The genome of a steinernematid-associated Pseudomonas piscis bacterium encodes the biosynthesis of insect toxins
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Several species of soil-dwelling Steinernema nematodes are used in the biocontrol of crop pests, due to their natural capacity to kill diverse lepidopteran species. Although this insect-killing trait is known to be augmented by the nematodes’ Xenorhabdus endosymbionts, the role of other steinernematid-associated bacterial genera in the nematode lifecycle remains unclear. This genomic study aimed to determine the potential of Pseudomonas piscis to contribute to the entomopathogenicity of its Steinernema host. Insect larvae were infected with three separate Steinernema cultures. From each of the three treatments, the prevalent bacteria in the haemocoel of cadavers, four days post-infection, were isolated. These three bacterial isolates were morphologically characterised. DNA was extracted from each of the three bacterial isolates and used for long-read genome sequencing and assembly. Assemblies were used to delineate species and identify genes that encode insect toxins, antimicrobials, and confer antibiotic resistance. We assembled three complete genomes. Through digital DNA–DNA hybridisation analyses, we ascertained that the haemocoels of insect cadavers previously infected with Steinernema sp. Kalro, Steinernema sp. 75, and Steinernema sp. 97 were dominated by Xenorhabdus griffiniae Kalro, Pseudomonas piscis 75, and X. griffiniae 97, respectively. X. griffiniae Kalro and X. griffiniae 97 formed a subspecies with other X. griffiniae symbionts of steinernematids from Kenya. P. piscis 75 phylogenetically clustered with pseudomonads that are characterised by high insecticidal activity. The P. piscis 75 genome encoded the production pathway of insect toxins such as orfamides and rhizoxins, antifungals such as pyrrolnitrin and pyoluteorin, and the broad-spectrum antimicrobial 2,4-diacetylphloroglucinol. The P. piscis 75 genome encoded resistance to over ten classes of antibiotics, including cationic lipopeptides. Steinernematid-associated P. piscis bacteria hence have the biosynthetic potential to contribute to nematode entomopathogenicity.
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This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community.# Prior to publication I would ask you to make some minor changes: Please change the Antismash reference to the most up-to-date one https://pubmed.ncbi.nlm.nih.gov/37140036/ Please include a reference/link/supplier for the Medeka tool you used for polishing one of the gen
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This study would be a valuable contribution to the existing literature. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. Please make sure you address the concerns raised by the reviewers. In particular some sections are hard to read and need clarification (eg the introduction). Parts of the methods need more details. More details can be found in the comments from the reviewer. For the statistical comment from reviewer 2, I disagree with this request since you have not done Amplicon/Metagenomics and hence those tests are not appropiate for the data you present in the manuscript. Please write a short response to the reviewer comment regarding this.
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Comments to Author
In their article entitled 'The genome of a steinernematid-associated Pseudomonas piscis bacterium encodes the biosynthesis of insect toxins' Awori et al., elaborate identification of non-Xenorhabdus bacteria from Steinernema soil nematodes and identification of genes and gene-clusters contributing to entomopathogenicity of the nematode. The report is a kind of succession to their earlier report on Xenorhabdus strains isolated from soil-dwelling nematodes in Kenya. The methodology is well described. The article is well drafted and includes genetic features linked to nematode-bacteria interaction and interaction among bacteria. Apart from some concerns listed here, the article can be recommended. 1. For Fig. 2: a more elegant presentation is warranted. Each molecules can be labelled as 'a', 'b' and …
Comments to Author
In their article entitled 'The genome of a steinernematid-associated Pseudomonas piscis bacterium encodes the biosynthesis of insect toxins' Awori et al., elaborate identification of non-Xenorhabdus bacteria from Steinernema soil nematodes and identification of genes and gene-clusters contributing to entomopathogenicity of the nematode. The report is a kind of succession to their earlier report on Xenorhabdus strains isolated from soil-dwelling nematodes in Kenya. The methodology is well described. The article is well drafted and includes genetic features linked to nematode-bacteria interaction and interaction among bacteria. Apart from some concerns listed here, the article can be recommended. 1. For Fig. 2: a more elegant presentation is warranted. Each molecules can be labelled as 'a', 'b' and so on and the identity (and description) can be disclosed in the figure legend. 2. Fig. 3: It seems that some apparent discrepancy are needed to get sorted- "arnBCADTEF-ugd operon" or "ara…." ? (as in figure label), and also the gene-order 'BCA…' or 'CBA…' ? 3. The authors described the colony characteristics of the cultivable bacteria isolated from haemoceol of Gallria, It would have been helpful if a high-quality image for the colonies would have been provided. 4. Phenotypic characterization of the isolated bacteria like comparative analysis of growth kinetics and biofilm forming potential of P. piscis 75 with other pseudomonads is warranted. 5. The absence of fitD should have been validated by PCR with the genomic DNA with proper control. 6. A graphical presentation for the functional annotation for CDS with known functions (with Gene Ontology, may be) would have made the genome-analysis part more discernible. Minor comments: 1. The introduction segment is quite extended. It should be more concise and focused. 2. An intricate discussion on environmental impact of the BCGs identified or the resistome revealed would further highlight significance of the study.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
Generally, English language and grammar needs a lot of attention, and you ethics approval needs to be provided in a lot more detail. There is also no statistical analysis or significance described throughout the manuscript. Major revisions are required as the conclusions are only partially supported by absent analysis. Line 12: Too general. Are all insects effected? Line 35: Define environmentally friendly Line 62: Check grammar Line 68: You use 'thus' far too many times and in the wrong context Line 68: Why are you giving aims part way through an introduction? This is poor practice. Line 91: What are the strains? You've listed species. Line 131: You ethics statement is not good enough. What ethics were considered for the use of insects? Who approved the project ethics? Do you have a certificate to …
Comments to Author
Generally, English language and grammar needs a lot of attention, and you ethics approval needs to be provided in a lot more detail. There is also no statistical analysis or significance described throughout the manuscript. Major revisions are required as the conclusions are only partially supported by absent analysis. Line 12: Too general. Are all insects effected? Line 35: Define environmentally friendly Line 62: Check grammar Line 68: You use 'thus' far too many times and in the wrong context Line 68: Why are you giving aims part way through an introduction? This is poor practice. Line 91: What are the strains? You've listed species. Line 131: You ethics statement is not good enough. What ethics were considered for the use of insects? Who approved the project ethics? Do you have a certificate to prove you have ethics approval? Line 137: Correct the reference formatting Line 140: 'about' was it 10 or not? Be specific. Using 'about' is not appropriate for publishable literature. Line 162: How long was the nanopore run? Where samples multiplexed? The results sections need to be addressed. There is a lot of discussion, however, no statistical analysis. This needs to be majorly addressed. Line 458: Make the conclusion stand out with it's own heading The conclusion could be made more specific to address the title of the manuscript Line 165: How large were your sequencing files? 250MB is next to nothing
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
No: No ethics considerations have been described for the use of insects.
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Comments to Author
In this manuscript, Awori et al., present the genome analysis of two Xenorhabdus strains and one Pseudomonas piscis strains associated to Steinerema nematodes. Furthermore, they functionally analyse the genome of P. piscis 75 and report the presence of genes associated to the production of entomopathogenic compounds and antibiotic resistance genes. Overall, the manuscript is coherent and most conclusions are well justified based on the results. However, I think several clarifications and modifications would be needed to improve the quality of the manuscript. Major considerations * With respect to the writing, there are many parts of the manuscript that are hard to read. This is due to the use of very long, complicated sentences that make the message very difficult to understand. Ideally, I would advise …
Comments to Author
In this manuscript, Awori et al., present the genome analysis of two Xenorhabdus strains and one Pseudomonas piscis strains associated to Steinerema nematodes. Furthermore, they functionally analyse the genome of P. piscis 75 and report the presence of genes associated to the production of entomopathogenic compounds and antibiotic resistance genes. Overall, the manuscript is coherent and most conclusions are well justified based on the results. However, I think several clarifications and modifications would be needed to improve the quality of the manuscript. Major considerations * With respect to the writing, there are many parts of the manuscript that are hard to read. This is due to the use of very long, complicated sentences that make the message very difficult to understand. Ideally, I would advise the authors to convey one concept per sentence to make it easier for the reader. Thus, some sentences would need to be shortened and split. Specific examples (not the only one throughout the text) are: L17-20, L70-76, L229-232, L236-238, L240-245, L258-262, L289-292, L302-307, L325-329, L403-406. * Ideally, the abbreviations shown n the Abbreviations section should be alphabetically ordered. Also, with this section at the beginning of the text, it would not be necessary to define the acronyms later in the text. Other abbreviations are not defined (e.g., "ca." in L59 and L288, EPN in L134 and L202). * L65: "other bacteria genera…". This is repeated later. Please avoid redundancy. * L68-70: The aim of the study would look better at the end of the Intro. * L108-110: this hypothesis is not much supported by what has been explained so far in the manuscript. It should be moved to later, where appropriate. * L130: The ethics statements can be put outside of the Methods section. * L 146: The usual LB broth recipe is tryptone, yeast extract and salt. Why was casein used? That would not be LB broth in theory. * L147-151: This is more Results than Methods. * Were the three bacterial isolates of study the only ones obtained after infecting Galleria? I would expect to see more isolates, even if they are minoritarian, and then the authors had to select based on predominance. If this is the case, could the authors provide any numbers on the diversity of isolates? Do the authors have any control with non-infected Galleria to observe the usual isolates you can get from them? * L158: "bacterial isolate Kalro, 75 and 97". Are this not Steinerema? * L175-176, L180: "as previously described". Please describe very briefly. * L183: "extra features" need to be explained. * L185: did the fitD gene appear in the genome annotation or was it a targeted search? If this is the case, it should be explained why this gene was targeted. * L206-216: this phenotypic description would look better and be more easily understood in a table. * L217-226: This might be just an impression, but this paragraph does not seem to link very well with the previous one. Please consider to move it or to integrate it better (or feel free to ignore this comment if you completely disagree). * L289, L290: 71% and 77% seem an unusually high number of genes with a known function. Could you please clarify this? Does this mean they have a gene name given by orthology, a description, a specific domain with a known function at least? In this regard, maybe the authors find useful to run an analysis to cluster genes according to the COG classification, which has a specific category for "unknown function" and "function predicted only" genes. * L311: How are the levels of entomopathogenicity defined? Is it based on the time Galleria took to die from the infection? * L338: why this range of aa length? * Table 2: among the unknown BGCs, there are some of them producing homoserine lactone and terpenes, which are relatively well-known compounds. What do the authors mean by "unknown"? Is it because they are produced by novel pathways? If that is the case, how was it figured out they produced these compounds in particular? This needs a bit more of specificity and clarity. * L371 (and others): here, cyanide is proposed as an insect toxin. However, this is produced by many organisms and it results toxic to others different from insects. Maybe the presentation of this compound as an insect toxin should be reconsidered. * L374: "biosynthetic basis of this lapidated nature" sounds like a too baroque sentence. Maybe an easier writing can be found. * L375, L376: gene names need to be italicised, please make sure this is revised throughout the manuscript. * L396: As a reflection to consider, can these be considered insect toxins if they result toxic to other organisms? * L407-412: Does this mean that X. griffiniae produces some of these antimicrobials (apart from the PAX lipopeptide)? * Figure 3C: arn genes are labelled wrong. * Figure legend 3 can be summarised as most of the text is either repeated in the main text or can be moved there. * L435: 1.18 Mb is a bit of a large distance between the arn gene cluster and the pmrAB operon (nearly 1/5 of the chromosome) to be relevant location-wise. I would recommend to simply mention they are located elsewhere in the chromosome. * L448-452: This is not supported by the results. It is risky to propose resistance to PAX based on a different species, but it is even riskier to propose it based on pmrB mutations that are not the same as shown in the cited papers. The presence of the arn genes undoubtedly confers the potential to be resistant to cationic peptides, but the evidence presented does not allow to conclude if they are expressed to a level that allows that resistance constitutively. This possibility can be discussed and argued, but it cannot be taken as a fact unless it is experimentally validated. Minor changes * L15: replace "genome" by "genomic" * L17: replace "one of three" by "three separate" * L27: replace "production" by "production pathway" * L28: "broad-spectrum antimicrobial". Can the authors specify? * L100: replace "it's" by "it is". Please avoid this sort of contractions. * L116: "Thika", please add the country. * L116 and throughout the manuscript: please keep one single reference format. * L140-142: Please rephrase this sentence, it is a bit convoluted. * L192: do the authors mean "identified and analysed"? * L196: replace "pmrA,B" by "pmrAB," * L230: replace "for" by "for example" * L239: "time" means "time after infection"? "cultivable cultures" is redundant * L258: "TK1" means Kalro? * L270 and throughout the text: replace "&" by "and" * L271: the definition of G+C content can be skipped, but this is up to the authors * L336: replace "too" by "either" at the end of the sentence. * L465: replace "encode" by "confer" I hope the authors find these comments helpful to improve their manuscript.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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