Genetic Manipulations of Staphylococcal Chromosomal DNA.

Performing genetic manipulation is often key to understanding bacterial gene function. In this chapter, we present the method of allelic exchange using temperature-sensitive plasmids to generate mutations in Staphylococcus, including single-nucleotide mutations, insertions, and gene deletions. In addition, this chapter summarizes other key genetic technologies used for the manipulation of S. aureus, including the CRISPR/Cas9 system and complementation.

Contribution of YjbIH to Virulence Factor Expression and Host Colonization in Staphylococcus aureus.

To persist within the host and cause disease, Staphylococcus aureus relies on its ability to precisely fine-tune virulence factor expression in response to rapidly changing environments. During an unbiased transposon mutant screen, we observed that disruption of a two-gene operon, yjbIH, resulted in decreased levels of pigmentation and aureolysin (Aur) activity relative to the wild-type strain. Further analyses revealed that YjbH, a predicted thioredoxin-like oxidoreductase, is predominantly responsible for the observed yjbIH mutant phenotypes, though a minor role exists for the putative truncated hemoglobin YjbI. These differences were due to significantly decreased expression of crtOPQMN and aur Previous studies found that YjbH targets the disulfide- and oxidative stress-responsive regulator Spx for degradation by ClpXP. The absence of yjbH or yjbI resulted in altered sensitivities to nitrosative and oxidative stress and iron deprivation. Additionally, aconitase activity was altered in the yjbH and yjbI mutant strains. Decreased levels of pigmentation and aureolysin (Aur) activity in the yjbH mutant were found to be Spx dependent. Lastly, we used a murine sepsis model to determine the effect of the yjbIH deletion on pathogenesis and found that the mutant was better able to colonize the kidneys and spleens during an acute infection than the wild-type strain. These studies identified changes in pigmentation and protease activity in response to YjbIH and are the first to have shown a role for these proteins during infection.

Aconitase Functions as a Pleiotropic Posttranscriptional Regulator in Helicobacter pylori.

UNLABELLED: Posttranscriptional regulation in bacteria has increasingly become recognized as playing a major role in response to environmental stimuli. Aconitase is a bifunctional protein that not only acts enzymatically but also can be a posttranscriptional regulator. To investigate protein expression regulated by Helicobacter pylori AcnB in response to oxidative stress, a global proteomics study was conducted wherein the ΔacnB strain was compared to the parent strain when both strains were O2 stressed. Many proteins, including some involved in urease activity, in combating oxidative stress, and in motility, were expressed at a significantly lower level in the ΔacnB strain. A bioinformatics prediction tool was used to identify putative targets for aconitase-mediated regulation, and electrophoretic mobility shift assays demonstrated that apo-AcnB is able to bind to RNA transcripts of hpn (encoding a nickel-sequestering protein), ahpC (encoding alkyl hydroperoxide reductase), and flgR (encoding flagellum response regulator). Compared to the wild type (WT), the ΔacnB strain had decreased activities of the nickel-containing enzymes urease and hydrogenase, and this could be correlated with lower total nickel levels within ΔacnB cells. Binding of apo-AcnB to the hpn 5' untranslated region (UTR) may inhibit the expression of Hpn. In agreement with the finding that AcnB regulates the expression of antioxidant proteins such as AhpC, ΔacnB cells displayed oxidative-stress-sensitive phenotypes. The ΔacnB strain has a lesser motility ability than the WT strain, which can likely be explained by the functions of AcnB on the FlgRS-RpoN-FlgE regulatory cascade. Collectively, our results suggest a global role for aconitase as a posttranscriptional regulator in this gastric pathogen. IMPORTANCE: Bacterial survival depends on the ability of the cell to sense and respond to a variety of environmental changes. For Helicobacter pylori, responding to environmental stimuli within the gastric niche is essential for persistence and host colonization. However, there is much to be learned about the regulatory mechanisms that H. pylori employs to orchestrate its response to different stimuli. In this study, we explore the role of aconitase, a bifunctional protein that has been found to act as a posttranscriptional regulator in several other bacteria. Our results shed light on the magnitude of aconitase-mediated regulation in H. pylori, and we propose that aconitase acts as a global regulator of key genes involved in virulence.

Aconitase-mediated posttranscriptional regulation of Helicobacter pylori peptidoglycan deacetylase.

Some bacterial aconitases are bifunctional proteins that function in the citric acid cycle and act as posttranscriptional regulators in response to iron levels and oxidative stress. We explore the role of aconitase (AcnB) in Helicobacter pylori as a posttranscriptional regulator of the cell wall-modifying enzyme peptidoglycan deacetylase, PgdA. Under oxidative stress, PgdA is highly expressed and confers resistance to lysozyme in wild-type cells. PgdA protein expression as well as transcript abundance is significantly decreased in an acnB mutant. In the wild type, pgdA mRNA half-life was 13 min, whereas the half-life for the acnB strain was 7 min. Based on electrophoretic mobility shift assays and RNA footprinting, the H. pylori apo-AcnB binds to the 3'-untranslated region of the pgdA RNA transcript. Some of the protected bases (from footprinting) were localized in proposed stem-loop structures. AcnB-pgdA transcript binding was abolished by the addition of iron. The acnB strain is more susceptible to lysozyme-mediated killing and was attenuated in its ability to colonize mice. The results support a model whereby apo-AcnB directly interacts with the pgdA transcript to enhance stability and increase deacetylase enzyme expression, which impacts in vivo survival.