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. 2020 Nov 27;8(12):1876.
doi: 10.3390/microorganisms8121876.

Dichloromethane Degradation Pathway from Unsequenced Hyphomicrobium sp. MC8b Rapidly Explored by Pan-Proteomics

Karim Hayoun  1 , Emilie Geersens  2   3 , Cédric C Laczny  4 , Rashi Halder  4 , Carmen Lázaro Sánchez  2 , Abhijit Manna  2 , Françoise Bringel  2 , Michaël Ryckelynck  3 , Paul Wilmes  4 , Emilie E L Muller  2 , Béatrice Alpha-Bazin  1 , Jean Armengaud  1 , Stéphane Vuilleumier  2
Affiliations

Dichloromethane Degradation Pathway from Unsequenced Hyphomicrobium sp. MC8b Rapidly Explored by Pan-Proteomics

Karim Hayoun et al. Microorganisms. .

Abstract

Several bacteria are able to degrade the major industrial solvent dichloromethane (DCM) by using the conserved dehalogenase DcmA, the only system for DCM degradation characterised at the sequence level so far. Using differential proteomics, we rapidly identified key determinants of DCM degradation for Hyphomicrobium sp. MC8b, an unsequenced facultative methylotrophic DCM-degrading strain. For this, we designed a pan-proteomics database comprising the annotated genome sequences of 13 distinct Hyphomicrobium strains. Compared to growth with methanol, growth with DCM induces drastic changes in the proteome of strain MC8b. Dichloromethane dehalogenase DcmA was detected by differential pan-proteomics, but only with poor sequence coverage, suggesting atypical characteristics of the DCM dehalogenation system in this strain. More peptides were assigned to DcmA by error-tolerant search, warranting subsequent sequencing of the genome of strain MC8b, which revealed a highly divergent set of dcm genes in this strain. This suggests that the dcm enzymatic system is less strongly conserved than previously believed, and that substantial molecular evolution of dcm genes has occurred beyond their horizontal transfer in the bacterial domain. Our study showed the power of pan-proteomics for quick characterization of new strains belonging to branches of the Tree of Life that are densely genome-sequenced.

Keywords: Hyphomicrobium; Nanopore; dcm genes; dehalogenation; dichloromethane; differential proteomics; genome sequencing; pan-proteomics.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Pan-proteomics workflow to decipher key protein determinants of the dichloromethane utilisation pathway from the Hyphomicrobium sp. MC8b strain. DCM: dichloromethane.
Figure 2
Figure 2
Comparative pan-proteomics analysis of Hyphomicrobium sp. MC8b. Volcano plot of protein abundances under dichloromethane versus methanol growth conditions. Proteins are distributed depending on their abundance fold-change and p-value. Blue proteins show significant fold-change (≥1.5) and are significantly differentially abundant (p value ≤ 0.05). Proteins in orange (p-value ≤ 0.05 but fold-change ≤ 1.5), green (fold-change ≥ 1.5 but p-value ≥ 0.05), and red (fold-change ≤ 1.5, p-value ≥ 0.05) were not further considered.
Figure 3
Figure 3
Sequence alignment of selected DCM dehalogenases covering the known sequence diversity of DCM dehalogenases, highlighting the highly divergent sequence of Hyphomicrobium sp. MC8b DcmA sequence detected and partially predicted using pan-proteomics analysis, and verified by sequencing of the MC8b genome. All peptides detected using the pan-proteomics Hypomicrobium database analysed in normal or error-tolerant mode are shown in red and green, respectively. Additional peptides detected by analysis of obtained spectra using the predicted proteome of strain MC8b based on the genome sequence of the strain are shown by black lines. A star denotes a sequence difference in the DcmA sequence of strain MC8b compared to that of strain H. sp. GJ21 and/or H. denitrificans ATCC 51888 (HDEN). Green stars indicate sequence variations in the MC8b DcmA sequence that prevented detection of peptides by analysis of the Hyphomicrobium database in the absence of error tolerance, and black stars indicate sequence variations extending beyond the one-mismatch threshold for peptide detection in error-tolerant mode, respectively.
Figure 4
Figure 4
Comparison of dcm genes of Hyphomicrobium strains MC8b, ATCC 51888, and GJ21, and of the reference DCM-degrading Alphaproteobacterial strain Methylobacterium extorquens DM4. Homologous genes are shown in the same colour, with percentage identities to strain MC8b given at the protein level. Transposase genes are shown in grey, with homologous genes showing the same fill pattern. Numbers under the arrows for Hyphomicrobium strains ATCC 51888 and GJ21 and Methylobacterium extorquens strain DM4 refer to corresponding gene identifiers in Genbank.
Figure 5
Figure 5
Hyphomicrobium sp. MC8b differential proteomics using the genome sequence of the strain. Volcano plot of protein abundances in dichloromethane versus methanol growth conditions. Colour code is as in Figure 2, with open blue symbols highlighting proteins with significant differential abundance that were additionally identified from the proteome of strain MC8b predicted from the genome sequence.
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