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Nature (2022)
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We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.
The microbial cell wall is essential for cell shape maintenance and resistance to external stressors1. The primary structural component of the cell wall is peptidoglycan (PG), a glycopolymer with peptide crosslinks located outside of the cell membrane1. PG biosynthesis and structure are responsive to shifting environmental conditions such as pH and salinity2–6, but mechanisms underlying such adaptations are incompletely understood. Precursors of PG and other cell surface glycopolymers are synthesized in the cytoplasm and then delivered across the cell membrane bound to the recyclable lipid carrier undecaprenyl phosphate (C55-P)7. The transporter protein(s) that return C55-P to the cytoplasmic face of the cell membrane have been elusive. Here, we identify the DUF368-containing and DedA transmembrane protein families as candidate C55-P translocases, filling a critical gap in knowledge of the proteins required for the biogenesis of microbial cell surface polymers. Gram-negative and -positive bacteria lacking their cognate DUF368-containing protein exhibited alkaline-dependent cell wall and viability defects, along with increased cell surface C55-P levels. pH-dependent synthetic genetic interactions between DUF368-containing proteins and DedA family members suggest that C55-P transporter usage is dynamic and modulated by environmental inputs. C55-P transporter activity was required by the cholera pathogen for growth and cell shape maintenance in the intestine. We propose that conditional transporter reliance provides resilience in lipid carrier recycling, bolstering microbial fitness within and outside of the host.
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Brandon Sit
Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, USA
Veerasak Srisuknimit
Present address: Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
These authors contributed equally: Brandon Sit, Veerasak Srisuknimit and Emilio Bueno
Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, USA
Brandon Sit, Veerasak Srisuknimit, Franz G. Zingl, Karthik Hullahalli & Matthew K. Waldor
Department of Microbiology, Harvard Medical School, Boston, USA
Brandon Sit, Veerasak Srisuknimit, Franz G. Zingl, Karthik Hullahalli & Matthew K. Waldor
Laboratory for Molecular Infection Medicine Sweden (MIMS), Department of Molecular Biology, Umeå Centre for Microbial Research (UCMR), Umeå University, Umeå, Sweden
Emilio Bueno & Felipe Cava
Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, USA
Matthew K. Waldor
Howard Hughes Medical Institute, Bethesda, USA
Matthew K. Waldor
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Correspondence to Felipe Cava or Matthew K. Waldor.
Bacterial and archaeal clades with PF04018-containing proteins. Annotree was used to plot and identify species with an annotated DUF368 domain. Each tab of the spreadsheet is a different level of bacterial classification (Phylum>Order>Family>Genus>Species). Archaeal clades are combined on the tab “Archaea”.
Homologues of VCA0040 in bacteria. HMMER was used to identify homologous sequences to VCA0040 in the Uniprot database. Each species is listed once.
Notable domain architectures of DUF368- and DedA-containing proteins. Sequences were obtained by searching InterProScan with the PF family designations for DUF368 (PF04018) and DedA (PF09335).
RNAseq of Δvca0040 V. cholerae during sphere formation. For the top 40 up-regulated genes in Δvca0040, manual curation of subcellular localization was performed with SignalP and pSortb. P-values were obtained through the Wald test performed by DESeq2 with adjustments for multiple comparisons.
MIC data for V. cholerae, S. aureus and E. coli. Data for each organism is separated into individual worksheets. Note that S. aureus data is split into two worksheets, one for WT versus Δ0846 characterization (“SA”) and one for WT versus Δ0846, Δ2816 and Δ0901 (“SA_dedA”). All V. cholerae data is under “VC” and all E. coli lptd4213 data is under “EC_lptd4213”.
Synthetic transposon-insertion screening in Δvca0040 V. cholerae. Note that intergenic regions (IG_) and regions with <5 informative sites (possible transposon insertion sites) were not analyzed. Hits on the second tab were thresholded at an inverse MWU p-value >100 before sorting by mean log2 fold change.
Synthetic transposon-insertion screening in ΔyghB V. cholerae. Note that this dataset has not been filtered as for Supplementary Table 6.
Multiplexed comparative proteomics of whole-cell WT and ΔsecDF1 V. cholerae. Fold change was calculated by dividing the average normalized relative abundance of proteins in ΔsecDF1 by that of WT.
Conservation of PF04018, PF09335, and PF02763 in bacterial species. Spreadsheet was generated by Annotree queries for all possible combinations of the three protein families. The “Master” sheet lists all species-level organisms with the indicated combination of domains. The “Model Species” sheet lists selected microbes of particular interest and can be used to look up any given species in the reference table. Species of interest should be manually confirmed for presence or absence by BLAST to guard against misannotation or non-annotation issues.
Strains and vectors and genomic information used in this study. Any listed strains or vectors may be requested from the lead contact (mwaldor@research.bwh.harvard.edu).
BLAST dictionary for assignment of putative VC names to HaitiWT V. cholerae loci. All coding sequences from HaitiWT were used as queries in a batch BLAST of the N16961 V. cholerae proteome. The top hit by e-value was selected and VC name automatically assigned. However, as batch BLAST outputs a hit regardless of confidence, manual curation is still required for specific loci when using this dictionary.
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Sit, B., Srisuknimit, V., Bueno, E. et al. Undecaprenyl phosphate translocases confer conditional microbial fitness. Nature (2022). https://doi.org/10.1038/s41586-022-05569-1
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Received: 02 March 2022
Accepted: 17 November 2022
Published: 30 November 2022
DOI: https://doi.org/10.1038/s41586-022-05569-1
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