Genetic evidence for the involvement of the S-layer protein gene sap and the sporulation genes spo0A, spo0B, and spo0F in Phage AP50c infection of Bacillus anthracis.

In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles associated with the surface of bacilli of the Sterne strain but not with the surfaces of Δsap, Δspo0A, Δspo0B, or Δspo0F mutants. The amount of Sap in the S layer of each of the spo0 mutant strains was substantially reduced compared to that of the parent strain, and incubation of AP50c with purified recombinant Sap led to a substantial reduction in phage activity. Phylogenetic analysis based on whole-genome sequences of B. cereus sensu lato strains revealed several closely related B. cereus and B. thuringiensis strains that carry sap genes with very high similarities to the sap gene of B. anthracis. Complementation of the Δsap mutant in trans with the wild-type B. anthracis sap or the sap gene from either of two different B. cereus strains that are sensitive to AP50c infection restored phage sensitivity, and electron microscopy confirmed attachment of phage particles to the surface of each of the complemented strains. Based on these data, we postulate that Sap is involved in AP50c infectivity, most likely acting as the phage receptor, and that the spo0 genes may regulate synthesis of Sap and/or formation of the S layer.

The role of the cytoplasmic heme-binding protein (PhuS) of Pseudomonas aeruginosa in intracellular heme trafficking and iron homeostasis.

The cytoplasmic heme-binding protein PhuS, encoded within the Fur-regulated Pseudomonas heme utilization (phu) operon, has previously been shown to traffic heme to the iron-regulated heme oxygenase (HO). We further investigate the role of PhuS in heme trafficking to HO on disruption of the phuS and hemO genes in a Pseudomonas aeruginosa siderophore-deficient and wild-type background. Previous studies have shown that deletion of hemO prevents the cells from utilizing heme as the sole source of iron. However, disruption of phuS alone resulted in a slow growth phenotype, consistent with its role as a heme-trafficking protein to HO. Furthermore, in contrast to the hemO and hemO/phuS deletion strains, the phuS knockout prematurely produced pyocyanin in the presence of heme. Western blot analysis of PhuS protein levels in the wild-type strain showed that Fur-regulation of the phu operon could be derepressed in the presence of heme. In addition the premature onset of pyocyanin production requires both heme and a functional HO. Suppression of the phenotype on increasing the external heme concentration suggested that the decreased heme-flux through HO results in premature production of pyocyanin. The premature production of pyocyanin was not due to lower intracellular iron levels as a result of decreased heme flux through HO. However, transcriptional analysis of the phuS mutants indicates that the cells are sensing iron deprivation. The present data suggest that PhuS has a dual function in trafficking heme to HO, and in directly or indirectly sensing and maintaining iron and heme homeostasis.

Heme inhibits the DNA binding properties of the cytoplasmic heme binding protein of Shigella dysenteriae (ShuS).

Heme uptake and utilization by pathogenic bacteria are critical for virulence and disease, since heme and heme proteins are a major source of iron within the host. Although the role of outer membrane heme receptors in this process has been extensively characterized at the genetic and biochemical level, the role of the cytoplasmic heme binding proteins is not yet clear. The Shigella dysenteriae cytoplasmic heme binding protein, ShuS, has previously been shown to promote utilization of heme as an iron source at low to moderate heme concentrations and to protect against heme toxicity at high heme concentrations. Herein, we provide evidence that ShuS of S. dysenteriae sequesters DNA non-sequence-specifically with a binding affinity of 3.6 microM as determined by fluorescence anisotropy studies. The ability to bind DNA was observed to be restricted to the apoprotein only. The molecular mass of the apo-ShuS-DNA complex was estimated to be approximately 700 kDa by size exclusion chromatography. Atomic force microscopy (AFM) revealed that apo-ShuS forms aggregates in the presence of DNA and provides a scaffolding matrix from which DNA is observed to loop outward. The AFM images of apo-ShuS-DNA complexes were strikingly similar to the AFM images of the stress-induced Escherichia coli protein, Dps, when complexed with DNA; however, unlike the Dps protein, ShuS failed to protect DNA against oxidative stress in vitro and in vivo. Since free heme can generate reactive oxygen species which are damaging to cellular DNA, the ability of ShuS to physically sequester DNA may provide a molecular basis for its role in preventing toxicity associated with high heme concentrations.