Reconstruction of mreB Expression in Staphylococcus aureus via a Collection of New Integrative Plasmids

Abstract

Protein localization has been traditionally explored in unicellular organisms, whose ease of genetic manipulation facilitates molecular characterization. The two rod-shaped bacterial models Escherichia coli and Bacillus subtilis have been prominently used for this purpose and have displaced other bacteria whose challenges for genetic manipulation have complicated any study of cell biology. Among these bacteria is the spherical pathogenic bacterium Staphylococcus aureus. In this report, we present a new molecular toolbox that facilitates gene deletion in staphylococci in a 1-step recombination process and additional vectors that facilitate the insertion of diverse reporter fusions into newly identified neutral loci of the S. aureus chromosome. Insertion of the reporters does not add any antibiotic resistance genes to the chromosomes of the resultant strains, thereby making them amenable for further genetic manipulations. We used this toolbox to reconstitute the expression of mreB in S. aureus, a gene that encodes an actin-like cytoskeletal protein which is absent in coccal cells and is presumably lost during the course of speciation. We observed that in S. aureus, MreB is organized in discrete structures in association with the membrane, leading to an unusual redistribution of the cell wall material. The production of MreB also caused cell enlargement, but it did not revert staphylococcal shape. We present interactions of MreB with key staphylococcal cell wall-related proteins. This work facilitates the use S. aureus as a model system in exploring diverse aspects of cellular microbiology. A significant number of studies of protein localization has been traditionally performed by using bacterial models, which are relatively simple and tractable haploid organisms (1-3). However, only a few bacterial species, such as the model organisms Escherichia coli and Bacillus subtilis, are generally considered for these studies, mostly because of the availability of a large number of molecular tools. In an attempt to have a broader and more general idea of the fundamental processes within the microbial world, there is growing interest in exploring other bacterial species that differ in shape, developmental properties, or infective potential from conventional bacterial models (4-6). Among these species is the spherical bacterium Staphylococcus aureus. It is an opportunistic pathogen that causes hard-to-treat acute and chronic infections in humans (7). It is indeed the study of its pathogenic attributes that makes this bacterium very attractive. Besides the interest in S. aureus as a major human pathogen, it is also an appealing model to explore questions related to cell shape, cell division, and cell proliferation, because it propagates very efficiently under laboratory conditions and is morphologically different from the conventional models E. coli and B. subtilis.In order to genetically manipulate S. aureus in the laboratory, several batteries of plasmids have been constructed over the past years (8-12), yet performance of p...

Nfu facilitates the maturation of iron‐sulfur proteins and participates in virulence in Staphylococcus aureus

“…In contrast, returning the gene under its native promoter via an episome resulted in full genetic complementation (Figs A, A, 10B and http://onlinelibrary.wiley.com/doi/10.1111/mmi.12860/suppinfo). The reason for this discrepancy is currently unknown, but similar problems have been reported for the pLL chromosomal integration system (Mainiero et al ., ; Yepes et al ., ).…”

Cell differentiation defines acute and chronic infection cell types in Staphylococcus aureus

“…Due to their surfactant properties, PSMα and PSMβ act as cytolytic toxins that contribute to bacterial dispersion and play an important role in acute staphylococcal infections ( Li et al, 2009a ; Peschel and Otto, 2013 ) ( Figure 1A ). These reporters were introduced into neutral loci in the S. aureus chromosome to ensure expression of a single reporter copy in each cell ( Yepes et al, 2014 ); we monitored their expression at the single-cell level in S. aureus aggregates using quantitative analysis of fluorescence microscopy images ( Figure 1B and Figure 1—figure supplement 1A–B ). All reporters showed bimodal expression and indicated the bifurcation of two cell subpopulations in S. aureus aggregates, one with lower and another with higher fluorescence levels.…”

“…To generate the S. aureus strains single-labeled with P ica -yfp , P spa -yfp , P psmα -yfp , P psmα -mars, P psmβ -yfp , P dnaA -yfp and double-labeled with P spa -yfp P ica -mars , P psmβ -yfp P psmα -mars , P psmα -yfp P ica -mars , P spa -yfp P psmα -mars , P ica -yfp P clfA -mars , P ica -yfp P isdA -mars , P spa -yfp P clfA -mars and P spa -yfp P isdA -mars transcriptional fusions, the respective promoter region comprising 200 to 500 bp upstream of the start codon was fused to the yfp reporter-gene using the plasmid pKM003 or to the rfp (mars) reporter-gene using the plasmid pKMmars. These fusions were subcloned into the plasmids pAmy and pLac ( Yepes et al, 2014 ). The plasmids were integrated into the neutral amy and lac loci of S. aureus chromosome to ensure a uniform and chromosome-equivalent copy number of the reporters in all the cells within the microbial community.…”

“…The plasmids were integrated into the neutral amy and lac loci of S. aureus chromosome to ensure a uniform and chromosome-equivalent copy number of the reporters in all the cells within the microbial community. The integration of reporters in amy and lac neutral loci occurs in a two-step recombination process, as described in ( Yepes et al, 2014 ). Briefly, integration of the plasmid into the chromosome of S. aureus occurs via a single recombination event.…”

LipidII: Just Another Brick in the Wall?

“…MreB, heterologously expressed in S . aureus , forms patches at discrete regions in the membrane (not at the septum, the normal location of LipidII), and these MreB patches organize new call wall synthesis, resulting in misshapen cells ( Fig 4 ) [ 54 ]. This experiment supports the notion that MreB recruits LipidII, either directly or indirectly.…”