The C-terminal domain of the novel essential protein Gcp is critical for interaction with another essential protein YeaZ of Staphylococcus aureus

Abstract

Previous studies have demonstrated that the novel protein Gcp is essential for the viability of various bacterial species including Staphylococcus aureus; however, the reason why it is required for bacterial growth remains unclear. In order to explore the potential mechanisms of this essentiality, we performed RT-PCR analysis and revealed that the gcp gene (sa1854) was co-transcribed with sa1855, yeaZ (sa1856) and sa1857 genes, indicating these genes are located in the same operon. Furthermore, we demonstrated that Gcp interacts with YeaZ using a yeast two-hybrid (Y2H) system and in vitro pull down assays. To characterize the Gcp-YeaZ interaction, we performed alanine scanning mutagenesis on the residues of C-terminal segment of Gcp. We found that the mutations of the C-terminal Y317-F322 region abolished the interaction of Gcp and YeaZ, and the mutations of the D324-N329 and S332-Y336 regions alleviated Gcp binding to YeaZ. More importantly, we demonstrated that these key regions of Gcp are also necessary for the bacterial survival since these mutated Gcp could not complement the depletion of endogenous Gcp. Taken together, our data suggest that the interaction of Gcp and YeaZ may contribute to the essentiality of Gcp for S. aureus survival. Our findings provide new insights into the potential mechanisms and biological functions of this novel essential protein.

Figure 1. The co-transcription of the sa1857, sa1856 (yeaZ), sa1855, and sa1854 (gcp) genes.

(A) Schematic representation of sa1857, sa1856 (yeaZ), sa1855, and sa1854 (gcp) genes in S. aureus. (B) RT-PCR using different primer pairs. RT (−), total RNA of S. aureus used as the template; RT (+), cDNA used as the template; positive controls, the genomic DNA used as the template (lane 1, 4, 7); M, 1 kb DNA ladder.

Figure 2. The determination of Gcp binding to YeaZ.

(A) Yeast two hybrid analysis of interaction between Gcp and YeaZ on histidine, leucine, and tryptophan drop out synthetic complete (SC-His, -Leu, -Trp) plates with 3-amino-1,2,4,-triazole (3-AT) and SC plates lacking adenine, leucine, and tryptophan (SC-Ade, -Leu, -Trp). Gcp was fused with the activation domain and YeaZ was fused to binding domain. The minus signs indicate empty vector controls. (B) In vitro immunoprecipitation analysis of interaction between Gcp and YeaZ. His-tagged Gcp and GST-tagged YeaZ were purified from E. coli. Gcp and YeaZ were incubated with Glutathione Sepharose 4B beads together or separately. Western blotting was carried out with rabbit-anti-Gcp serum to detect Gcp. Lane 1: purified Gcp protein was loaded as a positive control.

Figure 3. Diagrams represent different deletion mutations of Gcp (A); and interactions between S. aureus Gcp and YeaZ detected by yeast two-hybrid analyses (B).

Yeast cells containing the indicated plasmids were serially diluted, and 5 µl of diluted cells was spotted on the plate with media lacking histidine, leucine, and tryptophan; or lacking adenine, leucine, and tryptophan to score for interactions after incubation at 30°C.

Figure 4. Diagrams represent different alanine substitutions of amino acids in Gcp (A); and interactions between S. aureus Gcp and YeaZ detected by yeast two-hybrid analyses (B).

Yeast cells containing the indicated plasmids were serially diluted, and 5 µl of diluted cells was spotted on the plate with media lacking histidine, leucine, and tryptophan (SC-His, -Leu, -Trp); or lacking adenine, leucine, and tryptophan (SC-Ade, -Leu, -Trp) to score for interactions after incubation at 30°C.

Figure 5. The impact of the expression of wild type or mutated Gcp in trans on the growth of the Pspac-regulated gcp expression strain.

(A) Construction of Gcp complementation system using the Pspac-regulated gcp expression strain and Western blot analysis of gcp expression in trans. Lane 1, purified recombinant Gcp; lane 2, negative control (JRN0210); lane 3, Gcp complementary strain (JNR0110). Without an inducer, ATc, Gcp is constitutively expressed due to the leakage of Pxyl/tetO promoter. (B–H) Growth curves of Gcp complementary strains. The Pspac-regulated gcp expression S. aureus strain carrying the staphylococcal wild-type gcp complementary plasmid, pXL108 was used as positive control (B); carrying parental plasmid, pMY1107, as a negative control (C); carrying the gcpseg1 complementary plasmid, pLT109 (D); carrying the gcpseg2 complementary plasmid, pLT209 (E); carrying the gcp1-1 complementary plasmid, pLT309 (F); carrying the gcp1-2 complementary plasmid, pLT409 (G); carrying the gcp1-3 complementary plasmid, pLT509 (H). The above strains were incubated overnight in TSB in the presence of appropriate antibiotics and 0.2 mM IPTG. Bacteria were diluted and incubated in fresh TSB containing appropriate antibiotics and different concentration of IPTG at 37°C with shaking. The cell growth was monitored by measuring OD_{600 nm} every 15 min for 18 h with 1 min mixing before each reading.

Figure 6. SDS-PAGE and Western blot analysis of Gcp and YeaZ's protease activity.

(A) Western blot results showing hydrolysis of Glycophorin A. GPA2 and GPA represent dimer and monomer of glycophorin A. 10 µg glycophorin A was incubated with PBS (lane 1), 5 µg O-sialoglycoprotein endopeptidase (Gcp) of P. haemolytica (Cedarlane Laboratories, lane 2), 5 µg Gcp (lane 3), 25 µg Gcp (lane 4), 50 µg Gcp (lane 5), 33 µg YeaZ (lane 6) and 5 µg Gcp and 3.3 µg YeaZ (lane 7). (B) SDS-PAGE analysis of the impact of the recombinant staphylococcal YeaZ on Gcp.