PMID:22636772

Citation	Fidopiastis, PM, Rader, BA, Gerling, DG, Gutierrez, NA, Watkins, KH, Frey, MW, Nyholm, SV and Whistler, CA (2012) Characterization of a Vibrio fischeri aminopeptidase and evidence for its influence on an early stage of squid colonization. J. Bacteriol. 194:3995-4002
Abstract	Vibrio fischeri cells are the sole colonists of a specialized light organ in the mantle cavity of the sepiolid squid Euprymna scolopes. The process begins when the bacteria aggregate in mucus secretions outside the light organ. The cells eventually leave the aggregate, enter the light organ, and encounter a rich supply of peptides. The need to dissociate from mucus and presumably utilize peptides led us to hypothesize that protease activity is integral to the colonization process. Protease activity associated with whole cells of Vibrio fischeri strain ES114 was identified as the product of a putative cell membrane-associated aminopeptidase (PepN). To characterize this activity, the aminopeptidase was cloned, overexpressed, and purified. Initial steady-state kinetic studies revealed that the aminopeptidase has broad activity, with a preference for basic and hydrophobic side chains and k(cat) and K(m) values that are lower and smaller, respectively, than those of Escherichia coli PepN. A V. fischeri mutant unable to produce PepN is significantly delayed in its ability to colonize squid within the first 12 h, but eventually it establishes a wild-type colonization level. Likewise, in competition with the wild type for colonization, the mutant is outcompeted at 12 h postinoculation but then competes evenly by 24 h. Also, the PepN-deficient strain fails to achieve wild-type levels of cells in aggregates, suggesting an explanation for the initial colonization delay. This study provides a foundation for more studies on PepN expression, localization, and role in the early stages of squid colonization.
Links	PubMed PMC3416539 Online version:10.1128/JB.00108-12
Keywords	Aliivibrio fischeri/enzymology; Aliivibrio fischeri/growth & development; Aliivibrio fischeri/physiology; Amino Acid Sequence; Aminopeptidases/genetics; Aminopeptidases/metabolism; Animal Structures/microbiology; Animals; Cloning, Molecular; Decapodiformes/microbiology; Escherichia coli/genetics; Gene Expression; Kinetics; Molecular Sequence Data; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Sequence Homology, Amino Acid; Substrate Specificity
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
VIBF1:Q5E5B9	enables	GO:0070006: metalloaminopeptidase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
VIBF1:Q5E5B9		GO:0004177: aminopeptidase activity	ECO:0000315:		F		Fig. 3 shows the pepN mutant strains had delayed colonization of the squid light organ, which was corrected when pepN was expressed on a plasmid.	complete CACAO 8614
VIBF1:Q5E5B9		GO:0070006: metalloaminopeptidase activity	ECO:0000314:		F		Fig. 1 shows the enzyme's activity was much greater in whole cells compared to cell-free supernatant, thus suggesting activity is cell-associated and not secreted.	complete CACAO 8616
VIBF1:Q5E5B9		GO:0005737: cytoplasm	ECO:0000314:		C		Table 2 shows the majority of the protein's activity is in the cytoplasm with substantial activity in the cell membrane. The protein is considered to be cytosolic with close association to the membrane.	complete CACAO 8618
VIBF1:Q5E5B9		GO:0070006: metalloaminopeptidase activity	ECO:0000315:		F		Table 2. demonstrates the impaired ability of the pepN mutant to metabolize an aminopeptidase substrate. It also shows that expression of pepN on pVSV105 restored aminopeptidase activity of the mutant cells to higher degree than the wild-type.	complete CACAO 8684
VIBF1:Q5E5B9	part_of	GO:0005737: cytoplasm	ECO:0000314: direct assay evidence used in manual assertion		C		Seeded From UniProt	complete
VIBF1:Q5E5B9	enables	GO:0070006: metalloaminopeptidase activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		Seeded From UniProt	complete
VIBFM:B5FE14		GO:0004177: aminopeptidase activity	ECO:0000315:		F		FIG 5 Aggregation defect of the pepN mutant. Newly hatched squid were incubated with 106 GFP-labeled wild-type (A) or pepN mutant (B) V. fischeri for 3 h,counterstained with Cell Tracker Orange (red) to visualize the light organ surface, and then examined by confocal microscopy. (C) The number of bacteria per aggregate per light organ lobe was determined by direct counting, and averages, � standard deviations are shown.	complete CACAO 8727

References

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