As a result of analyzing the internal transcribed spacer (ITS) and 5 0 end of the EF-1a sequence of 145 isolates of Metarhizium spp. isolated from soil in Japan using selective agar medium, eight species were identified. ITS sequence analysis divided the isolates into three clades. Two were identified as M. flavoviride var. pemphigi and M. lepidiotae, respectively. EF-1a sequence analysis identified the other clades as six species: M. anisopliae, M. brunneum, M. guizhouense, M. majus, M. pingshaense and M. robertisii. The distribution of M. flavoviride var. pemphigi was restricted to forest or wood soil, and conidial sizes of M. guizhouense and M. majus were incongruent with the phylogeny based on the sequence of the 5 0 end of EF-1a.

To investigate the role of superoxide dismutase (SOD) in virulence against the silkworm, Bombyx mori, mutants of Pseudomonas aeruginosa PAO1 lacking manganese-SOD (PAO1sodM), iron-SOD (PAO1sodB), or both (PAO1sodMB) were generated. The mutants were injected into the hemocoel of B. mori. The virulence decreased in the order PAO1 ‫؍‬ PAO1sodM > PAO1sodB > PAO1sodMB. In particular, PAO1sodMB was avirulent at a dose of 10 5 cells or less. The sod double mutant PAO1sodMB was then complemented with either pSodM or pSodB in trans. In both the complemented strains, the virulence was partially restored. Of the two plasmids, pSodB contributed more to the virulence of P. aeruginosa against B. mori. The results of growth in B. mori hemolymph broth and microscopic analysis suggested that a longer lag phase and superoxide sensitivity correlated with decreased virulence in sod mutants. In conclusion, the SODs are required for full virulence of P. aeruginosa against B. mori and Fe-SOD is more important than Mn-SOD in the infection process.Pseudomonas aeruginosa is ubiquitous and is found in diverse environments including soil, freshwater, and marine environments. It is also an opportunistic pathogen in three distinct groups of organisms: vertebrates, invertebrates, and plants (17,18).Similar to other pathogens, P. aeruginosa must overcome the oxidative stress response generated by the host for successful infection. During the process of active infection, the primary source of exogenous oxidative stress for pathogenic bacteria is attack by host phagocytic cells. Phagocytes utilize the cytotoxic effects of many of the reactive oxygen species, such as superoxide, hydrogen peroxide, and the highly toxic hydroxyl radical. These reactive oxygen species can damage the nucleic acids, proteins, and cell membranes of pathogens. On the other hand, pathogens have effective enzymatic pathways of oxidant inactivation, including those catalyzed by superoxide dismutase (SOD), catalase/peroxidase, and glutathione in combination with glutathione peroxidase and glutathione reductase (12, 13). SOD represents the first line of defense against superoxide stress by converting superoxide into hydrogen peroxide and oxygen, thereby protecting cells from the toxic effects of superoxide.P. aeruginosa possesses both manganese-cofactored SOD (Mn-SOD) and iron-cofactored SOD (Fe-SOD), and the genes encoding these proteins (sodM, previously referred to as sodA, and sodB, respectively) have been cloned and characterized (14,15,16). The expression of these SODs is controlled by environmental factors. In the presence of relatively high concentrations of extracellular iron, Fe-SOD is preferentially expressed, whereas the expression of Fe-SOD decreases and Mn-SOD is produced under iron-limited conditions (5, 14). For example, P. aeruginosa PAO1 produces mainly Fe-SOD in Luria-Bertani (LB) broth. In contrast Mn-SOD is produced in low-phosphate succinate (LPS) broth (16).Since superoxide is endogenously produced under aerobic conditions, inactivation of SOD often re...

To investigate the pathogenicity of Pseudomonas aeruginosa in insects, a gacA mutant of P. aeruginosa PA01 was constructed by site-directed mutagenesis. The mutant was designated as C1. C1 was less virulent to Bombyx mori than the parent strain. To complement the gacA gene, P. aeruginosa C1 was transformed with the broad host range plasmid pJB3Km1 carrying a 3.9-kbp gacA fragment. The expression of the gacA mRNA in C1 (pgacA) was detected. In addition, the complemented mutant restored the level and timing of pyocyanin production, indicating that functional GacA is produced in the complemented strain. However, no significant difference was observed between C1 and C1 (pgacA) with respect to the killing of B. mori larvae.

The role of the alkaline protease (AprA) in pyocyanin production in Pseudomonas aeruginosa was investigated. AprA was overproduced when a plasmid carrying the aprA gene was introduced to an aprA-deletion mutant strain, EG03; thus, aprA-complemented EG03 was used as an overproducing strain. The complemented strain produced higher pyocyanin than the mutant strain in all commercially available media evaluated. Particularly, pyocyanin production was higher in the complemented than in the parental strain in brain-heart infusion and tryptic soy broths. These results suggested that protein degradation products by AprA were utilized for pyocyanin production. Protein-rich media were used in subsequent validation studies. Similar results were obtained when the basal medium was supplemented with casein or skim milk as the sole organic nitrogen source. However, gelatin failed to induce abundant pyocyanin production in the complemented strain, despite the presence of protein degradation products by AprA as assessed by SDS-PAGE. Thus, gelatin degradation products may not be suitable for pyocyanin synthesis. In conclusion, AprA could contribute to pyocyanin production in the presence of several proteins or peptides.

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