Analysis of growth phase regulated KatA and CatE and their physiological roles in determining hydrogen peroxide resistance in Agrobacterium tumefaciens

Agrobacterium tumefaciens possesses two catalases, a bifunctional catalase-peroxidase, KatA and a homologue of a growth phase regulated monofunctional catalase, CatE. In stationary phase cultures and in cultures entering stationary phase, total catalase activity increased 2-fold while peroxidase activity declined. katA and catE were found to be independently regulated in a growth phase dependent manner. KatA levels were highest during exponential phase and declined as cells entered stationary phase, while CatE was detectable at early exponential phase and increased during stationary phase. Only small increases in H2O2 resistance levels were detected as cells entering stationary phase. The katA mutant was more sensitive to H2O2 than the parental strain during both exponential and stationary phase. Inactivation of catE alone did not significantly change the level of H2O2 resistance. However, the katA catE double mutant was more sensitive to H2O2 during both exponential and stationary phase than either of the single catalase mutants. The data indicated that KatA plays the primary role and CatE acts synergistically in protecting A. tumefaciens from H2O2 toxicity during all phases of growth. Catalase-peroxidase activity (KatA) was required for full H2O2 resistance. The expression patterns of the two catalases in A. tumefaciens reflect their physiological roles in the protection against H2O2 toxicity, which are different from other bacteria.     

Inhibitory effects of basic or neutral phospholipid on acidic phospholipid-mediated dissociation of adenine nucleotide bound to DnaA protein,the initiator of chromosomal DNA replication

DnaA protein activity, the initiator of chromosomal DNA replication in bacteria, is regulated by acidic phospholipids such as phosphatidylglycerol (PG) or cardiolipin (CL) via facilitation of the exchange reaction of bound adenine nucleotide. Total lipid isolated from exponentially growing Staphylococcus aureus cells facilitated the release of ATP bound to S. aureus DnaA protein, whereas that from stationary phase cells was inert. Fractionation of total lipid from stationary phase cells revealed that the basic phospholipid, lysylphosphatidylglycerol (LPG), inhibited PG- or CL-facilitated release of ATP from DnaA protein. There was an increase in LPG concentration during the stationary phase. A fraction of the total lipid from stationary phase cells of an integrational deletion mprF mutant, in which LPG was lost, facilitated the release of ATP from DnaA protein. A zwitterionic phospholipid, phosphatidylethanolamine, also inhibited PG-facilitated ATP release. These results indicate that interaction of DnaA protein with acidic phospholipids might be regulated by changes in the phospholipid composition of the cell membrane at different growth stages. In addition, the mprF mutant exhibited an increased amount of origin per cell in vivo, suggesting that LPG is involved in regulating the cell cycle event(s).     

Evaluation of Staphylococcus aureus virulence factors using a silkworm model

Previous studies have indicated that the silkworm model is useful for identifying virulence genes of Staphylococcus aureus, a human pathogenic bacterium. Here we examined the scope of S.?aureus virulence factors that can be evaluated using the silkworm model. Gene-disrupted mutants of the agr locus, arlS gene and saeS gene, which regulate the expression of cell surface adhesins and hemolysins, exhibited attenuated virulence in silkworms. Mutants of the hla gene encoding α-hemolysin, the hlb gene encoding β-hemolysin, and the psmα and psmβ operons encoding cytolysins, however, showed virulence in silkworms indistinguishable from that of the parent strain. Thus, these S.?aureus cytolysins are not required for virulence in silkworms. In contrast, the gene-disrupted mutants of clfB, fnbB and sdrC, which encode cell-wall-anchored proteins, attenuated S.?aureus virulence in silkworms. In addition, the mutant of the srtA gene encoding sortase A, which anchors cell-wall proteins, showed attenuated virulence in silkworms. These findings suggest that the silkworm model can be used to evaluate S.?aureus cell-wall proteins and regulatory proteins as virulence factors.     

Identification and characterization of a second superoxide dismutase gene (sodM) from Staphylococcus aureus

A gene encoding superoxide dismutase (SOD), sodM, from S. aureus was cloned and characterized. The deduced amino acid sequence specifies a 187-amino-acid protein with 75% identity to the S. aureus SodA protein. Amino acid sequence comparisons with known SODs and relative insensitivity to hydrogen peroxide and potassium cyanide indicate that SodM most likely uses manganese (Mn) as a cofactor. The sodM gene expressed from a plasmid rescued an Escherichia coli double mutant (sodA sodB) under conditions that are otherwise lethal. SOD activity gels of S. aureus RN6390 whole-cell lysates revealed three closely migrating bands of activity. The two upper bands were absent in a sodM mutant, while the two lower bands were absent in a sodA mutant. Thus, the middle band of activity most likely represents a SodM-SodA hybrid protein. All three bands of activity increased as highly aerated cultures entered the late exponential phase of growth, SodM more so than SodA. Viability of the sodA and sodM sodA mutants but not the sodM mutant was drastically reduced under oxidative stress conditions generated by methyl viologen (MV) added during the early exponential phase of growth. However, only the viability of the sodM sodA mutant was reduced when MV was added during the late exponential and stationary phases of growth. These data indicate that while SodA may be the major SOD activity in S. aureus throughout all stages of growth, SodM, under oxidative stress, becomes a major source of activity during the late exponential and stationary phases of growth such that viability and growth of an S. aureus sodA mutant are maintained.     

The production of extracellular proteins is regulated by ribonuclease III via two different pathways in Staphylococcus aureus

Staphylococcus aureus ribonuclease III belongs to the enzyme family known to degrade double-stranded RNAs. It has previously been reported that RNase III cannot influence cell growth but regulates virulence gene expression in S. aureus. Here we constructed an RNase III inactivation mutant (Δrnc) from S. aureus 8325-4. It was found that the extracellular proteins of Δrnc were decreased. Furthermore, we explored how RNase III regulated the production of the extracellular proteins in S. aureus. We found during the lag phase of the bacterial growth cycle RNase III could influence the extracellular protein secretion via regulating the expression of secY2, one component of accessory secretory (sec) pathway. After S. aureus cells grew to exponential phase, RNase III can regulate the expression of extracellular proteins by affecting the level of RNAIII. Further investigation showed that the mRNA stability of secY2 and RNAIII was affected by RNase III. Our results suggest that RNase III could regulate the pathogenicity of S. aureus by influencing the level of extracellular proteins via two different ways respectively at different growth phases.     

Early expression of SCIN and CHIPS drives instant immune evasion by Staphylococcus aureus

Chemotaxis inhibitory protein of staphylococci (CHIPS) and Staphylococcal complement inhibitor (SCIN) are small, excreted molecules that play a crucial role in the staphylococcal defence against the human innate immune system. Here we show that they both counteract crucial acute responses of our immune system such as complement activation, neutrophil chemotaxis and neutrophil activation. By studying gene expression via promoter-green fluorescent protein fusions, Northern blots and protein expression analyses, we show that SCIN and CHIPS are produced during the early (exponential) growth stages. Although the SCIN and CHIPS genes are expressed simultaneously, they are differently regulated by various Staphylococcus aureus regulatory loci. However, the sae locus is crucial for upregulation of both SCIN and CHIPS. This is the first study that presents the expression of two extracellular S. aureus proteins early during growth. Because SCIN and CHIPS are both efficient modulators of neutrophil chemotaxis, phagocytosis and killing, their early expression is necessary for efficient modulation of the early immune response.