Scarless deletion of up to seven methyl-accepting chemotaxis genes with an optimized method highlights key function of CheM in <i>Salmonella</i> Typhimurium

<div><p>Site-directed scarless mutagenesis is an essential tool of modern pathogenesis research. We describe an optimized two-step protocol for genome editing in <i>Salmonella enterica</i> serovar Typhimurium to enable multiple sequential mutagenesis steps in a single strain. The system is based on the λ Red recombinase-catalyzed integration of a selectable antibiotics resistance marker followed by replacement of this cassette. Markerless mutants are selected by expressing the meganuclease I-SceI which induces double-strand breaks in bacteria still harboring the resistance locus. Our new dual-functional plasmid pWRG730 allows for heat-inducible expression of the λ Red recombinase and tet-inducible production of I-SceI. Methyl-accepting chemotaxis proteins (MCP) are transmembrane chemoreceptors for a vast set of environmental signals including amino acids, sugars, ions and oxygen. Based on the sensory input of MCPs, chemotaxis is a key component for <i>Salmonella</i> virulence. To determine the contribution of individual MCPs we sequentially deleted seven MCP genes. The individual mutations were validated by PCR and genetic integrity of the final seven MCP mutant WRG279 was confirmed by whole genome sequencing. The successive MCP mutants were functionally tested in a HeLa cell infection model which revealed increased invasion rates for non-chemotactic mutants and strains lacking the MCP CheM (Tar). The phenotype of WRG279 was reversed with plasmid-based expression of CheM. The complemented WRG279 mutant showed also partially restored chemotaxis in swarming assays on semi-solid agar. Our optimized scarless deletion protocol enables efficient and precise manipulation of the <i>Salmonella</i> genome. As demonstrated with whole genome sequencing, multiple subsequent mutagenesis steps can be realized without the introduction of unwanted mutations. The sequential deletion of seven MCP genes revealed a significant role of CheM for the interaction of <i>S</i>. Typhimurium with host cells which might give new insights into mechanisms of <i>Salmonella</i> host cell sensing.</p></div

Plasmids used in this study.

<p>Plasmids used in this study.</p

Strains used in this study.

<p>Strains used in this study.</p

Functional characterization of the WRG279 mutant.

<p>(A) HeLa cells were infected with different STM strains and relative invasion rates compared to STM WT were calculated after one hour of infection. An <i>invC</i> mutant lacking a functional T3SS-1 was used as a negative control for invasion and a motile but non-chemotactic <i>cheY</i> mutant was included to evaluate the impact of directed motility. The Δ1 to Δ7 strains represent sequential MCP deletions as follows: Δ1 = WRG246 Δ<i>aer</i>; Δ2 = WRG255 Δ<i>aer</i>, Δ<i>tcp</i>; Δ3 = WRG260 Δ<i>aer</i>, Δ<i>tcp</i>, Δ<i>tsr</i>; Δ4 = WRG264 Δ<i>aer</i>, Δ<i>tcp</i>, Δ<i>tsr</i>, Δ<i>trg</i>; Δ5 = WRG269 Δ<i>aer</i>, Δ<i>tcp</i>, Δ<i>tsr</i>, Δ<i>trg</i>, Δ<i>cheM</i>; Δ6 = WRG277 Δ<i>aer</i>, Δ<i>tcp</i>, Δ<i>tsr</i>, Δ<i>trg</i>, Δ<i>cheM</i>, Δ<i>mcpC</i>; Δ7 = WRG279 Δ<i>aer</i>, Δ<i>tcp</i>, Δ<i>tsr</i>, Δ<i>trg</i>, Δ<i>cheM</i>, Δ<i>mcpC</i>, Δ<i>mcpB</i>. The right panel shows the invasion rates of the Δ7 strain complemented with pCheM (pWRG847) or transformed with the empty vector pWSK29 (vector). Statistical significance was calculated using a one sample <i>t</i> test against the hypothetical value 1.0 and was defined as ** for <i>p</i> < 0.01 and *** for <i>p</i> < 0.001. (B) Swarming phenotypes of different <i>Salmonella</i> strains as indicated on LB soft agar plates. Depicted is one representative out of three similar experiments. The diagram in the lower right panel shows the diameter of the swarming rings of <i>S</i>. Typhimurium WT and the Δ7 MCP mutant complemented with a CheM expression plasmid or a vector control as described in (B). Data of three independent biological replicates including means and standard deviations are shown. Statistical significance was calculated using a two-tailed paired Student’s <i>t</i> test and was defined as *** for <i>p</i> < 0.001.</p

Expected fragment sizes of verification PCRs.

<p>Expected fragment sizes of verification PCRs.</p

Genomic characterization of the WRG279 (Δ7 MCP) mutant.

<p>(A) Agarose gel showing PCR fragments generated using the primers as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172630#pone.0172630.t001" target="_blank">Table 1</a> with either wild type (W) or WRG279 (Δ7) chromosomal DNA as a template. Shorter PCR products of the expected sizes confirmed gene deletion for the seven loci in WRG279. M = DNA marker, band sizes are shown in kbp (B) Next generation sequencing coverage data of strain WRG279 (black line) and NCTC 12023 WT (dotted gray line) is shown for the targeted MCP genes (gray rectangles) including 300 nucleotides before and after each coding sequence. For strain WRG279 no sequencing reads were obtained for the deleted genes and thus a lack of coverage was observed at the respective nucleotide positions of the wild type sequence.</p

Overview of the method.

<p>(A) Schematic representation of the functional units of plasmid pWRG730. The operon containing λ Red recombinase functions is under control of the heat-inducible phage-derived promoter <i>p</i>_<i>L</i>. Expression of the I-SceI meganuclease is controlled by a tetracycline-inducible promoter (P_<i>tet</i>). A chloramphenicol resistance cassette (<i>cat</i>) is used for selection purposes. Due to its temperature-sensitive origin of replication (<i>repA</i>_<i>ts</i>) the plasmid can be easily cured at elevated growth temperatures. (B) Representation of the two-step scarless deletion methodology. A kanamycin resistance cassette (<i>aph</i>) is amplified together with an I-SceI cleavage site (grey triangle) from pWRG717 with two 60-mer primers each containing site-specific homology extensions at their 5’-ends (striped squares). Chromosomal integration of this first targeting construct (TC) is achieved by λ Red recombinase expression from pWRG730. The 2^nd TC is also generated by PCR using chromosomal DNA as template and contains a direct fusion of up- and downstream homology regions. After genomic integration of the 2^nd TC using λ Red recombinase, successful recombinants are selected by I-SceI expression from pWRG730. A detailed description of the method can be found in the main text.</p

Expected fragment sizes of verification PCRs.

<p>Expected fragment sizes of verification PCRs.</p

Distribution of single nucleotide polymorphisms unique for WRG279.

<p>Distribution of single nucleotide polymorphisms unique for WRG279.</p

Integrating AlN with GdN Thin Films in an in Situ CVD Process: Influence on the Oxidation and Crystallinity of GdN

The application potential of rare earth nitride (REN) materials has been limited due to their high sensitivity to air and moisture leading to facile oxidation upon exposure to ambient conditions. For the growth of device quality films, physical vapor deposition methods, such as molecular beam epitaxy, have been established in the past. In this regard, aluminum nitride (AlN) has been employed as a capping layer to protect the functional gadolinium nitride (GdN) from interaction with the atmosphere. In addition, an AlN buffer was employed between a silicon substrate and GdN serving as a seeding layer for epitaxial growth. In pursuit to grow high-quality GdN thin films by chemical vapor deposition (CVD), this successful concept is transferred to an in situ CVD process. Thereby, AlN thin films are included step-wise in the stack starting with Si/GdN/AlN structures to realize long-term stability of the oxophilic GdN layer. As a second strategy, a Si/AlN/GdN/AlN stacked structure was grown, where the additional buffer layer serves as the seeding layer to promote crystalline GdN growth. In addition, chemical interaction between GdN and the Si substrate can be prevented by spatial segregation. The stacked structures grown for the first time with a continuous CVD process were subjected to a detailed investigation in terms of structure, morphology, and composition, revealing an improved GdN purity with respect to earlier grown CVD thin films. Employing thin AlN buffer layers, the crystallinity of the GdN films on Si(100) could additionally be significantly enhanced. Finally, the magnetic properties of the fabricated stacks were evaluated by performing superconducting quantum interference device measurements, both of the as-deposited films and after exposure to ambient conditions, suggesting superparamagnetism of ferromagnetic GdN grains. The consistency of the magnetic properties precludes oxidation of the REN material due to the amorphous AlN capping layer