Thermal Endurance by a Hot-Spring-Dwelling Phylogenetic Relative of the Mesophilic Paracoccus

ABSTRACT High temperature growth/survival was revealed in a phylogenetic relative (SMMA_5) of the mesophilic Paracoccus isolated from the 78 to 85°C water of a Trans-Himalayan sulfur-borax spring. After 12 h at 50°C, or 45 min at 70°C, in mineral salts thiosulfate (MST) medium, SMMA_5 retained ~2% colony forming units (CFUs), whereas comparator Paracoccus had 1.5% and 0% CFU left at 50°C and 70°C, respectively. After 12 h at 50°C, the thermally conditioned sibling SMMA_5_TC exhibited an ~1.5 time increase in CFU count; after 45 min at 70°C, SMMA_5_TC had 7% of the initial CFU count. 1,000-times diluted Reasoner’s 2A medium, and MST supplemented with lithium, boron, or glycine-betaine, supported higher CFU-retention/CFU-growth than MST. Furthermore, with or without lithium/boron/glycine-betaine, a higher percentage of cells always remained metabolically active, compared with what percentage formed single colonies. SMMA_5, compared with other Paracoccus, contained 335 unique genes: of these, 186 encoded hypothetical proteins, and 83 belonged to orthology groups, which again corresponded mostly to DNA replication/recombination/repair, transcription, secondary metabolism, and inorganic ion transport/metabolism. The SMMA_5 genome was relatively enriched in cell wall/membrane/envelope biogenesis, and amino acid metabolism. SMMA_5 and SMMA_5_TC mutually possessed 43 nucleotide polymorphisms, of which 18 were in protein-coding genes with 13 nonsynonymous and seven radical amino acid replacements. Such biochemical and biophysical mechanisms could be involved in thermal stress mitigation which streamline the cells’ energy and resources toward system-maintenance and macromolecule-stabilization, thereby relinquishing cell-division for cell-viability. Thermal conditioning apparently helped inherit those potential metabolic states which are crucial for cell-system maintenance, while environmental solutes augmented the indigenous stability-conferring mechanisms. IMPORTANCE For a holistic understanding of microbial life’s high-temperature adaptation, it is imperative to explore the biology of the phylogenetic relatives of mesophilic bacteria which get stochastically introduced to geographically and geologically diverse hot spring systems by local geodynamic forces. Here, in vitro endurance of high heat up to the extent of growth under special (habitat-inspired) conditions was discovered in a hot-spring-dwelling phylogenetic relative of the mesophilic Paracoccus species. Thermal conditioning, extreme oligotrophy, metabolic deceleration, presence of certain habitat-specific inorganic/organic solutes, and potential genomic specializations were found to be the major enablers of this conditional (acquired) thermophilicity. Feasibility of such phenomena across the taxonomic spectrum can well be paradigm changing for the established scopes of microbial adaptation to the physicochemical extremes. Applications of conditional thermophilicity in microbial process biotechnology may be far reaching and multifaceted.

/ complete genomes), there is a lot of excellent data presented and I think researchers who think about thermal adaptation will be interested in the work.
There are a couple references to memory / memorize when the authors actually mean evolution / adaptation / genetic change (e.g. lines 41 and 365). I don't think this anthropomorphism is particularly useful.
It is unclear from the data presented in Figure 2 if SMMA_5_TC is actually capable of growth at 50C or if the strain survives this condition better than the parent strain (SMMA_5). The selection regime shown in Figure 4A doesn't necessarily require that the strain grow at 50C, but rather survive for 12 hours prior to shifting down to 37C. The authors are clear in the manuscript and do not outright claim to have shown growth. A growth experiment could clear this up, but I don't think is required.
Comparisons of these strains is hampered by the incomplete genomes generated by short Illumina reads. For future work the authors should consider coupling some low-cost long-read sequencing (such as Nanopore) to combine with their short-read sequences to generate finished genomes.
Specific comments for consideration.
Line 146 -perhaps mention where LMG 4218 was isolated from for comparison purposes?
Line 188 -please rephrase this. I think I know what you mean, but the heading is awkward.
Line 208 -is this release due to cell lysis?
Line 505 -this section is quite speculative and does not add meaningfully to the manuscript, in my opinion.
Line 531 -does addition of Li lead to 'metabolic decelaration' as well? Might this account for the enhanced thermal tolerance when the medium was amended with Li?
Line 566 -was the SMMA_5_TC variant also deposited in the culture collection? Line 617 -please clarify the medium names here as 4 conditions are given but only 3 names are provided.

Reviewer comments:
Reviewer #2 (Comments for the Author): Title: Thermal endurance by a hot-spring-dwelling phylogenetic relative of the mesophilic Paracoccus Summary: A Paracoccus strain isolated from high-temperature environments was studied. Cell viability was examined after incubation at varying temperatures. The genome was sequenced. Comments: The authors may have isolated an interesting member of the genus Paracoccus. There are cases in which phylogenetically related organisms display significantly different growth temperatures, and this study may demonstrate such a case in the Paracoccus genus. However, some basic biochemical properties are missing, and should be provided. Although the authors focus on temperature endurance, a growth temperature analysis at varying temperatures is essential to understand the newly isolated strain and interpret the results of experiments on temperature endurance. Specific comments 1. Cells of the newly isolated strain(s), along with a conventional Paracoccus strain, should be grown at 37,40,45,50,55,60,65 degrees in liquid culture. The specific growth rates must be calculated and compared. 2. Searching for links between metabolism and thermal endurance may be interesting, but the metabolism should be described in a more specific manner. The authors should distinguish between catabolism and anabolism for metabolism such as for amino acids, and compare the features with conventional Paracoccus strains with genome sequences. The connection to increased/decreased thermal endurance should be explained (eg. Some amino acids might act as compatible solutes. Are their biosynthesis pathways strengthened?). 3. The number and types of heat shock proteins and the chaperone proteins should be compared with those from conventional Paracoccus strains. There may be an increase compared to conventional Paracoccus strains. 4. As suggested in the discussion, the authors should compare the number and types of ion-channels in SMMA_5 with those from conventional Paracoccus strains. The authors could also include ABC transporters. 5. There seems to be discussion on aspects of cell growth, such as DNA replication, with temperature in the discussion. This makes the growth experiments (specific growth rate analyses) indicated in comment 1 all the more important.

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Response to the comments of Editor / Reviewer #1
Comment 1: The manuscript by Mondal et al. seeks to understand and explore thermal endurance of a Paracoccus strain (SMMA_5) isolated from a hot spring. The authors evolve a variant of SMMA_5 through a regime of high-temperature (50C) growth conditions, yielding a strain denoted as SMMA_5_TC. The authors have done a large amount of work to determine thermal endurance characteristics of these strains, with comparisons to a mesophilic Paracoccus isolate and a lab strain of E. coli. Partial genomes were also generated for both strains and some differences noted by the authors. The authors describe the differences between the strains appropriately as potential mechanisms for enhancing survival at 50C, but these hypotheses were not experimentally validated. While there are some additional experiments that would help the authors tell a clearer story (growth vs survival at 50C complete genomes), there is a lot of excellent data presented and I think researchers who think about thermal adaptation will be interested in the work. RESPONSE: We thank you for these comments, and believe you appreciated the underlying science of this study. We also fully agree with your concerns, so have now dealt with each one of them in course of this revision.
Comment 2: There are a couple references to memory memorize when the authors actually mean evolution adaptation genetic change (e.g. lines 41 and 365). I don't think this anthropomorphism is particularly useful.

RESPONSE:
We agree that the current paper does not have any direct data to substantiate such remarks in the context of the evolution / adaptation of the hot spring Paracoccus, so we have now removed the words memory, memorize, etc. from wherever it had been used previously with reference to SMMA_5 or SMMA_5_TC (please see the changes made in lines 47, 414, 444 and 673 of the Track Changes file).
However, in the discussion, we have retained the word memory for those comparator cases (lines 422 and 425 of the Track Changes file) where similar phenomena reported for other organisms have been referred to as bacterial memory in the concerned papers. Notably, most of the papers cited for those comparator instances (references numbered as 33, 34, 36, and 37  Comment 3: It is unclear from the data presented in Figure 2 if SMMA_5_TC is actually capable of growth at 50C or if the strain survives this condition better than the parent strain (SMMA_5). The selection regime shown in Figure 4A doesn't necessarily require that the strain grow at 50C, but rather survive for 12 hours prior to shifting down to 37C. The authors are clear in the manuscript and do not outright claim to have shown growth. A growth experiment could clear this up, but I don't think is required. RESPONSE: We thank you for appreciating the clarity of our data presentation details.
We also agree with your point that a growth experiment could resolve the actual extent of cell division in SMMA_5_TC at 50°C.
But, as you have aptly noted at the end of your comment, such data are better kept out from the purview of the current manuscript because the paper is already loaded with huge data sets of physiology, and once we present a full-fledged growth curve (kinetic data) for SMMA_5_TC, to put things into their right perspective, we shall have to present corresponding data for SMMA_5 as well as all the comparator strains (that too under the different media and temperature conditions which are already in question).
Having said that, we are indeed happy to convey that the extensive (multiconditional) transcriptome analyses, which we are already carrying out for SMMA_5 and SMMA_5_TC, begin with in-depth growth experiments under different media and temperature conditions. Comment 4: Comparisons of these strains is hampered by the incomplete genomes generated by short Illumina reads. For future work the authors should consider coupling some low-cost long-read sequencing (such as Nanopore) to combine with their short-read sequences to generate finished genomes. RESPONSE: We agree, and have already progressed considerably on the Phase II study involving genome completion (indeed by combining long ONT reads with short Illumina reads), followed by extensive transcriptome analysis for SMMA_5 and SMMA_5_TC (we earnestly plan to communicate that paper to this journal very soon). So far as the present paper is concerned, the two, approximately 98.5% complete, shotgun genomes are sufficiently reliable for the limited molecular biological conclusions that have been currently drawn (off the records, the ONT-completed genomes added ~30 kb sequence, i.e. only 20-25 genes, to the existing genomes). RESPONSE: We agree, so have now fixed the problem by removing the subjective words "cell viability" and "divisibility" from the heading, and replacing them by the data-centric terminologies "proportion of metabolically active cells" and "proportion of CFUs" (please see lines 207-208 of the Track Changes file).

RESPONSE:
The data presented in the Results sections just above this line showed that the cells of both SMMA_5 and SMMA_5_TC remain in a metabolically active, but non-dividing or hardly dividing, state after 12 h incubation in MST at 50°C. Under this condition, therefore, the cells are healthy, and capable of synthesizing and transporting essential biomolecules. In order to provide a subtle clarification on this issue we have now added a small clause to the existing sentence on glycine-betaine release by SMMA_5 and SMMA_5_TC to the spent medium (please see lines 228-

of the Track Changes file).
Comment 4 Line 505: this section is quite speculative and does not add meaningfully to the manuscript, in my opinion.
RESPONSE: We agree that the discussion pertaining to the predicted pI values of all putative proteins of the different Paracoccus species (including SMMA_5) is largely theoretical. We have now edited the text to contextualize the issue in a more straight forward and logical way (please see lines 612-616 and 624-643 of the Track

Changes file).
As for the relevance and necessity of this section, the following points are noteworthy.
It has been shown previously that at equivalent temperature levels, hot spring waters having neutral to moderately alkaline pH generally harbor greater microbial diversities than their acidic counterparts. In view of this, and also in consideration of the circum-neutral pH (diurnal range: 7.2-8.0) of the Lotus Pond vent-water (the habitat of the test organism), we investigated the effects of pH 7.0-9.0 on the high temperature growth/survival of SMMA_5.
This point was missing from the previous manuscript, so we have now added this justification to the Introduction (please see lines 113-117 of the Track Changes file).
Again, from a molecular perspective, pH of the chemical milieu is a key biophysical parameter for protein stability, and thereby overall microbial adaptation to different temperature regimes. Consequently, it was essential for the present study to investigate the pH optima for thermal endurance by the hot spring Paracoccus: we did so and found that pH 7.5 was best for growth at 37°C and CFU-retention at 50°C and 70°C. As an aftermath of these data, the biophysical discussions centered on pI values of proteins became necessary.

In the previous manuscript, this contextual explanation was missing from the section of the Discussion in question; we have now added it (please see lines 612-616 of the Track Changes file).
Additionally, during the current revision, this Discussion section on biophysical adaptations also turned out to be very useful in accommodating the fresh critical discourse (please see lines 581-611 of the Track Changes file) emanating from the new analyses pertaining to amino acid frequencies in Paracoccus proteomes, which in turn were warranted by Reviewer 2.
Comment 5 Line 531: does addition of Li lead to 'metabolic decelaration' as well Might this account for the enhanced thermal tolerance when the medium was amended with Li RESPONSE: We agree with your postulation, but the data available at this point of time does not allow us to be very specific about lithium; or for that matter any of three solutes which enhanced thermal tolerance.
Based on the results in hand, only this much can be inferred safely: metabolic deceleration is intrinsic to the SMMA_5 strategy for thermal endurance, and the environmental solutes plausibly augment that intrinsic metabolic plan.
The whole issue was discussed in the section starting at line 531 of the previous manuscript (now, line number 645 of Track Changes file).
Comment 6 Line 566: was the SMMA_5_TC variant also deposited in the culture collection RESPONSE: No, SMMA_5_TC has not yet been deposited to any culture collection center (CCC). The main issue here is that CCCs generally maintain strains via lyophilization and long-term storage, with viability checks and renewals of the biomass at long intervals. But to maintain the characteristic features of the strain SMMA_5_TC the culture needs to be transferred every 14 days (via incubation at 50°C for 12 h and then at 37°C for another 12 h) and stored as a running culture at 25°C; we are in talks with a few CCCs to figure out whether any one of them agrees to undertake this tedious transfer regime. Line number 745-751 (of the Track Changes file) goes on to add "Growth or decline in the CFU-count of SMMA_5 and SMMA_5_TC at different temperatures was further tested in MST supplemented with 4 mM Na 2 B 4 O 7 .10H 2 O (i.e., 16 mM B), 1 mM LiOH.H 2 O (i.e., 1 mM Li) and/or 10 mM glycine-betaine or N,N,N trimethylglycine (C 5 H 11 NO 2 ); the three solute-fortified variants of MST were referred to as MSTB, MSTL and MSTG respectively."

Response to the comments of Reviewer #2
Title Thermal endurance by a hot-spring-dwelling phylogenetic relative of the mesophilic Paracoccus Summary A Paracoccus strain isolated from high-temperature environments was studied. Cell viability was examined after incubation at varying temperatures. The genome was sequenced. Comments The authors may have isolated an interesting member of the genus Paracoccus. There are cases in which phylogenetically related organisms display significantly different growth temperatures, and this study may demonstrate such a case in the Paracoccus genus. However, some basic biochemical properties are missing, and should be provided. Although the authors focus on temperature endurance, a growth temperature analysis at varying temperatures is essential to understand the newly isolated strain and interpret the results of experiments on temperature endurance.

RESPONSE:
We thank the Reviewer for appreciating the phenomenology underlying the present study, and helping improve the manuscript technically and in terms of interpretations. We have taken positive actions with respect to all your suggestions and each of them are narrated below.
As warranted by you, we have now carried out a growth analysis at varying temperatures for the newly isolated strain SMMA_5 alongside the conventional Paracoccus strain LMG 4218 and E. coli K-12. We also valued your suggestion pertaining to the delineation of basic biochemical properties for the new isolate, as that would help us know the new strain better in the context of its close phylogenetic relatives. However, we just stopped short of presenting a comparative dataset of biochemical attributes in this paper because such a dataset (which holds major implications for the exact taxonomic identification of the strain) might just shift the focus aside from the thermal endurance aspect which is central to the current narrative (we would surely present these information in a subsequent paper of taxonomy in Int J Syst Evol Microbiol).

Specific comments
Comment 1: Cells of the newly isolated strain(s), along with a conventional Paracoccus strain, should be grown at 37,40,45,50,55,60,65 degrees in liquid culture. The specific growth rates must be calculated and compared.

RESPONSE:
We agree: the organisms have now been grown at 37°C, 40°C and 45°C, and their specific growth rates calculated at these temperatures. The newly incorporated data, discussions and methods which pertain to this aspect can be seen in lines 154-173, 646-653 and 734-739

of the Track Changes file.
A comparison between the generation times of SMMA_5 and P. pantotrophus LMG 4218, recorded at 37°C, 40°C and 45°C (in MST medium), indeed showed that with increasing temperature growth decelerated in both the strains, but the slowdown was less in SMMA_5, as compared to LMG 4218. Albeit no growth was observed for either strain at 50°C under the routine culture conditions, a small increase in CFUcount, reflective of extremely slow growth rate, was observed for the hot spring Paracoccus alone after certain habitat-inspired manipulations were rendered in the strain's maintenance history and/or the chemical composition of the culture media.
Comment 2: Searching for links between metabolism and thermal endurance may be interesting, but the metabolism should be described in a more specific manner. The authors should distinguish between catabolism and anabolism for metabolism such as for amino acids, and compare the features with conventional Paracoccus strains with genome sequences. The connection to increased decreased thermal endurance should be explained (eg. Some amino acids might act as compatible solutes. Are their biosynthesis pathways strengthened). RESPONSE: We agree, and have now added the precise identities the unique SMMA_5 genes for the enriched COG categories (i) amino acid transport and metabolism, (ii) cell wall/membrane/envelope biogenesis, and (iii) lipid transport and metabolism, in relation to -all 44 Paracoccus species having near-complete genome sequences available, as well as -the 14 phylogenomically closest relatives. The newly incorporated data and discussions which pertain to this aspect can be seen in lines 338-347 and 539-546 of the Track Changes file, alongside the new Table S5.
Furthermore, we have also analyzed the relative abundances of the 20 individual amino acids (expressed as the percentage of all amino acids present) across the putative (in silico translated) proteomes of SMMA_5 and its 14 closest phylogenomic relatives (new data and discussions pertaining to this aspect can be seen in lines 347-361 and 581-611 of the Track Changes file, alongside the new Table S10).

Comment 3:
The number and types of heat shock proteins and the chaperone proteins should be compared with those from conventional Paracoccus strains. There may be an increase compared to conventional Paracoccus strains.

RESPONSE:
We agree, and have now worked more on this. A comparison of SMMA_5 with a few of its phylogenomically closest relatives, in relation to the types and numbers of their genes for heat shock proteins and chaperones, was already there in the previous manuscript (previous Table S5); in the revised manuscript we have expanded that analysis by including all the 14 Paracoccus species that occupied the same clade as SMMA_5 in the phylogenomic tree. The updated comprehensive data can be found in the new Table S6, while corresponding changes to the text can be seen in lines 374-382 and 537-546 of the Track Changes file.
Comment 4: As suggested in the discussion, the authors should compare the number and types of ion-channels in SMMA_5 with those from conventional Paracoccus strains. The authors could also include ABC transporters. RESPONSE: We agree, and have now compared the number and types of genes for channel proteins and porins / aquaporins; ABC transport systems; cobalt, copper, magnesium and/or nickel transporters; uni-/ sym-/ antiporters; ECF class transporters; Ton and Tol transporters; and TRAP transporters in SMMA_5 with those present in the 14 phylogenomically closest Paracoccus species.
The new data and discussions pertaining to this aspect can be seen in lines 428-437 of the Track Changes file, alongside the new Table S9.
Comment 5: There seems to be discussion on aspects of cell growth, such as DNA replication, with temperature in the discussion. This makes the growth experiments (specific growth rate analyses) indicated in comment 1 all the more important.

RESPONSE:
We agree, so have now grown SMMA_5 alongside the conventional Paracoccus strain LMG 4218 (and also E. coli strain K-12) at different temperatures and calculated their specific growth rates at those temperatures. As already mentioned in our response to your Comment 1, these new experiments, data and discussions (incorporated in lines 154-173, 646-653 and 734-739 of the Track Changes file) have indeed substantiated our inferences on metabolic deceleration as central to thermal endurance.