Fig 1.
Workflow for the identification of K. pneumoniae surface and secreted proteins.
Fig 2.
Enrichment of K. pneumoniae outer membrane proteins.
(A) Protein profile at each step in the outer membrane (OM) extraction process. WC = whole cell lysate from K. pneumoniae isolate 30660, Mem = total membrane fraction, N-LS = N-lauroylsarcosine treatment, SC = sodium carbonate wash, TSE = Tris-sucrose-EDTA. (B) Comparison of outer membrane proteins in different media. AUM = artificial urine media. (C) Growth of K. pneumoniae isolate 30660 in LB or RPMI. Arrows with solid lines indicate sample collection at mid-exponential (ME) phase of growth and arrows with dashed lines indicate sample collection at stationary (S) phase of growth.
Fig 3.
Identification of K. pneumoniae outer membrane proteins by 2DE.
(A) Two-dimensional map of outer membrane proteins from isolate 30660 cultured to stationary phase of growth in LB. Numbers refer to the extracted spots that were processed for identification by LC-MS/MS. The pI and molecular weight markers are shown on the top and right, respectively. (B) Categorical representation by function of predicted outer membrane proteins. (C) Categorical representation by function of predicted outer membrane proteins.
Fig 4.
Comparison of outer membrane protein expression in LB and RPMI growth media.
Representative 2D gels of stationary-phase outer membrane proteins expressed in LB (green) or RPMI (purple) were overlayed in Samespots software (Nonlinear Dynamics). White spots indicate colocalized proteins.
Table 1.
Comparison of outer membrane protein abundance in LB and RPMI.
Fig 5.
(A) Culture supernatant proteins from K. pneumoniae at late-exponential phase of growth in M63 minimal media supplemented with 0.4% glycerol and 200 μM FeSO4 or in TSB. Numbers refer to the extracted spots that were processed for identification by LC-MS/MS. (B) Exoproteins from culture supernatants at late-exponential phase of growth for K. pneumoniae isolate 30684 in different culture media. (+) = bacteria present in culture; (−) = media without bacteria. (C) Categorical representation by function of proteins identified from spent media.
Table 2.
Phyre structural predictions for K. pneumoniae hypothetical proteins.
Fig 6.
Predicted structures of porin-like hypothetical proteins.
The amino acid sequence for each hypothetical membrane protein was modeled in silico using Phyre. A representative three-dimensional structure was selected for each porin-like protein and the orientation of each protein with respect to the membrane was predicted using TM-DET. First Glance Jmol Viewer was used to capture high resolution images of each structure, and these images were incorporated manually into a representative outer membrane. The outer membrane (OM) is in grey, beta sheets are yellow arrows, alpha helices are purple or magenta rockets, white lines are coiled regions, and blue lines are turns.