The Staphylococcus aureus Membrane Protein SA2056 Interacts with Peptidoglycan Synthesis Enzymes

Abstract

The yet uncharacterized membrane protein SA2056 belongs to the ubiquitous RND (Resistance-Nodulation-cell Division) family of transmembrane efflux transporters. The sa2056 gene is located downstream of femX, the gene encoding the essential, non-ribosomal peptidyl-transferase adding the first glycine in the staphylococcal cell wall pentaglycine interpeptide. Due to its proximity to and weak co-transcription with femX, we assumed that sa2056 may somehow be involved in peptidoglycan synthesis. Specific antibodies against SA2056 showed that this protein is expressed during growth and present in the membrane fraction of cell preparations. Using a bacterial two hybrid system, SA2056 was shown to interact (i) with itself, (ii) with FemB, which adds glycines 4 and 5 to the peptidoglycan interpeptide and (iii) with the essential penicillin binding proteins, PBP1 and PBP2, required for cell division and incorporation of the peptidoglycan into the cell wall. Unexpectedly, deletion of sa2056 led to no phenotype regarding growth, antibiotic resistances or cell morphology; nor did sa2056 deletion in combination with femB inactivation alter b-lactam and lysostaphin sensitivity and resistance, respectively, pointing to possible redundancy in the cell wall synthesis pathway. These results suggest an accessory role of SA2056 in S. aureus peptidoglycan synthesis, broadening the range of biological functions of RND proteins.

Keywords: FemABX; PBP; RND protein; Staphylococcus aureus; bacterial two-hybrid system; peptidoglycan.

Figure 1

Expression of sa2056 in strain Newman and its sa2056 mutant. Genetic organization of the femX-sa2056 region in (a) the wild-type and (b) the sa2056 mutant. Construction of the sa2056 mutant is detailed in supplementary figure S1. (c) Growth curves from Luria-Bertani broth (LB) cultures monitored during 9 h. (d) Northern blot analyses of RNA samples taken after 1, 3, 5 and 7 h of growth. Digoxigenin (DIG)-labeled probes for femX (left panel) and sa2056 (right panel) were used. Relevant bands are indicated. Ethidium bromide-stained 16S rRNA is shown as an indication of RNA loading. Bands that might be caused by interference of bulk 16S and 23S rRNA are designated by asterisks. Specific antibodies against SA2056 (114.7 kDa) were used for Western blot analyses of (e) cell wall (CW), cell membrane (CM) and cytoplasmic (CP) fractions isolated from exponentially growing cells and (f) membrane preparations from samples taken after 1, 3, 5 and 7 h of growth.

Figure 2

Analysis of SA2056 topology. (a) Model of SA2056 topology depicting cytoplasmic (white), membrane (black) and exoplasmic (blue) segments. The N- and C-terminus of the protein are both predicted to be located in the cytoplasm. (b) SA2056 fragments F1–14 cloned to the N-terminus of PhoA. (c) Activity of fusion proteins was measured in biological and technical triplicates; mean values for each clone are given, and the standard deviation is indicated. SA2056 fragments directing PhoA to the exoplasm were expected to produce values at least five times higher than the background levels (dashed line) measured in the phoA-negative E. coli strain CC118 (control).

Figure 3

SA2056 interactions determined using the bacterial two-hybrid system. (a) SA2056 interactions with itself and the FemABX factors. (b) SA2056 interactions with penicillin binding proteins (PBPs). Three representative co-transformants containing the plasmids indicated were analyzed regarding β-galactosidase activity, which was determined by measuring the formation of o-nitrophenol (top). Means of three technical replicates and their standard deviation are shown. The threshold corresponding to the highest negative control value multiplied by five is indicated by a dashed line. Alternatively, the ability of co-transformants to degrade lactose to lactate was tested on MacConkey agar (bottom), where acidification of the medium leads to pink colonies.

Figure 4

Pull-down experiments. Recombinant glutathion-S-transferase (GST)-tagged SA2056 and FemABX proteins were incubated with SA2056-His₆ and aliquots of bound proteins were separated on denaturing polyacrylamide gels for Coomassie staining (a) or (b) transferred to a polyvinylidene fluoride (PVDF)-membrane for Western blot analysis using specific antibodies against SA2056. Relevant bands are indicated. GST (26 kDa), GST-FemA (74.1 kDa), GST-FemB (74 kDa), GST-FemX (74.3 kDa), SA2056-His₆ (115 kDa). Probably due to degradation, an additional band (~70 kDa; GST-SA2056’) was visible in the preparation of GST-SA2056 (147.2 kDa) and detected with the antibodies. For appropriate separation, a 10%-(a) and a 7.5%-polyacrylamide gel (b) were used. GST-tagged proteins had a tendency to run slightly faster than expected from their predicted mass.