The Caulobacter crescentus paracrystalline S-layer protein is secreted by an ABC transporter (type I) secretion apparatus

Abstract

Caulobacter crescentus is a gram-negative bacterium that produces a two-dimensional crystalline array on its surface composed of a single 98-kDa protein, RsaA. Secretion of RsaA to the cell surface relies on an uncleaved C-terminal secretion signal. In this report, we identify two genes encoding components of the RsaA secretion apparatus. These components are part of a type I secretion system involving an ABC transporter protein. These genes, lying immediately 3' of rsaA, were found by screening a Tn5 transposon library for the loss of RsaA transport and characterizing the transposon-interrupted genes. The two proteins presumably encoded by these genes were found to have significant sequence similarity to ABC transporter and membrane fusion proteins of other type I secretion systems. The greatest sequence similarity was found to the alkaline protease (AprA) transport system of Pseudomonas aeruginosa and the metalloprotease (PrtB) transport system of Erwinia chrysanthemi. The prtB and aprA genes were introduced into C. crescentus, and their products were secreted by the RsaA transport system. Further, defects in the S-layer protein transport system led to the loss of this heterologous secretion. This is the first report of an S-layer protein secreted by a type I secretion apparatus. Unlike other type I secretion systems, the RsaA transport system secretes large amounts of its substrate protein (it is estimated that RsaA accounts for 10 to 12% of the total cell protein). Such levels are expected for bacterial S-layer proteins but are higher than for any other known type I secretion system.

FIG. 1

Sample colony immunoblot using anti-S antibody. NA1000 has an S-layer, JS1003 does not, and JS1001 sheds the S-layer, forming a halo around the colony. B1, B2, and B5 represent samples of the S-layer-negative Tn5 mutants that were found.

FIG. 2

Graphic representation of the DNA examined in this study. A triangle represents the approximate location of a Tn5 insertion, a T indicates a terminator, and an arrow indicates a promoter. Restriction enzyme sites: B, BamHI; E, EcoRI; H, HindIII; S, SstI. Boxes containing patterns are ORFs or genes, as indicated below the diagram.

FIG. 3

Complementation of Tn5 mutants with rsaA. Protein was extracted from the surfaces of the Tn5 mutants and JS1003 carrying plasmid pRK415rsaAΔPK, which expresses rsaA under the control of the lac promoter. Protein was also extracted from the surfaces of the wild type and rsaA knockout mutants that did not contain any plasmid to demonstrate differences in expression. Equal amounts of surface extracts were loaded onto the gel, and a Western blot was performed by using a polyclonal antibody against RsaA. Lanes: 2 through 10, surface extracts from cells containing plasmid pRK415rsaAΔPK (PK); 1, purified RsaA; 2, JS1003 (PK); 3, B9 (PK); 4, B13 (PK); 5, B1 (PK) (a Tn5 insertion in rsaA); 6, B5 (PK); 7, B15 (PK); 8, B17 (PK); 9, B2 (PK); 10, B12 (PK) (a random Tn5 insertion); 11, JS1003 (rsaA); 12, NA1000 (wild type). The arrow indicates full-length RsaA.

FIG. 4

Alignment of RsaD with AprD and PrtD using the ClustalW algorithm implemented by the program MacVector 6.0. Identical and similar amino acids are boxed. Identical amino acids are shaded. Similar amino acids are in boldface. The ABC transporter protein is represented by PrtD for metalloprotease transport in E. chrysanthemi and AprD for alkaline protease transport in P. aeruginosa.

FIG. 5

Alignment of RsaE with AprE and PrtE by using the ClustalW algorithm implemented by the program MacVector 6.0. Identical and similar amino acids are boxed. Identical amino acids are shaded. Similar amino acids are in boldface. The MFP is represented by PrtE in E. chrysanthemi and by AprE in P. aeruginosa.

FIG. 6

Complementation of transport-deficient mutants using rsaD and rsaE. Western blots of surface-extracted protein using anti-S antibody. (A) Lanes: 1, B17 (DE); 2, B15 (DE); 3, B1 (DE); 4, B17 (17A7); 5, B15 (17A7); 6, JS1003; 7, NA1000. DE indicates that the cells carried plasmid pRAT5:pRK415 containing the genes rsaD and rsaE. 17A7 indicates that the cells carry cosmid 17A7 containing the entire RSA operon. Equal amounts of surface extract were loaded in all lanes. (B) Lanes: 1, B1 (DE); 2, B5 (DE); 3, B9 (DE); 4, B15 (DE); 5, B17 (DE); 6; NA1000. DE indicates that the cells carry plasmid pRAT5:pBBR5 expressing the genes rsaD and rsaE. Equal amounts of surface extract were loaded in all lanes except 6, where there was only one-quarter of the amount loaded in the other lanes. The arrow indicates full-length RsaA.

FIG. 7

Expression of prtB in C. crescentus. PrtB was expressed in all of the colonies shown by using plasmid pRK415:pRUW500. The cells were spotted onto PYE plates containing 1% skim milk. Halos around colonies indicate that active PrtB is being secreted. Note that NA1000 and B12 cells are producing RsaA, as well as PrtB, and that the halos surrounding these colonies are smaller.