Proteases influence colony aggregation behavior in Vibrio cholerae

Tyler C. Detomasi, Allison E. Batka, Julie S. Valastyan, Molly A. Hydorn, Charles S. Craik, Bonnie L. Bassler, Michael A. Marletta

Research output: Contribution to journalArticlepeer-review


Aggregation behavior provides bacteria protection from harsh environments and threats to survival. Two uncharacterized proteases, LapX and Lap, are important for Vibrio cholerae liquid-based aggregation. Here, we determined that LapX is a serine protease with a preference for cleavage after glutamate and glutamine residues in the P1 position, which processes a physiologically based peptide substrate with a catalytic efficiency of 180 ± 80 M-1s-1. The activity with a LapX substrate identified by a multiplex substrate profiling by mass spectrometry screen was 590 ± 20 M-1s-1. Lap shares high sequence identity with an aminopeptidase (termed VpAP) from Vibrio proteolyticus and contains an inhibitory bacterial prepeptidase C-terminal domain that, when eliminated, increases catalytic efficiency on leucine p-nitroanilide nearly four-fold from 5.4 ± 4.1 × 104 M−1s−1 to 20.3 ± 4.3 × 104 M−1s−1. We demonstrate that LapX processes Lap to its mature form and thus amplifies Lap activity. The increase is approximately eighteen-fold for full-length Lap (95.7 ± 5.6 × 104 M−1s−1) and six-fold for Lap lacking the prepeptidase C-terminal domain (11.3 ± 1.9 × 105 M−1s−1). In addition, substrate profiling reveals preferences for these two proteases that could inform in vivo function. Furthermore, purified LapX and Lap restore the timing of the V. cholerae aggregation program to a mutant lacking the lapX and lap genes. Both proteases must be present to restore WT timing, and thus they appear to act sequentially: LapX acts on Lap, and Lap acts on the substrate involved in aggregation.

Original languageEnglish (US)
Article number105386
JournalJournal of Biological Chemistry
Issue number12
StatePublished - Dec 2023

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry
  • Cell Biology


  • Vibrio cholerae
  • aggregation
  • biofilm
  • proteolysis


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