reads, respectively.

Genome assembly’s quality statistics generated by different assemblers for B. altitudinis 19RS3 and B. altitudinis T5S-T4 are summarized in Tables 2 and 3, respectively. The complete quality statistics obtained by all the assemblers using different k-mer values are available as S1–S12 Tables.

Table 2. Comparison of assembled genome quality statistics generated by different assemblers for Bacillus altitudinis 19RS3 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil.

Assembler	SPAdes	ABySS	Velvet	SOAPdenovo2	Geneious (Velvet)	CLC Genomics Workbench
K-mer	79	87	93	89	91	64
^# contigs (≥ 500 bp)	16	18	15	43	-	43
^# contigs (≥ 1000 bp)	14	14	12	39	37	18
Largest contig (bp)	966.271	1.184.276	1.943.801	492.824	-	966.324
N50 (bp)	931.914	928.348	1.943.801	227.675	155.382	895.161

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K-mer: k value used to execute the assemblers.

# contigs ≥ 500 bp: number of contigs larger or equal to 500 bp.

# contigs ≥ 1000 bp: number of contigs larger or equal to 1000 bp.

N50: length of the contig overlapping the midpoint of the length-order concatenation of contigs.

Table 3. Comparison of assembled genome quality statistics generated by different assemblers for Bacillus altitudinis T5S-T4 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil.

Assembler	SPAdes	ABySS	Velvet	SOAPdenovo2	Geneious (Velvet)	CLC Genomics Workbench
K-mer	97	95	89	91	89	20
^# contigs (≥ 500 bp)	34	28	24	61	-	79
^# contigs (≥ 1000 bp)	31	22	23	51	44	74
Largest contig (bp)	805.153	807.963	805.135	656.867	-	534.586
N50 (bp)	344.108	552.057	344.151	131.452	222.742	214.550

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K-mer: k value used to execute the assemblers.

# contigs ≥ 500 bp: number of contigs larger or equal to 500 bp.

# contigs ≥ 1000 bp: number of contigs larger or equal to 1000 bp.

N50: length of the contig overlapping the midpoint of the length-order concatenation of contigs.

In the assembly of B. altitudinis 19RS3 genome, ABySS and Velvet generated the contigs with the highest N50 value. These two assemblies produced N50 values that are more than five times higher than the worst assemblies. Velvet also generated the fewest number of contigs and performed considerably better than the other assemblers. Geneious generated the worst assembly with the fewest N50 value and the highest number of contigs.

For the assembly of B. altitudinis T5S-T4 genome, ABySS had the highest N50 value, followed by Velvet. This last assembler also generated the fewest number of contigs. The CLC Genomics Workbench assembly, despite its large N50 contig size, had more contigs than any other assembler.

The best assembly for both genomes was obtained using the Velvet software. The B. altitudinis 19RS3 genome was assembled into 15 contigs (≥ 500 bp) with an N50 value of 1,943,801 bp and the longest contig length of 1,943,801 bp. The B. altitudinis T5S-T4 genome was assembled into 24 contigs (≥ 500 bp) with an N50 of 344,151 bp and the longest contig length of 805,135 bp. The B. altitudinis 19RS3 and B. altitudinis T5S-T4 assembled contigs were deposited in Genbank under accession numbers JACAAH01 and JACAAI01 respectively.

Genomic features of B. altitudinis 19RS3 (Fig 1) and B. altitudinis T5S-T4 (Fig 2) presented similar size, noncoding sequences, ribosomal RNA sequences, and transfer RNA (Table 4).

Table 4. General genome features of Bacillus altitudinis strain 19RS3 and T5S-T4 plant growth-promoting bacteria isolated from Ilex paraguariensis St. Hil.

Features	19RS3 chromosome	T5S-T4 chromosome
Genome size	3,794,712	3,738,127
G + C (%)	41.2	41.2
Predicted CDS	3861	3801
rRNAs	6	7
tRNAs	74	60
Genbank accession	JACAAH01	JACAAI01

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C+G (%): guanine and cytosine content; CDS: protein-coding genes; rRNAs: ribosomal RNA; tRNAs: transfer RNA.

In the chromosome sequence of B. altitudinis 19RS3 a total of 3861 CDSs and 80 RNAs genes were predicted (Table 4). Among these CDSs 2762 (68.43%) genes were classified into 469 functional subsystems. Similarly, in the chromosome sequence of B. altitudinis T5S-T4 a total of 3801 CDSs and 67 RNAs genes were predicted (Table 4). Among these CDSs 2750 (69.54%) genes were classified into 472 functional subsystems. There is a high similarity among the number of genes in each category between B. altitudinis 19RS3 and B. altitudinis T5S-T4; but the former has more genes in several metabolism-related functions in cellular processes such as cell wall formation and capsule formation, regulation and cellular signaling, and genes related to phages, prophages, transposable elements, plasmids (Table 5). Most of the genes were associated with the metabolism of carbohydrates and amino acids derivates.

Table 5. Annotation of genes involved in the metabolism and other cellular processes of Bacillus altitudinis 19RS3 and Bacillus altitudinis T5S-T4 plant growth-promoting bacteria isolated from Ilex paraguariensis St. Hil.

Genes functions		B. altitudinis 19RS3	B. altitudinis T5S-T4
Genes related to metabolism	Fatty acids, lipids, and isoprenoids	114	112
	Amino acids derivatives	434	426
	Sulfur	34	34
	Carbohydrates	447	444
	Cofactors, vitamins, prosthetic groups, pigments	195	196
	Aromatics compounds	6	8
	DNA	74	100
	Phosphorus	26	26
	Iron	48	47
	Secondary metabolism	4	4
	Nitrogen-proteins	277	262
	Nucleosides and nucleotides	109	109
	Potassium	7	7
	RNA	153	154
Genes related to cellular processes	Division and cellular cycle	58	58
	Dormancy and sporulation	120	120
	Cellular wall and capsule formation	152	148
	Photosynthesis	0	0
	Miscellaneous	40	40
	Motility and chemostasis	87	88
	Regulation and cellular signaling	67	59
	Related to phages, prophages, transposable elements, plasmids	20	14
	Respiration	64	64
	Response to stress	93	95
	Membrane transport	78	78
	Virulence, disease, and defense	55	57

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The presence of related genes to PGPR mechanisms or the metabolic pathway prediction of RAST was found from the gene annotation. The production of enzymes involved in the metabolism of indole acetic acid (IAA) via the tryptophan pathway coded by the gene cluster trp(ABD) was predicted, suggesting that B. altitudinis 19RS3 and B. altitudinis T5S-T4 have the potential to biosynthesize auxin. The gene cluster that encodes to produce bacilibactin, dhb(ACEBF), was also found in both genomes showing the potential for the production of siderophore.

The pst(SCAB) genes, coding for Pi-specific transporter, were found in the genome of B. altitudinis 19RS3 and B. altitudinis T5S-T4 suggesting the capacity of both strains for inorganic phosphate solubilization. Finally, the genes nif(U) and nif(S), were present in both strains which are involved in nitrogenase enzymatic activity responsible for the biological fixation of nitrogen. However, the presence of the complete gene cluster which is essential for the nitrogenase activity was not found.

Volatile compounds as 2,3-butanediol and acetoin might be produced by B. altitudinis 19RS3 and B. altitudinis T5S-T4 given that it has the potential to produce the enzymes α−acetolactate synthetase, α−acetolactate decarboxylase, and acetoin utilization protein. Coding regions for surfactin production were also found and the complete gene cluster srf(ABCD) was annotated in each genome. Genes responsible for flagellar motility, chemotaxis, and biofilm synthesis, which allow B. altitudinis 19RS3 and T5S-T4 to move toward root-exudates facilitating adhesion to plant surfaces, were encountered. Also, some genes related to stress response, such as implicated in osmotic stress, oxidative stress, cold and heat shock, and detoxification, in addition to genes related to sporulation were present in both genomes, indicating a possible protection mechanism to extreme environmental conditions.

The genomes comparison revealed specific gene clusters involved in PGP capabilities (Table 6). All the genomes presented genes associated with the production of volatile compounds such as 2,3-butanediol and acetoin. Only B. altitudinis 19RS3, B. altitudinis T5S-T4 and B. velezensis FZB42 showed the presence of genes associated with surfactin production. This commercial strain also presented genes for phytase and iturin production. Interestingly, other genes coding for bacilibactin, IAA production, Pi-specific transporter, and PHO regulon were discovered only in our studied strains.

Table 6. Comparative genomics of Bacillus altitudinis 19RS3 and Bacillus altitudinis T5S-T4 with the reported genomes of Bacillus altitudinis W3, Bacillus altitudinis GQYP101 and Bacillus velezensis FZB42.

Compound	Genes	Gen (kpb)	B. altitudinis 19RS3	B. altitudinis T5S-T4	B. altitudinis W3	B. altitudinis GQYP101	B. velezensis FZB42
Bacilibactin	dhb(ACEBF)	11.7	+	+	-	-	-
Surfactin	srf(ABCD)	26.2	+	+	-	-	+
2,3 butanediol dehydrogenase		1.04	+	+	+	+	+
Acetoin utilization protein	acu(C)	1.16	+	+	+	+	+
Acetolactate decarboxylase	bud(A)	0.77	+	+	+	+	+
Acetolactate synthase	als(S)	1.7	+	+	+	+	+
IAA production	trp(ABD)	3.02	+	+	-	-	-
Pi-specific transporter	pst(SCAB)	3.54	+	+	-	-	-
PHO regulon	pho(RP)	2.47	+	+	-	-	-
Phytase	phy(C)	1.15	-	-	-	-	+
Iturin	bmy(DABC)	37.2	-	-	-	-	+

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+ presence of the complete gene cluster

- absence of the complete gene cluster

Discussion

In the present study the genome of two PGP strains isolated from I. paraguariensis St. Hil., B. altitudinis 19RS3 and B. altitudinis T5S-T4, were sequenced and assembled. We compared the assembled genome quality statistics generated by different de novo assemblers available for Windows and Linux operating systems. Although no assembler was the best in all the various metrics simultaneously, the Velvet assembler generated the fewest contig number and the higher N50 value. We also annotated both genomes, detected the genes associated with PGP properties, and determinate the presence of these sequences in two B. altitudinis genomes reported in the NCBI.

The prokaryotic genomic structure characteristics were considered to select the sequencing platform, as well as the construction of the library. Some authors [62] indicate it may be useful to try different strategies for de novo assembly of a newly sequenced organism. They propose to evaluate the strategies for the construction of contigs and analyze their effect on the assembly when choosing the best parameters. They also emphasize that knowing the characteristics of the genomic structure of an organism, the sequencing platform, and the construction of the library can be especially useful when choosing assembly tools.

The raw reads obtained for both genomes were processed to eliminate adapters and possible contaminants that can affect the quality of the results, creating a problem when comparing the efficiency of the assemblers. Some authors [28] recommend a trimming step to ensure the high quality of the data. We agree and highlight the importance of filtering and trimming to generate better results because in a previous study we evaluated the effect of the use of raw and filtered reads as input files, in the assembly of the genome of B. altitudinis 19RS3 and obtained a better assembly using the filtered reads [63].

When considering the number of contigs, the longest contig length, and the N50 value in the assemblies of both genomes, the software Velvet and ABySS generated the best results. As in our study, other authors [64] evaluated de novo assemblers using reads of prokaryotic genomes. In their work, Velvet showed a greater number of contigs and a lower value of N50, while ABySS generated a lower number of contigs in the paired data sets and showed a higher N50 value. The authors associated these results variation to factors such as the quality of the data and the k-mer size. About this last item, we decided to use different k-mer values considering their effect in genome assemblies. For SPAdes assembler, we used a k-mer value of 63 greater than the average size of the reads and we sought to gradually increase the values, getting to obtain more precise assemblies with k-mer values of 79 and 97. Large k-mers often result in larger contigs, but excessively large k-mers can cause a fragmented graph with a higher number of contigs. SPAdes, ABySS, and SOAPdenovo2 generated their best assemblies with the highest k-mer value, however, they also produced the most fragmented assemblies. Several authors [65, 66], showed SPAdes stands out as one of the best assemblers for the assembly of Illumina data, due to its quality and high precision. Although in our study, assemblies with a fewer number of contigs were obtained using other software, SPAdes produced very good results for the assembly of both genomes.

As showed in the assemblies obtained in this work, the value that presented the greatest variation was the number of contigs. We agree with some authors [49] that the wide variety of assemblers’ available use different heuristic approaches to meet the challenges of genome assembly and this results in significant differences when comparing the number of contigs they generated. For this reason, we consider necessary a thorough and complete evaluation of the assembled genome quality statistics generated by different assemblers before selecting the best assembly.

In the present study, we predicted genes and enzymes associated with PGP mechanisms in the B. altitudinis 19RS3 and B. altitudinis T5S-T4 genomes. We detected genes related to the conversion pathway of tryptophan to indole, which is consistent with the determined indole production observed in the in vitro assays [41]. The presence of the bacillibatin gene cluster showed the potential of siderophore production, while the detection of Pi transporters and the Pho regulon indicated a possibility for inorganic phosphate solubilization. The presence of nif(U) and nif(S) was also determined in both genomes suggesting the possibility of the strain to fix environmental nitrogen. The properties mentioned above are consistent with the in vitro and in vivo PGP activities determined experimentally in previous studies for both strains [41].

The results obtained for the assembly of B. altitudinis 19RS3 and B. altitudinis T5S-T4 genomes are like the reported for other Bacillus PGP strains such as B. flexus KLBMP 4941 [15–67], B. pumilus GM3FR [68], B. mycoides GM6LP [69], B. vallismortis NBIF-001 [70], B. velezensis 2A-2B [71], and B. velezensis UCMB5140 [14]. Particularly the genome of B. altitudinis FD48 [72] comprises several genes related to plant growth promotion mechanisms, such as those for the biogenesis of organic acids involved in inorganic phosphorus solubilization, iron, and siderophore uptake systems, and nitrogen metabolism. Perhaps of this, genome annotation isn´t available to realize a deeper genome comparison. The PGP genes reported for B. subtilis EA-CB0575 [28] related to IAA, siderophore production, acetoin, 2,3-butanediol, and LPs production, nitrogen fixation, and phosphate solubilization are like those detected in B. altitudinis 19RS3 and B. altitudinis T5S-T4 genomes. The comparison realized with B. altitudinis W3, B. altitudinis GQYP101, and B. velezensis FZB42 genomes indicated that our strains present some unique genes able to promote I. paraguariensis growth. The five genomes present genes associated with the production of volatile compounds as 2,3-butanediol and acetoin, but the other PGP gene clusters were only detected in our studied strains. Also, we determinate the presence of loci for surfactins codification in the genomes of B. altitudinis 19RS3, B. altitudinis T5S-T4, and B. velezensis FZB42. The commercial strain FZB42 also presents genes to the phytase and iturin production. Each Bacillus PGP strain provides a subtle difference in terms of their plant growth-promoting and biocontrol activities. Future design of an effective bioinoculant should be based on combinations of PGP strains supplementing each other.

Conclusion

The best assembly for B. altitudinis 19RS3 and B. altitudinis T5S-T4 was obtained using the Velvet software. A great number of genes associated with PGP mechanisms were annotated and analyzed. It was found genes involved in auxin biosynthesis, siderophore production, phosphate metabolism, and nitrogen fixation. Also, other genes associated with acetoin and 2,3-butanediol production, motility, chemotaxis, adhesion, sporulation, and defense functions were encountered. The gene detection realized in the present study supports the PGP properties observed in previous assays.

The results obtained offer the basis for a better understanding of B. altitudinis 19RS3 and T5S-T4 biology and make them promissory for the development of novel strategies in the biotechnological application of these bacteria as bioinoculant. The information presented here will allow in-depth functional and comparative genome analyses to provide a better understanding of beneficial plant-bacteria associations.

Supporting information

S1 Table. Assembled genome quality statistics obtained for Bacillus altitudinis 19RS3 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using ABySS assembler.

(DOCX)

Click here for additional data file.^{(15.4KB, docx)}

S2 Table. Assembled genome quality statistics obtained for Bacillus altitudinis T5S-T4 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using ABySS assembler.

(DOCX)

Click here for additional data file.^{(15.3KB, docx)}

S3 Table. Assembled genome quality statistics obtained for Bacillus altitudinis 19RS3 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using SPAdes assembler.

(DOCX)

Click here for additional data file.^{(15.2KB, docx)}

S4 Table. Assembled genome quality statistics obtained for Bacillus altitudinis T5S-T4 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using SPAdes assembler.

(DOCX)

Click here for additional data file.^{(15.3KB, docx)}

S5 Table. Assembled genome quality statistics obtained for Bacillus altitudinis 19RS3 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using Velvet assembler.

(DOCX)

Click here for additional data file.^{(15.3KB, docx)}

S6 Table. Assembled genome quality statistics obtained for Bacillus altitudinis T5S-T4 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using Velvet assembler.

(DOCX)

Click here for additional data file.^{(15.2KB, docx)}

S7 Table. Assembled genome quality statistics obtained for Bacillus altitudinis 19RS3 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using SOAPdenovo2 assembler.

(DOCX)

Click here for additional data file.^{(14.9KB, docx)}

S8 Table. Assembled genome quality statistics obtained for Bacillus altitudinis T5S-T4 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using SOAPdenovo2 assembler.

(DOCX)

Click here for additional data file.^{(15.4KB, docx)}

S9 Table. Assembled genome quality statistics obtained for Bacillus altitudinis 19RS3 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using geneious assembler with the Velvet algorithm.

(DOCX)

Click here for additional data file.^{(13.7KB, docx)}

S10 Table. Assembled genome quality statistics obtained for Bacillus altitudinis T5S-T4 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using geneious assembler with the Velvet algorithm.

(DOCX)

Click here for additional data file.^{(13.8KB, docx)}

S11 Table. Assembled genome quality statistics obtained for Bacillus altitudinis 19RS3 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using CLC workbench assembler.

(DOCX)

Click here for additional data file.^{(12.3KB, docx)}

S12 Table. Assembled genome quality statistics obtained for Bacillus altitudinis T5S-T4 a plant-growth-promoting bacteria isolated from Ilex paraguariensis St. Hil. using CLC workbench assembler.

(DOCX)

Click here for additional data file.^{(12.3KB, docx)}

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This study was supported by Proyecto del Instituto Nacional de la Yerba Mate (INYM, Argentina) "Biofertilizantes: validación a campo y estudios de trazabilidad de la utilización de Bacillus sp. como fertilizante para yerba mate” Res. n° 274/17 (INYM - PRASY) granted to ML.

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PLoS One. doi: 10.1371/journal.pone.0248274.r001

Decision Letter 0

Ying Ma

16 Dec 2020

PONE-D-20-28962

De novo assembly of Bacillus altitudinis 19RS3 and Bacillus altitudinis T5S-T4, two Plant Growth-Promoting Bacteria isolated from Ilex paraguariensis St. Hil. (yerba mate)

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: No

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4. 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 #1: No

Reviewer #2: Yes

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5. 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 #1: This manuscript describes the de novo sequencing and assembly of genomes from two isolates of Bacillus altitudinis that were recovered from plant tissue. The authors have used various computational tools to obtain the best assembly, and have annotated the genomes of both isolates. Part of the annotation identified putative functions that may be related to the plant growth promotion capabilities of the isolates. The sequencing and analysis is pretty routine, but the results are pretty clear and this provides helpful information to the body of information of genome analysis of plant growth promoting rhizobacteria (PGPR). I have two important points to be addressed, plus a few minor comments, as follows:

1. The paper would be strengthened by examining the relationship between these two new genomes with other published Bacillus genomes, especially those with PGPR activity. When compared, what genes are conserved or are novel? This would be helpful to know in order to more closely connect potential genes with PGPR mechanisms.

2. Related to the above, the current analysis identifies broad categories of genes potentially involved in PGPR activity, such as iron metabolism, but some more in depth analysis of specific genes would be helpful. In particular, identify if genes are present that have been identified in other Bacillus species.

3. Line 130, what tissues were used for DNA isolation? Leaves, etc.?

4. Please have someone carefully proofread for proper English, including use of 'a', 'an', and 'the'. Often one of these words is present when not needed, or absent when it is needed. See for example in lines 38, 73 and 86.

5. Line 32 should read 'Assembly evaluation was done...'

6. line 66, should read '...great diversity of species...', 'Kepping' should be 'Keeping'

7. line 68, 'perspective' seems to be the wrong word here.

8. line 82, should be 'revealed'

9. lines 105, 106, remove 's' from the ends of words where it is not needed.

10. line 142, 'in' should be 'by'

11. line 176, 'bigger' is an awkward word here, better to say 'higher' or 'larger'

12. lines 181-183, the end of the sentence after the last comma is redundant and should be removed.

13. line 190 and table, are the accession numbers really all zeros after the initial letters, or are these placeholders to be updated?

Reviewer #2: General comments: This manuscript described the assembly and annotation of two plant growth-promoting bacteria. The authors describe multiple different assemblies constructed using various different software programs and choose the assembly they believe is the best. This is an interesting dataset and reporting that could be of interest to bacterial researchers and researchers interested in using more natural means of beneficials for plant growth and overall health.

Manuscript concerns:

1. One main concern of the manuscript is what this adds to the community. Many previous studies have looked at bacterial genome assemblies across multiple software types. It seems for the most part this manuscript agrees with basically all previous findings. The authors should really focus and point out what their results are adding to the community. Adding the assemblies of PGPB is great and justified, but the manuscript focuses so much on the assembly of multiple softwares that it dilutes down the importance of having these genomes without really adding much to the space of assembly software decisions.

2. The methods are insufficient in details. It would be really difficult for anyone to reproduce your results and assemblies. We are not told the types of reads used, how each of the assembly software was run and utilized (what parameters were used or changed or prioritized).

3. Something isn’t right with the T5S-T4 CLC workbench results in Table 3. The largest contain is 178bp but the N50 is 895bp?

4. I’m questioning the availability of the data for this manuscript. The only thing available seems to be contigs assemblies from Velvet. Where is the rest of the data used and generated in the manuscript that would be useful to the community? As far as I can tell the raw read information isn’t available and either is the annotations done for the assemblies. These exclusions don’t really adhere to the data availability guidelines of the journal.

5. Why are there such differences in the assemblies? It seems reasonable for one to think that the T5S-T4 genome might have a better assembly as there is more input data, but the findings show the opposite with the T5S-T4 genome having substantially more contigs and much smaller N50. Is there an underlying data difference, characteristic of the genome, or other possible reason for this?

6. For the T5S-T4 assembly, why was the Velvet assembly picked as the best over the ABySS assembly? Going by metrics of # of contigs it is very close and the ABySS assembly has a larger contig and a much larger N50.

7. It seems a bit more could be done to choose which assembly is the ‘best’ than just contig number, largest contig size, and N50. Other measures that could be considered would be map the reads back to the assemblies to determine the number that align and at what mapping quality. Also, the annotation is not assessed at all, but could also be used to help determine the assembly quality by using a BUSCO or similar software to check for gene completeness. These would really help strengthen the reasoning for picking an assembly over others.

8. Figure 3 and 4: Is there are better way the authors can think of to present this data? The pie chart does’t really add anything and it is difficult to match up colors of categories and the chart. The authors could perhaps at least order the output and categories by size or some other manner for easier comparisons for the readers.

9. pg 4 line 88-89: What region are you referring too? The reader isn’t familiar with where you are.

10. pg 5 line 108-11: What type of insights? Could expand this to help the reader understand what is known in the genomics of PGPB to see how your work is important and fits and adds to the community.

11. pg 14 line 249-250: There is no highlighting the importance of filtering in this manuscript. There are no results of assemblies without trimming to compare to assemblies with trimming, so this statement can’t really be made based on the data presented in this manuscript.

12. pg 14 line 259-262: This sentence is confusing. It is unclear how kmers were picked and how you would use kmers that are larger than some, or most, of the read lengths. If the kmer length was larger then the read was the read removed from the analysis?

13. Grammar and tense usage throughout the manuscript needs to be checked.

**********

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PLoS One. 2021 Mar 11;16(3):e0248274. doi: 10.1371/journal.pone.0248274.r002

Author response to Decision Letter 0

12 Jan 2021

Response to reviewers

Title: De novo genome assembly of Bacillus altitudinis 19RS3 and Bacillus altitudinis T5S-T4, two Plant Growth-Promoting Bacteria isolated from Ilex paraguariensis St. Hil. (yerba mate)

Authors: Iliana Julieta Cortese, María Lorena Castrillo, Andrea Liliana Onetto, Gustavo Ángel Bich, Pedro Darío Zapata, Margarita Ester Laczeski.

First, we want to express our sincere thanks to the reviewers for their great work, their notes have allowed us not only to improve the manuscript but also to reflect on future research.

After a careful review of our proposed article, based on the suggestions made by the reviewers, we have processed your submission for a new evaluation. In the new manuscript, we have used the track changes mode in Word for the modifications made to the original text as you suggested.

We hope that the work done will achieve the final approval of the Editorial Team. If not, all authors are available to resolve any issue or proceed with further revisions, as necessary.

We then respond to each of the reviewers' observations.

Reviewer Comments:

Reviewer 1

This manuscript describes the de novo sequencing and assembly of genomes from two isolates of Bacillus altitudinis that were recovered from plant tissue. The authors have used various computational tools to obtain the best assembly,and have annotated the genomes of both isolates. Part of the annotation identified putative functions that may be related to the plant growth promotion capabilities of the isolates. The sequencing and analysis is pretty routine, but the results are pretty clear and this provides helpful information to the body of information of genome analysis of plant growth promoting rhizobacteria (PGPR). I have two important points to be addressed, plus a few minor comments, as follows:

Response: Following your suggestion we have incorporated in the manuscript the conserved genes of both bacterial strains related to PGP properties.

Response: As suggested, new relationships between these two new genomes and other published Bacillus genomes, especially of bacteria with PGPR activity, were examined. A table was added to add information and improve analysis in the results section. In addition, related information was added to the discussion.

3. Line 130, what tissues were used for DNA isolation? Leaves, etc.?

Response: We changed tissues for roots.

Response: English proofread was checked

5. Line 32 should read 'Assembly evaluation was done...'

Response: We changed the text.

6. line 66, should read '...great diversity of species...', 'Kepping' should be 'Keeping'

Response: We corrected the word.

7. line 68, 'perspective' seems to be the wrong word here.

Response: The expression has been improved.

8. line 82, should be 'revealed'

Response: We changed the text.

9. lines 105, 106, remove 's' from the ends of words where it is not needed.

Response: We removed “s” from the ends of words where it wasn´t needed.

10. line 142, 'in' should be 'by'

Response: We changed the word.

11. line 176, 'bigger' is an awkward word here, better to say 'higher' or 'larger'

Response: We change bigger to higher.

12. lines 181-183, the end of the sentence after the last comma is redundant and should be removed.

Response: We removed the sentence.

13. line 190 and table, are the accession numbers really all zeros after the initial letters, or are these placeholders to be updated?

Response: We thank the reviewer for this particularly important remark. We corrected the accession numbers of both genomes.

Reviewer 2

General comments: This manuscript described the assembly and annotation of two plant growth-promoting bacteria. The authors describe multiple different assemblies constructed using various different software programs and choose the assembly they believe is the best. This is an interesting dataset and reporting that could be of interest to bacterial researchers and researchers interested in using more natural means of beneficials for plant growth and overall health.

Response: We thank the Reviewer 2 for her/his careful reading of the manuscript and for her/his constructive remarks, which were useful in improving our paper. We followed this important comment. In the new version of our manuscript, we added more information about the PGP genes detected in both genomes. We considered this information should be useful for future comparative genome analyses to provide a better understanding of beneficial plant-bacteria associations.

Response: We added the type of reads used. About the parameters applied for each software we used the pre-determinate options indicated in the manual user instructions and we only vary the k-mer value. In the new version of our manuscript, we include the genome quality statistics generated by different assemblers as Supplementary material.

3. Something isn’t right with the T5S-T4 CLC workbench results in Table 3. The largest contain is 178bp but the N50 is 895bp?

Response: We checked and corrected the values.

Response: We report in Genbank all the information related to this project: Bioproject, Biosample, and WGS data, which are the focus of our manuscript and put this information available to the community. In the Genbank server, we considered reporting only the best data of assemblies (contigs from Velvet) to avoid errors or confusion. Also, related to this query, in the results section, we included a sentence making explicit the availability of the assembled genome. Now we also add as supporting information the other assemblies statistics obtained in this work.

As for the raw sequence information, we are still working with that data. We appreciate your understanding of their reserve until we finish with their processing. If more information is needed for the scientific community at this stage, we ask the reviewers to convey the concern again.

Response: Although the genome of B. altitudinis T5S-T4 has more reads, it doesn´t mean that it will generate a better assembly. The data of both genomes were obtained in the same way, and the characteristics of the reads generated were remarkably similar, so we don´t identified any underlying difference.

Response: About this item, we decided to select the assembly with a lower number of contigs ≥ 500 bp as better. Velvet assembler is commonly used in prokaryotic genome assembly, so we take the decision mentioned above.

Response: As we answered in the commentary 2, in the new version of our manuscript we include the genome quality statistics generated by different assemblers as Supplementary material.

Response: The information from Figures 3 and 4 were replaced and reorganized in Table 5 for better understanding.

9. pg 4 line 88-89: What region are you referring too? The reader isn’t familiar with where you are

Response: We clarified this sentence by adding: Ilex paraguariensis St. Hil., a plant that is also commonly called yerba mate, is one of the most economically important crops in the northeast of Argentina.

10. pg 5 line 108-11: What type of insights? Could expand this to help the reader understand what is known in the genomics of PGPB to see how your work is important and fits and adds to the community

Response: We added more information about the molecular and genetic mechanisms of plant growth promoting (PGP) activities.

Response: A citation of a previous work of our authorship was included in the discussion section to add information related to trimming and filtering steps and their effect on the genome assembly of B. altitudinis 19RS3. In that publication, we compare the assemblies obtained by using raw-reads and filtered-reads as input files.

Response: We added more information about the k-mer size selection in the materials and methods section. In general terms, the values were odd to avoid palindromes and were strictly inferior to read length.

13. Grammar and tense usage throughout the manuscript needs to be checked.

Response: We thank Reviewers 1 and 2 for their careful reading of the manuscript and for their constructive remarks, which were useful in improving our paper. We followed this important comment and the manuscript writing was completely checked.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(32.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0248274.r003

Decision Letter 1

Ying Ma

24 Feb 2021

De novo assembly of Bacillus altitudinis19RS3 and Bacillus altitudinis T5S-T4, two Plant Growth-Promoting Bacteria isolated from Ilex paraguariensis St. Hil. (yerba mate)

PONE-D-20-28962R1

Dear Dr. Laczeski,

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.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. 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.

Kind regards,

Ying Ma, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: No

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: Thank you for addressing the concerns mentioned in my previous review. The manuscript is considerably improved and I feel it now provides for a better comparison with other Bacillus strains with PGPB activity. There are still places in the text where the English or specific wording could be improved.

Line 72, genres should be genera

Line 116, delete the s to read biofertilizer production. There are other places where there is an unneeded s at the end of a word, and other similar word issues throughout.

Reviewer #2: Comments: I want to thank the authors for making improvements on most of the comments from the reviews. The revisions that where made improve the manuscript greatly. The authors did a commendable job of taking the reviews and improving the substance, structure, and digestibility of the manuscript.

**********

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 #1: No

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0248274.r004

Acceptance letter

Ying Ma

1 Mar 2021

PONE-D-20-28962R1

De novo genome assembly of Bacillus altitudinis 19RS3 and Bacillus altitudinis T5S-T4, two Plant Growth-Promoting Bacteria isolated from Ilex paraguariensis St. Hil. (yerba mate)

Dear Dr. Laczeski:

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

Dr. Ying Ma

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Assembled genome quality statistics obtained for Bacillus altitudinis 19RS3 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using ABySS assembler.

(DOCX)

Click here for additional data file.^{(15.4KB, docx)}

S2 Table. Assembled genome quality statistics obtained for Bacillus altitudinis T5S-T4 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using ABySS assembler.

(DOCX)

Click here for additional data file.^{(15.3KB, docx)}

S3 Table. Assembled genome quality statistics obtained for Bacillus altitudinis 19RS3 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using SPAdes assembler.

(DOCX)

Click here for additional data file.^{(15.2KB, docx)}

S4 Table. Assembled genome quality statistics obtained for Bacillus altitudinis T5S-T4 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using SPAdes assembler.

(DOCX)

Click here for additional data file.^{(15.3KB, docx)}

S5 Table. Assembled genome quality statistics obtained for Bacillus altitudinis 19RS3 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using Velvet assembler.

(DOCX)

Click here for additional data file.^{(15.3KB, docx)}

S6 Table. Assembled genome quality statistics obtained for Bacillus altitudinis T5S-T4 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using Velvet assembler.

(DOCX)

Click here for additional data file.^{(15.2KB, docx)}

S7 Table. Assembled genome quality statistics obtained for Bacillus altitudinis 19RS3 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using SOAPdenovo2 assembler.

(DOCX)

Click here for additional data file.^{(14.9KB, docx)}

S8 Table. Assembled genome quality statistics obtained for Bacillus altitudinis T5S-T4 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using SOAPdenovo2 assembler.

(DOCX)

Click here for additional data file.^{(15.4KB, docx)}

(DOCX)

Click here for additional data file.^{(13.7KB, docx)}

(DOCX)

Click here for additional data file.^{(13.8KB, docx)}

S11 Table. Assembled genome quality statistics obtained for Bacillus altitudinis 19RS3 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil. using CLC workbench assembler.

(DOCX)

Click here for additional data file.^{(12.3KB, docx)}

S12 Table. Assembled genome quality statistics obtained for Bacillus altitudinis T5S-T4 a plant-growth-promoting bacteria isolated from Ilex paraguariensis St. Hil. using CLC workbench assembler.

(DOCX)

Click here for additional data file.^{(12.3KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(32.9KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

De novo genome assembly of Bacillus altitudinis 19RS3 and Bacillus altitudinis T5S-T4, two plant growth-promoting bacteria isolated from Ilex paraguariensis St. Hil. (yerba mate)

Iliana Julieta Cortese

María Lorena Castrillo

Andrea Liliana Onetto

Gustavo Ángel Bich

Pedro Darío Zapata

Margarita Ester Laczeski

Roles

Abstract

Introduction

Materials and methods

Bacteria

DNA extraction

Library preparation and genome sequencing

Genome assembly and evaluation

Table 1. De novo assemblers used for the genome assemblies of Bacillus altitudinis 19RS3 and Bacillus altitudinis T5S-T4 plant growth-promoting bacteria isolated from Ilex paraguariensis St. Hil.

Genome annotation

Genome comparison

Results

Table 2. Comparison of assembled genome quality statistics generated by different assemblers for Bacillus altitudinis 19RS3 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil.

Table 3. Comparison of assembled genome quality statistics generated by different assemblers for Bacillus altitudinis T5S-T4 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil.

Fig 1. The chromosomal organization of Bacillus altitudinis 19RS3 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil.

Fig 2. The chromosomal organization of Bacillus altitudinis T5S-T4 a plant growth-promoting bacterium isolated from Ilex paraguariensis St. Hil.

Table 4. General genome features of Bacillus altitudinis strain 19RS3 and T5S-T4 plant growth-promoting bacteria isolated from Ilex paraguariensis St. Hil.

Table 5. Annotation of genes involved in the metabolism and other cellular processes of Bacillus altitudinis 19RS3 and Bacillus altitudinis T5S-T4 plant growth-promoting bacteria isolated from Ilex paraguariensis St. Hil.

Table 6. Comparative genomics of Bacillus altitudinis 19RS3 and Bacillus altitudinis T5S-T4 with the reported genomes of Bacillus altitudinis W3, Bacillus altitudinis GQYP101 and Bacillus velezensis FZB42.

Discussion

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Ying Ma

Roles

Author response to Decision Letter 0

Decision Letter 1

Ying Ma

Roles

Acceptance letter

Ying Ma

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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