The resuscitation-promoting factor (Rpf) from Micrococcus luteus and its putative reaction product 1,6-anhydro-MurNAc increase culturability of environmental bacteria

  1. Juan Guzman
  2. Dipansi Raval
  3. Dirk Hauck
  4. Alexander Titz
  5. Anja Poehlein
  6. Thomas Degenkolb
  7. Rolf Daniel
  8. Andreas Vilcinskas

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Abstract

Bacterial dormant cells do not divide and are not immediately culturable, but they persist in a state of low metabolic activity, a physiological state having clinical relevance, for instance in latent tuberculosis. Resuscitation promoting factors (Rpf) are proteins that act as signaling molecules mediating growth and replication. In this study we aimed to test the effect of Rpf  from Micrococcus luteus on the number and diversity of cultured bacteria using insect and soil samples, and to examine if the increase in culturability could be reproduced with the putative reaction product of Rpf, 1,6-anhydro-MurNAc. The rpf gene from M. luteus was amplified and cloned into a pET21b expression vector and the protein was expressed in E. coli BL21(DE3) cells and purified by affinity chromatography using a hexa-histidine tag. 1,6-anhydro-N-acetylmuramic acid (1,6-anhydro-MurNAc) was prepared using reported chemical synthesis methods. Recombinant Rpf protein or 1,6-anhydro-MurNAc were added to R2A cultivation media, and their effect examined on the culturability of bacteria from eight environmental samples including four cockroach guts and four soils. Colony forming units, 16S rRNA gene copies and Illumina amplicon sequencing of the 16S rRNA gene were measured for all eight samples subjected to three different treatments: Rpf, 1,6-anhydro-MurNAc or blank control. Both Rpf and 1,6-anhydro-MurNAc increased the number of colony forming units, and 16S rRNA gene copies across the samples although the protein was more effective. The Rpf and 1,6-anhydro-MurNAc promoted the cultivation of a diverse set of bacteria and specially certain clades of the phyla Actinomycetota and Bacillota. This study opens the path for improved cultivation strategies aiming to isolate and study yet undescribed living bacterial organisms.

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  1. Access Microbiology

    The work presented is clear and the arguments well formed. 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. You have not addressed the reviewers’ comments sufficiently after revision.

  2. Version published to 10.1099/acmi.0.000647.v3 on Access Microbiology
  3. Version published to 10.1099/acmi.0.000647.v4 on Access Microbiology
  4. Access Microbiology

    You have not addressed the reviewers’ comments sufficiently after revision. There are certain references that are missing such as Lane 1991, Klindworth et al 2013. Can the authors please add these and verify that other references have been included in the list. Line 163: Cockroach samples? Please clarify which samples (e.g. gut suspension) are authors referring to. Line 212-218: This section is a repetition of line 127 and can be removed. Line 255-265 : This section deals with species richness. However, no plots have been provided to assess ASV detected in each samples. In addition, Shannon and inverted Simpson indexes are described later without showing the data…”. Please elaborate on this section and provided respective plots to support ASV numbers. Line 279: The authors elaborated on the increase in abundance of certain specific phyla on addition of Rpf but missed describing how the addition of Rpf “decrease” the abundance of certain phyla e.g. Actinomycetota in soil 2 and soil 4, Pseudomonadota in cockroach LV. Can this be described here in this section. This is relevant as it affects the discussion in Line 355-358. The authors describe this as “not observe a sustained increase”. Can this be described as a “decrease” instead? Line 335: Please give full form of LPSN.

  5. Version published to 10.1099/acmi.0.000647.v2 on Access Microbiology
  6. Access Microbiology

    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. The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments. Please provide more detail in the Methods section and ensure that software is consistently cited and its version and parameters included.

  7. Access Microbiology

    Comments to Author

    The ACMI-D-23-00105 described the path for improved cultivation strategies by addition of recombinant M. luteus Rpf and 1,6-anhydro-MurNAc aiming to isolate and study yet undescribed living bacterial organisms, which was of great significance for the isolation of hard-to-culture microbial species. However, some revision are needed. 1.When bacteria were isolated from eight environmental samples, how many repetitions were performed for different treatments, including the addition of Rpf and 1, 6-Anhydro-murnac, as well as controls? It should be explained clearly in the text because this directly affected the accuracy of the conclusion. Moreover, there should be error bars in Figure 1cd. 2. The authors simply analyzed CFU and the copies of 16S rRNA gene at different treatments, as well as changes in phyla and order level species diversity from Amplicon sequencing results. For culturable species diversity, why only focus on the analysis of strains G173LV and G177LV? 3. It is suggested that the changes of culturable species diversity after the addition of Rpf and 1, 6-Anhydro-murnac should be analyzed emphatically. 4. The strain Streptomyces sp. G177LV showed high similarity(identity 99.71%) to the type species Streptomyces termitum NBRC 13087T. What is the basis for the author to suggest that G177LV is a new species of the genus Gordonia? In general, 98.65% similarity of 16S rRNA, 70% DDH value and 95% ANI value are used as the threshold for recognizing novel species. Please refer to PMID: 24505072. 5. Which pET expression vector was used should specify in the abstract. 6.All Latin species names of bacteria should be italicized. When the species name appears for the first time, it should indicate the genus name and the species addition, and when it appears again, the genus name can be abbreviated. For example, the full name of Micrococcus luteus is written in italics when it first appears, and can be shortened to M. luteus when it appears again. 7. Why was the pH of the medium adjusted to 8.8 when isolating bacteria? Some groups that are not tolerant to high pH may be overlooked.

    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?

    Yes

  8. Access Microbiology

    Comments to Author

    This interesting article presents convincing arguments that adding Rpf or 1-6-anhydro-N-acetyl-muramic acid to cultivation media can help culture new bacteria or resuscitate old cultures. It also opens the field for future research on the potential antibiotic resistance in cockroaches and the identification of new species. This is a significant work, and I strongly support its publication in Access Microbiology. Overall, the findings are well presented and have been analysed well in the discussion. Some issues regarding methodological reproducibility can be found in the article - I have raised these in my comments below. Please find my comments below. Line 111 - How were the soil samples collected? More details are necessary for replication. Lines 114-115 - I am unsure what "the top layer of every sample" means. Do you mean the supernatant? And does this include the cockroach samples as well or just the soils? Also, by "clean tube" do you mean 1.5 mL tubes? A few extra details are necessary here to fully understand your methodology. Line 118 - CASO broth, the full name of CASO is given in lines 172-173. The full name should be brought to line 118, where CASO broth is first mentioned. Line 119 - How was the rpf gene from M. luteus amplified? What were the reagents and PCR conditions? More details are necessary for replication. Line 120 - How was the vector linearised? More details are necessary for replication. Line 128 - How much LB broth was used for the overnight culture? Line 132 - How was the amplicon purified? Did you use a kit? Did you cut it from the agarose gel? More details are necessary for replication. Line 181 - Falcon is the name of the company. I recommend changing to "centrifuge tube" instead. Line 192 - Illumina instead of llumina Line 196 - Silva 138. Which SILVA 138? Was it SSU or LSU? Was it Ref N99 or just Ref? More details are necessary for replication. Lines 219-220 and 308-314 - I am curious about cockroach-AT and soil-2. It's interesting how different they are from the other samples. You discuss this briefly but I think it needs expanding. Was there anything different with those particular samples? Line 225 - qPCR. Reaction efficiency and sample replication numbers are not mentioned. Please add. Also, the actual copy numbers need to be added somewhere in the text. Line 244 - How many sequences and ASVs did you get in total? Knowing this would put the individual results into perspective. Line 253 - Was the higher species richness observed significant? No statistics are mentioned anywhere - were these carried out? Line 273 - "The increment in abundance…" should be "in relative abundance" Lines 279 and 285 - "at the expense of" This needs rephrasing because it implies the two taxa are correlated. Line 280 - Why are the results "unexpected"? Line 282 - I find it very interesting the soils vary so much - was there anything different between the sampling sites? Maybe this could be added to the discussion? Line 289 - Discussion. Did you have a treatment where you added both Rpf and 1,6-anhydro-MurNAc? It would be interesting to see what would grow.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Very 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

  9. Version published to 10.1099/acmi.0.000647.v1 on Access Microbiology