Construction of the Pseudomonas putida Strain with Low Motility and Reduced Biofilm Formation for Application in Fermentation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript explored the possibility of reducing biofilm formation and foam formation in Pseudomonas Putida Strain in order to improve fermentation processes. There are some issues that need to be addressed before this paper can be accepted for publishing.

 

Detailed comments:

 

Title:

The title is not a good representation of the research done in the paper

 

Introduction:

Line 45- 48: This sentence is confusing, please make it clear.

Line 48 - 50: Please provide any references to support this statement. Moreover, if “algA can be removed without the loss of the ability of the microorganism to form biofilms”, why did the authors choose to delete algA over algB, algC, or algD? Clarifying the differences among these genes and the reasoning behind the selection of algA for deletion can strengthen the argument.

 

Table 1: 

Please provide a rationale for not performing individual gene deletion tests. Only testing the combinations of three genes makes it unclear which specific component has the most significant impact. Individual deletions would help identify the relative contribution of each gene.

 

Line 230: The author cannot attribute the reduction in mobility to the deletion of flhA gene in the LN6160 strain. To confirm flhA’s role, they need to perform the same test on a strain with ∆flhA as the sole deletion

Author Response

Dear Colleague, we thank you for your review of our manuscript

The title is not a good representation of the research done in the paper

We changed the title of the article to «Construction Of The Pseudomonas putida Strain With Low Motility and Reduced Biofilm Formation For Its Application In Fermentation», which we assume is more proper for this manuscript. (Lines 2-4)

Introduction:

Line 45- 48: This sentence is confusing, please make it clear.

We are agree with the suggestion therefore the text was corrected to remove the confusions in the sentence (Line 52-54).

Line 48 - 50: Please provide any references to support this statement. Moreover, if “algA can be removed without the loss of the ability of the microorganism to form biofilms”, why did the authors choose to delete algA over algB, algC, or algD? Clarifying the differences among these genes and the reasoning behind the selection of algA for deletion can strengthen the argument.

We are agree with the remark of the reviewer the meaning which meant to be put in this sentence is better to express with the following sentence: “In fact, the genes algA, algB, algC and algD have no counterparts and none of them can be removed without decrease in the ability of the microorganism to form biofilms.” (Lines 52-54)

Table 1: 

Please provide a rationale for not performing individual gene deletion tests. Only testing the combinations of three genes makes it unclear which specific component has the most significant impact. Individual deletions would help identify the relative contribution of each gene.

The aim of our work was to obtain the strain suitable for fermentation purposes. We did not construct single mutants ΔflhA, ΔpilQ and double mutant ΔflhA-ΔpilQ, since the study describing influence of these gene deletion, including double mutant, has been already published for Pseudomonas aeruginosa strain (Klausen M, Heydorn A, Ragas P, Lambertsen L, Aaes-Jørgensen A, Molin S, Tolker-Nielsen T. Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol Microbiol. 2003 Jun;48(6):1511-24. doi: 10.1046/j.1365-2958.2003.03525.x. PMID: 12791135.). We have indeed obtained further insights into intermediate mutants by evaluating the biofilm formation capabilities of ΔalgA, ΔalgA ΔflhA, and ΔalgA ΔflhA ΔpilQ, and this data has been added to our manuscript. (see Figure 4, paragraph 3.5, Lines 276-272).

Line 230: The author cannot attribute the reduction in mobility to the deletion of flhA gene in the LN6160 strain. To confirm flhA’s role, they need to perform the same test on a strain with ∆flhA as the sole deletion

We supplied additional information (see Figure 3) which supports attribution the reduction in mobility to the deletion of flhA gene in the LN6160 strain. (Discussion Lines 257-258)

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, the effect of deleting genes involved in alginate accumulation ( algA ), flagellar output ( flhA ), and pilus formation ( pilQ ) on the behavior of Pseudomonas aeruginosa PCL1760 in a bioreactor was investigated. The final strain showed a significant reduction in biofilm and foam formation in small bioreactors, while displaying higher growth rates, enhancing its attractiveness for application in biotechnological processes. However, the article still needs to be revised in the following points:

1. 3.4 The results are not strictly described. The preamble mentions that the genes pilQ and flhA are important for biofilm formation, whereas the results only mention that the absence of the gene flhA significantly reduced the migration rate of the strain.

2. In the 3.6 results section, there is a lack of information about the specifications (volume, temperature, etc.) of the mini-bioreactor.

3. In addition to discussing the effects of knockouts on strain growth and biofilm formation, this study also suggests discussing the possible effects of these knockouts on other biological characteristics of the strains (e.g. drug resistance).

4. The graphics of the article need to be further improved. For example, the starting point of the vertical coordinate (0,1) in Fig. 2 is wrong, should it be correctly written as 0.1? Figure 6 could have been presented as a line graph, which would have been more aesthetically pleasing and provided a clearer view of the trend. While the X-axis of Fig. 6 shows the number of cells, what is the meaning of the Y-axis?

5.  Compared with the wild type, this strain has reduced biofilm and foam formation and better growth, so how effective is it in practical production applications?

6. In the conclusion, it is stated that the enhanced growth performance of the mutant strain may be associated with reduced energy burden, please add direct evidence to support this hypothesis.

7. In the paper, only the effect of triple knockout strains is shown, while the effect of single knockout strains is not presented. In addition, the authors mentioned in the Discussion, “We found that cells were able to form biofilms despite the deletion of the algA gene (Fig. 4, Fig. 5); we deleted the pilQ and flhA genes in Pseudomonas malodorata strain PCL1760 , and observed an almost complete inhibition of its cellular motility on semi-liquid agar plates (Fig. 3). These descriptions are based on the effects of three-gene knockouts and lack comparative analysis of the effects of single-gene knockouts and therefore lack rigor.

VIII. It is recommended to supplement the literature with relevant studies published in recent years to ensure that the latest research advances are understood and cited, especially in the areas of mechanisms of biofilm formation and metabolic engineering in bacterial strains.

Author Response

Dear Colleague, We thank you for your review of our manuscript

1. 3.4 The results are not strictly described. The preamble mentions that the genes pilQand flhA are important for biofilm formation, whereas the results only mention that the absence of the gene flhA significantly reduced the migration rate of the strain.

The aim of our work was to obtain the strain suitable for fermentation purposes. We did not construct single mutants ΔflhA, ΔpilQ and double mutant ΔflhA-ΔpilQ, since the study describing influence of these gene deletion, including double mutant, has been already published for Pseudomonas aeruginosa strain (Klausen M, Heydorn A, Ragas P, Lambertsen L, Aaes-Jørgensen A, Molin S, Tolker-Nielsen T. Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol Microbiol. 2003 Jun;48(6):1511-24. doi: 10.1046/j.1365-2958.2003.03525.x. PMID: 12791135.). We have indeed obtained further insights into intermediate mutants by evaluating the biofilm formation capabilities of ΔalgA, ΔalgA ΔflhA, and ΔalgA ΔflhA ΔpilQ, and this data has been added to our manuscript. (see Paragraph 3.5 Lines 269-274)

2. In the 3.6 results section, there is a lack of information about the specifications (volume, temperature, etc.) of the mini-bioreactor.

All specifications for mini-bioreactors are given in the section 2.7 of Materials and Methods.(see lines 191-196)

3. In addition to discussing the effects of knockouts on strain growth and biofilm formation, this study also suggests discussing the possible effects of these knockouts on other biological characteristics of the strains (e.g. drug resistance).

It is well known that biofilms are important for the resistance of the bacterial cells to antibiotics. In fact the process of mutant generation involves the steps with selection on kanamycin and ampicillin where we did not notice any difference in growth of our mutants in comparing to the wild type strain. We also considered that antibiotics are not frequently used for fermentations that is why we did not introduced additional experiments on drug resistance.

4. The graphics of the article need to be further improved. For example, the starting point of the vertical coordinate (0,1) in Fig. 2 is wrong, should it be correctly written as 0.1? Figure 6 could have been presented as a line graph, which would have been more aesthetically pleasing and provided a clearer view of the trend. While the X-axis of Fig. 6 shows the number of cells, what is the meaning of the Y-axis?

The figures 2 and 6 were corrected.

5. Compared with the wild type, this strain has reduced biofilm and foam formation and better growth, so how effective is it in practical production applications?

Our results demonstrate that the biofilm characteristics and foam formation of LN6160 are significantly reduced without compromising growth, which has clear implications for enhancing the efficiency and cost-effectiveness of bioreactors. Although we did not set experiments in big volume bioreactors we believe that

excessive biofilm formation can lead to problems such as clogged bioreactors and higher maintenance costs. Using strains with reduced biofilm production can improve operational stability and decrease downtime, resulting in more efficient production cycles.

6. In the conclusion, it is stated that the enhanced growth performance of the mutant strain may be associated with reduced energy burden, please add direct evidence to support this hypothesis.

We improved our discussion with additional literature data. (lines 355-357)

7. In the paper, only the effect of triple knockout strains is shown, while the effect of single knockout strains is not presented. In addition, the authors mentioned in the Discussion, “We found that cells were able to form biofilms despite the deletion of the algA gene (Fig. 4, Fig. 5); we deleted the pilQ and flhA genes in Pseudomonas malodorata strain PCL1760, and observed an almost complete inhibition of its cellular motility on semi-liquid agar plates (Fig. 3). These descriptions are based on the effects of three-gene knockouts and lack comparative analysis of the effects of single-gene knockouts and therefore lack rigor.

Thank you for your comments we agree that thorough investigation of the impact of all single mutations for biofilm formation and motility could provide more detail information on there abilities of bacterial cells. In the same time there are many publications on influence of deletion of pilQ and flhA genes оn motility and biofilm formation on closely related microorganisms (Klausen M, Heydorn A, Ragas P, Lambertsen L, Aaes-Jørgensen A, Molin S, Tolker-Nielsen T. Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol Microbiol. 2003 Jun;48(6):1511-24. doi: 10.1046/j.1365-2958.2003.03525.x. PMID: 12791135.). We did not see the reason to repeat the research of our colleagues and focused on construction of the strain fitting better with improving fermentation process.

VIII. It is recommended to supplement the literature with relevant studies published in recent years to ensure that the latest research advances are understood and cited, especially in the areas of mechanisms of biofilm formation and metabolic engineering in bacterial strains.

Thank you for your recommendation. We already supplemented our publication with literature about relevant studies published in recent years.

Reviewer 3 Report

Comments and Suggestions for Authors

The article, Construction of the Pseudomonas putida Strain with Attractive Properties for Its Application in Biotechnological Processes, introduces a modified Pseudomonas putida strain (LN6160) developed through targeted deletions of genes (algA, flhA, and pilQ) associated with biofilm formation, flagellar export, and pili assembly. These genetic alterations aim to enhance the strain's performance in biotechnological processes, specifically in bioreactors, by reducing biofilm formation, mobility, and foam production. While this study represents a valuable contribution to microbial biotechnology, particularly for bioreactor applications, certain methodological, theoretical, and practical limitations weaken the impact and comprehensiveness of the findings.

General Comments
The research addresses a critical challenge in biotechnological applications involving microbial cultivation in bioreactors—biofilm and foam formation, as well as unnecessary energy expenditure on bacterial motility structures. The study’s methodology is thorough, with detailed steps for plasmid construction, gene deletion, and comparative growth analyses. The results, which show that LN6160’s biofilm and motility characteristics are significantly reduced without compromising growth, hold clear implications for improving bioreactor efficiency and cost-effectiveness. The potential for using LN6160 in fine chemical production and protein expression systems is also well-stated and aligns with the current industry trend toward bioprocess optimization.

Areas for Improvement
Literature Review and Contextualization: While the article provides a foundational context for biofilm and motility challenges in bioreactors, more discussion on similar strains or previous modifications in Pseudomonas species would enrich the background. Specifically, mentioning studies on biofilm reduction in other industrially relevant bacteria could strengthen the relevance of LN6160’s characteristics. A clearer comparison with other methods (e.g., chemical anti-biofilm agents or mechanical interventions) could contextualize the advantages of the genetic approach more effectively.
Detailed Mechanistic Insights: The article attributes LN6160's enhanced growth rates to the energy savings from reduced biofilm, flagella, and pili formation. However, deeper mechanistic insights into how these deletions affect energy metabolism could enhance the discussion. Including data or literature supporting this metabolic shift would strengthen this conclusion. There is limited exploration of alternative pathways or compensatory mechanisms that could contribute to residual biofilm formation despite algA deletion. A discussion on other extracellular polysaccharides in Pseudomonas could provide a fuller understanding of the biofilm reduction process.
Addressing Potential Limitations: The authors should acknowledge potential limitations associated with the reduced motility and biofilm capabilities. In some biotechnological applications, such as soil bioremediation or certain fermentation processes, biofilm formation or motility may be advantageous. Discussion on the limitations and specific applications where LN6160’s modifications are ideal would provide a balanced view.
Future Directions for Further Optimization: The article could benefit from a section on potential gene targets for additional optimization of LN6160. For instance, further deletions or metabolic pathway modifications could be explored to improve the strain’s productivity for specific substrates or chemical production.

Specific Comments
Table and Figure Clarity: The figures showing biofilm formation and growth curves are clear, though they would benefit from additional annotations that clarify strain differences at various time points.
Statistical Analysis: The study includes appropriate statistical analyses Tukey's test for biofilm formation). However, more information on replicates, variance, and confidence intervals would enhance the reliability of the conclusions drawn. For instance, in comparing growth rates, confidence intervals for optical density measurements would support claims about significant differences.
Grammar and Syntax: The article is well-written overall, though minor grammatical errors and typographical issues were noted in some sections. A final review for language accuracy is recommended.

Author Response

Dear Colleague, We thank you for your review of our manuscript

General Comments
The research addresses a critical challenge in biotechnological applications involving microbial cultivation in bioreactors—biofilm and foam formation, as well as unnecessary energy expenditure on bacterial motility structures. The study’s methodology is thorough, with detailed steps for plasmid construction, gene deletion, and comparative growth analyses. The results, which show that LN6160’s biofilm and motility characteristics are significantly reduced without compromising growth, hold clear implications for improving bioreactor efficiency and cost-effectiveness. The potential for using LN6160 in fine chemical production and protein expression systems is also well-stated and aligns with the current industry trend toward bioprocess optimization.

Areas for Improvement Literature Review and Contextualization: While the article provides a foundational context for biofilm and motility challenges in bioreactors, more discussion on similar strains or previous modifications in Pseudomonas species would enrich the background. Specifically, mentioning studies on biofilm reduction in other industrially relevant bacteria could strengthen the relevance of LN6160’s characteristics. A clearer comparison with other methods (e.g., chemical anti-biofilm agents or mechanical interventions) could contextualize the advantages of the genetic approach more effectively.

We amended the Introduction section with additional information. Lines 37-42 and 78-80.

Detailed Mechanistic Insights: The article attributes LN6160's enhanced growth rates to the energy savings from reduced biofilm, flagella, and pili formation. However, deeper mechanistic insights into how these deletions affect energy metabolism could enhance the discussion. Including data or literature supporting this metabolic shift would strengthen this conclusion. There is limited exploration of alternative pathways or compensatory mechanisms that could contribute to residual biofilm formation despite algA deletion. A discussion on other extracellular polysaccharides in Pseudomonas could provide a fuller understanding of the biofilm reduction process.

We amended the Discussion section with additional information. Lines 305-308, 349-354, 355-357.

Addressing Potential Limitations: The authors should acknowledge potential limitations associated with the reduced motility and biofilm capabilities. In some biotechnological applications, such as soil bioremediation or certain fermentation processes, biofilm formation or motility may be advantageous. Discussion on the limitations and specific applications where LN6160’s modifications are ideal would provide a balanced view.

We are agree, that loss of the motility or decrease in biofilm formation can remarkably decrease efficacy of the mutants in some biotechnological applications, especially when it concerns fitness of the strains in the natural ecological niches. We introduced this view into discussion.

Future Directions for Further Optimization: The article could benefit from a section on potential gene targets for additional optimization of LN6160. For instance, further deletions or metabolic pathway modifications could be explored to improve the strain’s productivity for specific substrates or chemical production.

We are indeed planning further improvement of this strain for the fermentation, which we reflect in modified Conclusion sections.

We are going to continue with improving of our “platform” strain 1760 with the attention to energy saving mechanisms of the cell, on which we give a hint in conclusions.

Specific Comments
Table and Figure Clarity: The figures showing biofilm formation and growth curves are clear, though they would benefit from additional annotations that clarify strain differences at various time points

Statistical Analysis: The study includes appropriate statistical analyses Tukey's test for biofilm formation). However, more information on replicates, variance, and confidence intervals would enhance the reliability of the conclusions drawn. For instance, in comparing growth rates, confidence intervals for optical density measurements would support claims about significant differences.

Grammar and Syntax: The article is well-written overall, though minor grammatical errors and typographical issues were noted in some sections. A final review for language accuracy is recommended.

Thank you very much for your recommendation to our work. We performed correction of the text accordingly your comments.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

I would like to thank the authors for their considerable efforts in addressing the provided comments and improving the quality of the article to meet the journal's requirements for publication. The thoughtful revisions have significantly enhanced the study’s scientific rigor and practical relevance. These improvements reflect the authors' dedication to advancing the field of microbial biotechnology while ensuring the research aligns with the high standards expected by the journal. Their responsiveness to feedback has resulted in a well-rounded and impactful article that is now suitable for publication.