No abstract

SummaryPseudomonas aeruginosa has five terminal oxidases for aerobic respiration. Two of them, the bo 3 oxidase (Cyo) and the cyanide-insensitive oxidase (CIO), are quinol oxidases and the other three, the cbb 3-1 oxidase (Cbb3-1), the cbb 3-2 oxidase (Cbb3-2) and the aa 3 oxidase (Aa3), are cytochrome c oxidases. The expression pattern of the genes for these terminal oxidases under various growth conditions was investigated by using lacZ transcriptional fusions and some novel regulatory issues were found. The Aa3 genes were induced under starvation conditions. The Cyo genes were induced by exposure to the nitric oxide-generating reagent S-nitrosoglutathione. The CIO genes were induced by exposure to sodium nitroprusside as well as cyanide. The stationary phase sigma factor RpoS was found to be involved in the expression of the Aa3 and CIO genes. The role of two redox-responsive transcriptional regulators, ANR and RoxSR, was investigated using the anr and roxSR mutant strains. The ANR was involved in the repression of the CIO genes and induction of the Cbb3-2 genes. The other three terminal oxidase genes were not significantly regulated by ANR. On the other hand, all five terminal oxidase genes were shown to be directly or indirectly regulated by RoxSR. The Aa3 genes were repressed but the genes for the other four enzymes were induced by RoxSR. The transcriptome data also showed that some respiration-related genes were regulated by RoxSR, suggesting that this twocomponent regulatory system plays an important role in the regulation of respiration in P. aeruginosa.

A cellulose-degrading defined mixed culture (designated SF356) consisting of five bacterial strains (Clostridium straminisolvens CSK1, Clostridium sp. strain FG4, Pseudoxanthomonas sp. strain M1-3, Brevibacillus sp. strain M1-5, and Bordetella sp. strain M1-6) exhibited both functional and structural stability; namely, no change in cellulose-degrading efficiency was observed, and all members stably coexisted through 20 subcultures. In order to investigate the mechanisms responsible for the observed stability, "knockout communities" in which one of the members was eliminated from SF356 were constructed. The dynamics of the community structure and the cellulose degradation profiles of these mixed cultures were determined in order to evaluate the roles played by each eliminated member in situ and its impact on the other members of the community. Integration of each result gave the following estimates of the bacterial relationships. Synergistic relationships between an anaerobic cellulolytic bacterium (C. straminisolvens CSK1) and two strains of aerobic bacteria (Pseudoxanthomonas sp. strain M1-3 and Brevibacillus sp. strain M1-5) were observed; the aerobes introduced anaerobic conditions, and C. straminisolvens CSK1 supplied metabolites (acetate and glucose). In addition, there were negative relationships, such as the inhibition of cellulose degradation by producing excess amounts of acetic acid by Clostridium sp. strain FG4, and growth suppression of Bordetella sp. strain M1-6 by Brevibacillus sp. strain M1-5. The balance of the various types of relationships (both positive and negative) is thus considered to be essential for the stable coexistence of the members of this mixed culture.

In this study, the microbial community succession in a thermophilic methanogenic bioreactor under deteriorative and stable conditions that were induced by acidification and neutralization, respectively, was investigated using PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). The SSCP analysis indicated that the archaeal community structure was closely correlated with the volatile fatty acid (VFA) concentration, while the bacterial population was impacted by pH. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (i.e., a Methanoculleus sp. and a Methanothermobacter sp.) and one species of aceticlastic methanogen (i.e., a Methanosarcina sp.). The quantitative PCR of the 16S rRNA gene from each methanogen revealed that the Methanoculleus sp. predominated among the methanogens during operation under stable conditions in the absence of VFAs. Accumulation of VFAs induced a dynamic transition of hydrogenotrophic methanogens, and in particular, a drastic change (i.e., an approximately 10,000-fold increase) in the amount of the 16S rRNA gene from the Methanothermobacter sp. The predominance of the one species of hydrogenotrophic methanogen was replaced by that of the other in response to the VFA concentration, suggesting that the dissolved hydrogen concentration played a decisive role in the predominance. The hydrogenotrophic methanogens existed close to bacteria in aggregates, and a transition of the associated bacteria was also observed by FISH analyses. The degradation of acetate accumulated during operation under deteriorative conditions was concomitant with the selective proliferation of the Methanosarcina sp., indicating effective acetate degradation by the aceticlastic methanogen. The simple methanogenic population in the thermophilic anaerobic digester significantly responded to the environmental conditions, especially to the concentration of VFAs.

The ubiquitous opportunistic pathogen Pseudomonas aeruginosa has five aerobic terminal oxidases: bo 3 -type quinol oxidase (Cyo), cyanide-insensitive oxidase (CIO), aa 3 -type cytochrome c oxidase (aa 3 ), and two cbb 3 -type cytochrome c oxidases (cbb 3 -1 and cbb 3 -2). These terminal oxidases are differentially regulated under various growth conditions and are thought to contribute to the survival of this microorganism in a wide variety of environmental niches. Here, we constructed multiple mutant strains of P. aeruginosa that express only one aerobic terminal oxidase to investigate the enzymatic characteristics and in vivo function of each enzyme. The K m values of Cyo, CIO, and aa 3 for oxygen were similar and were 1 order of magnitude higher than those of cbb 3 -1 and cbb 3 -2, indicating that Cyo, CIO, and aa 3 are low-affinity enzymes and that cbb 3 -1 and cbb 3 -2 are high-affinity enzymes. Although cbb 3 -1 and cbb 3 -2 exhibited different expression patterns in response to oxygen concentration, they had similar K m values for oxygen. Both cbb 3 -1 and cbb 3 -2 utilized cytochrome c 4 as the main electron donor under normal growth conditions. The electron transport chains terminated by cbb 3 -1 and cbb 3 -2 generate a proton gradient across the cell membrane with similar efficiencies. The electron transport chain of aa 3 had the highest proton translocation efficiency, whereas that of CIO had the lowest efficiency. The enzymatic properties of the terminal oxidases reported here are partially in agreement with their regulatory patterns and may explain the environmental adaptability and versatility of P. aeruginosa.