Novel enzyme for dimethyl sulfide-releasing in bacteria reveals a missing route in the marine sulfur cycle
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Abstract
Dimethylsulfoniopropionate (DMSP) is an abundant and ubiquitous organosulfur molecule and plays important roles in the global sulfur cycle. Cleavage of DMSP produces volatile dimethyl sulfide (DMS), which has impacts on the global climate. Multiple pathways for DMSP catabolism have been identified. Here we identified yet another novel pathway, the ATP DMSP lysis pathway. The key enzyme, AcoD, is an ATP-dependent DMSP lyase. AcoD belongs to the acyl-CoA synthetase superfamily, which is totally different from other DMSP lyases, showing a new evolution route. AcoD catalyses the conversion of DMSP to DMS by a two-step reaction: the ligation of DMSP with CoA to form the intermediate DMSP-CoA, which is then cleaved to DMS and acryloyl-CoA. The novel catalytic mechanism was elucidated by structural and biochemical analyses. AcoD is widely distributed in many bacterial lineages including Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Firmicutes, revealing this new pathway plays important roles in global DMSP/DMS cycles.
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This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on December 1 2020, follows.
Summary
The study isolates bacteria from diverse Antarctic samples which utilise DMSP as the sole carbon source. It initially focuses on a Gammaproteobacterium, Psychrobacter sp.D2, which the authors establish lacks a known DMSP lyase enzyme despite having DMSP lyase activity (this needs to be quantified). Through RNA-seq and bioinformatics, they identify the gene cluster responsible for this activity and identify a novel DMSP lyase somewhat related to DddD in that it involves CoA, but critically also ATP, which distinguishes it from the pack of other known Ddd enzymes. This enzyme is a ATP-dependent DMSP CoA synthase required for growth on DMSP and its transcription is upregulated by DMSP availability. The …
This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on December 1 2020, follows.
Summary
The study isolates bacteria from diverse Antarctic samples which utilise DMSP as the sole carbon source. It initially focuses on a Gammaproteobacterium, Psychrobacter sp.D2, which the authors establish lacks a known DMSP lyase enzyme despite having DMSP lyase activity (this needs to be quantified). Through RNA-seq and bioinformatics, they identify the gene cluster responsible for this activity and identify a novel DMSP lyase somewhat related to DddD in that it involves CoA, but critically also ATP, which distinguishes it from the pack of other known Ddd enzymes. This enzyme is a ATP-dependent DMSP CoA synthase required for growth on DMSP and its transcription is upregulated by DMSP availability. The novel mechanism of this enzyme is proposed from a strong structural component to the study. The authors propose the downstream pathway for DMSP catabolism, which we find to be oversold and requiring gene mutagenesis to confirm, and to be preliminary in comparison with the authors' other findings. Finally, the study attempts to show how widespread the enzyme is in sequenced bacteria, confidently showing it to be functional in other related Gammaproteobacteria and some Firmicutes.
Essential Revisions
Title: "a missing route", was it really missing? We would suggest a more precise title. Would be better to say "that releases DMS" or an alternative.
This is a Ddd enzyme by definition and should be named as such.
Line 27- We disagree with the use of a new gene prefix when there is a strong precedent for the use of Ddd for "DMSP-dependent DMS". If this enzyme is a DMSP lyase and is in bacteria then its naming should follow protocol and be called Ddd"X"-X-being a letter not currently utilised in known systems. Deviating from this convention causes confusion and is not appropriate. Furthermore, AcoD is already assigned in some bacteria to acetaldehyde dehydrogenase II.
- As presented, the bioinformatics-based evidence regarding the broad distribution of this enzyme (as claimed e.g. in the Abstract, line 33) does not stand up. Currently as presented in the manuscript, especially Fig 6, we are led to believe the enzyme is more widespread than can be demonstrated based on the authors' evidence (i.e., the authors allow a very low threshold of sequence identity and claim function outside of the groups they have tested). Either more work is needed to show that claims of such a wide distribution are merited, or the authors should limit their claims to what can be substantiated by their work. Specifically, the authors cannot comment on the "functional" enzyme being widespread outside of the Gamma's and Firmicutes that were tested, let alone the importance of the role in DMSP cycling. Only three "AcoD" enzymes were ratified in this study, which are relatively closely related to each (Psychrobacter sp. D2 Sporosarcina sp. P33 and Psychrobacter sp. P11G5 that are > 77% identical to each other). As can be seen in Fig 6, these three proteins cluster together and are far removed from all the other sequences on the figure, for which we have no evidence of their function (i.e., nothing can realistically be said on Deltas, Actinos or Alphas or the MAGS). Just to be clear, these other proteins shown in clades above and below the functional "AcoDs" in fig 6 are only ~30% identical to ratified "AcoD". Furthermore, only strain D2 was shown to make DMS; none of the other strains were tested. Far more testing of the diverse enzymes and strains are needed to make these statements as this study only tests one strain and three of the closely related enzymes (defined on Fig 6). Additional specific comments on this issue:
Line 280. The sentence on MAGS and the environments containing them does not stand up for reasons summarised above. All MAGS shown on Fig 6 are not similar enough to "AcoD" to be termed as functional Ddd enzymes. More work has to be done on the strains and enzymes that are more divergent to true "AcoDs" before such a statement is supported. Please delete. Line 509-We agree with what the authors write about stringency. However, these parameters do not seem to have been utilised as stated here. Their stringency statement holds up for comparison between the D2 "AcoD" and two other tested "AcoD" enzymes and all those in the middle clade on Fig.6. But this is not the case for the proteins shown above and below this "AcoD" clade in Fig 6 which have at best around 30% identity to characterised enzymes. See below for examples. As the authors state in their methods, high-stringency methods are needed to exclude other acetyl-CoA synthetase family proteins. Thus, most of the genes shown on fig6 cannot be taken as having this Ddd activity.
"To further validate that these AcoD homologs" the authors examined the activity of two closely related enzymes from a group of nine homologs with > 65 % sequence identity (starting line 283, Figure 6). It is not surprising that these enzymes have the same activity. Homologs outside this group of nine (Figure 6) are far less related to the characterized AcoD (< 32 % seq. identity). Conservation of the phosphate-transferring His (His292) and an active site Trp (Trp391) does not seem to be strong evidence for functional conservation. The manuscript does not provide any additional evidence that these less related enzymes also degrade DMSP. Either more experimentation is necessary, or the paragraph on the "Distribution of the ATP DMSP lysis pathway in bacteria" must be revised.
For example: Psychrobacter AcoD (WP_068035783.1) is 31% identical to Bilophila sp. 4_1_30 (WP_009381183.1) in the below group of bacteria on Fig 6. Psychrobacter AcoD (WP_068035783.1) is 29% identical to Thermomicrobium roseum (WP_041435830.1) in the above group of bacteria on Fig 6. Line 283. This is not the case! The two sequences that were chosen to "validate" are far to close to the D2 "AcoD" than to MAGS and other potential "AcoDs" shown above and below the functional Ddd clade on Fig 6. This section design is weak and does not lend weight to the expansiveness of this family. More work on the more diverse enzymes and bacteria is needed to support the authors claims. Please delete or study the activity of the more diverse strains and their candidate "AcoDs". Fig. 6. This is a nicely presented figure that unfortunately slightly deceives the reader. The authors need to clearly show which strains they have shown to have Ddd activity (currently one as I understand it) and which enzymes they have shown to have the appropriate activity (currently three closely related enzymes as I understand it). If I am not wrong these are all confined to the middle clade of Gammas and Firmicutes. These stand clearly apart form the other strains (above and below) which have not been studied and which are only ~ 30% Identical to "AcoD" at the protein level. This is not clear on the figure and definitely misleads in the abstract and throughout the manuscript.
- We expect to see kinetics done on the new enzyme in line with what the authors have done in other related studies on Ddd and Dmd enzymes.
This is important to place the work in context with previously identified Ddd and Dmd enzymes, many of which have been analysed by these authors in previous publications. The characterization of the AcoD activity remains entirely qualitative. The authors only provide relative activities measured at a single substrate concentration. This data does not support the following statement: "Mutations of these two residues significantly decreased the enzymatic activities of AcoD, suggesting that these residues play important roles in stabilizing the DMSP-CoA intermediate" (l.223-225).
- The manuscript does provide unambiguous evidence for the activity of AcoD and its function during growth on DMSP. On the other hand, the description of the "ATP DMSP lysis pathway" is less clear.
Transcriptomics analysis (Figure 2C) suggest that growth on DMSP upregulate the genes 1696 (BCCT), 1697 (AcoD), 1698 and 1699. The function of the third and fourth protein remain unclear (line 253). Instead, a reductase (AcuI) encoded somewhere else on the same genome was shown to transform the acryloyl-CoA to propionate-CoA. What was the transcription profile of acuI acuH in the RNA-seq? were they induced by growth on DMSP? Is the 1696-1697-1698-1699 gene cluster conserved? What is the function of 1698 and 1699? These questions are only relevant if the authors plan to maintain the claim of having identified a new pathway. This pathway prediction component is very weak and could be supplemented by KO mutagenesis of the dddCB and acuI. Without such work this is speculation and needs to be written as such.
- Appropriate controls, units and quantification should be used:
Line 102- Please give a normalised value for the level of DMS produced from DMSP per time and protein/cells.
Figure 2. A. One would expect to see a growth curve of D2 on DMSP compared to acrylate, a conventional carbon source (e.g. pyruvate, glycerol or succinate) and a no carbon control. As "AcoD" is predicted to ligate CoA to DMSP it would be good to know if the strain grows on acrylate. It might be predicted to have different properties to e.g. Halomonas which does grow on acrylate. At least a no carbon and conventional carbon source should definitely be included.
B. The units for this figure are not appropriate. It would be more appropriate to show the actual amount of DMS that is produced by the strain, ideally normalised to protein, cells or absorbance and time. Detail in the figure what the control is.
C. Would like to see error bars on this figure. Also would have been sensible to colour code these to match panel D.
Figure 3. B and C. as with Figure 2 we need to see levels of DMS normalised to cells/protein and time.
Line 374 - No controls. Please include these as detailed above. No carbon, conventional carbon source, acrylate?
Quantitative data supporting Supplementary Fig. 12 would be helpful. After all this route would have to explain that the bacteria can use acrylate CoA as sole carbon source (or at least alternatives would have to be discussed). Is the identified activity sufficient for this task?
Line 388 - This method is/should be quantitative. It is standard practice to report DMS production normalised to time and cells/protein. Here we are only given peak area.
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