Function and redundancy of the chaplin cell surface proteins in aerial hypha formation, rodlet assembly, and viability in Streptomyces coelicolor

Abstract

The chaplins are a family of eight secreted proteins that are critical for raising aerial hyphae in Streptomyces coelicolor. These eight chaplins can be separated into two main groups: the long chaplins (ChpA to -C) and the short chaplins (ChpD to -H). The short chaplins can be further subdivided on the basis of their abilities to form intramolecular disulfide bonds: ChpD, -F, -G, and -H contain two Cys residues, while ChpE has none. A "minimal chaplin strain" containing only chpC, chpE, and chpH was constructed and was found to raise a substantial aerial mycelium. This strain was used to examine the roles of specific chaplins. Within this strain, the Cys-containing ChpH was identified as the major polymerization unit contributing to aerial hypha formation and assembly of an intricate rodlet ultrastructure on the aerial surfaces, and the two Cys residues were determined to be critical for its function. ChpC augmented aerial hypha formation and rodlet assembly, likely by anchoring the short chaplins to the cell surface, while ChpE was essential for the viability of wild-type S. coelicolor. Interestingly, the lethal effects of a chpE null mutation could be suppressed by the loss of the other chaplins, the inactivation of the twin arginine translocation (Tat) secretion pathway, or the loss of the rodlins.

FIG. 1.

The introduction of additional chaplin genes into the 7× chp mutant strain (J3149A), which contains only chpE, results in increased aerial hypha formation (plate images) and increased surface ultrastructure (scanning electron micrographs). (A) J3149A. (B) J3149A with chpH introduced. (C) J3149A with chpC and chpH introduced. (Inset) Starburst rodlet pattern on the spore surface. Images were taken after 7 days of incubation. Bars = 250 nm.

FIG. 2.

Scanning electron micrographs comparing the surface ultrastructure of wild-type S. coelicolor M600 (top) with that of a 7× chp mutant strain (containing only chpE) to which chpAD and chpCH were introduced on integrating plasmid vectors (bottom).

FIG. 3.

Alignment of the short-chaplin domains from S. coelicolor (sc), S. avermitilis (av), S. griseus (sg), and S. scabies (ss). The sequences shown represent the processed (signal peptide removed), mature chaplin form. Identical amino acid residues are highlighted in black, and similar amino acid residues are shown in gray. The arrows indicate the sites of conserved Cys residues.

FIG. 4.

The phenotypic effect of removing the two Cys residues in ChpH. (A) A strain producing only ChpE and ChpH* is largely incapable of raising aerial hyphae (top and middle) and exhibits no rodlet ultrastructure (bottom). (B) A strain producing ChpC, ChpE, and ChpH* is capable of raising slightly more hyphae than the strain shown in panel A but is still largely defective in aerial hypha formation (top and middle) and is also devoid of rodlet fibers on the aerial surfaces (bottom). Bars: middle, 5 μm; bottom, 200 nm.

FIG. 5.

Phenotypic comparison of wild-type S. coelicolor M600 with the constructed chpE null mutant (carrying an insertion element in tatB). The top panel shows the two strains grown on MS medium, while the bottom panel shows the two strains grown on rich R5 medium.

FIG. 6.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry of cell wall extracts isolated from the tatB chpE double mutant grown on MS medium. The peaks corresponding to ChpD, ChpH, ChpG, and ChpF are labeled. The x axis represents the mass (m)/charge (z) ratio, where z = 1.

FIG. 7.

(A) Agarase assay in S. lividans 10-164 comparing secretion of agarase with its native signal peptide (top left), RdlA signal peptide (top right), and RdlB signal peptide (bottom left). S. lividans 10-164 alone served as a negative control (bottom right). (B) Scanning electron micrographs showing the surfaces of the tatB chpE double mutant and the tatB (TP3) mutant. Abundant rodlet ultrastructure is evident for both strains. Bars = 100 nm.