Denitrification is a well-studied respiratory system that is also important in the biogeochemical nitrogen cycle. Environmental signals such as oxygen and N-oxides have been demonstrated to regulate denitrification, though how denitrification is regulated in a bacterial community remains obscure. Pseudomonas aeruginosa is a ubiquitous bacterium that controls numerous genes through cell-to-cell signals. The bacterium possesses at least two N-acyl-L-homoserine lactone (AHL) signals. In our previous study, these quorum-sensing signals controlled denitrification in P. aeruginosa. In addition to the AHL signals, a third cell-to-cell communication signal, 2-heptyl-3-hydroxy-4-quinolone, referred to as the Pseudomonas quinolone signal (PQS), has been characterized. In this study, we examined the effect of PQS on denitrification to obtain more insight into the respiratory regulation in a bacterial community. Denitrification in P. aeruginosa was repressed by PQS, which was partially mediated by PqsR and PqsE. Measuring the denitrifying enzyme activities indicated that nitrite reductase activity was increased by PQS, whereas PQS inhibited nitric oxide reductase and the nitraterespiratory chain activities. This is the first report to demonstrate that PQS influences enzyme activities, suggesting this effect is not specific to P. aeruginosa. Furthermore, when iron was supplied to the PQS-added medium, denitrifying activity was almost restored, indicating that the iron chelating property of PQS affected denitrification. Thus, our data indicate that PQS regulates denitrification primarily through iron chelation. The PQS effect on denitrification was relevant in a condition where oxygen was limited and denitrification was induced, suggesting its role in controlling denitrification where oxygen is present.Bacteria regulate their metabolism by sensing environmental signals in order to adapt to various environmental conditions. In the environment, a number of bacteria are capable of using N-oxides as alternative electron acceptors of oxygen. Denitrification is a mode of anaerobic respiration in which nitrate (NO 3 Ϫ ) or nitrite (NO 2 Ϫ ) is reduced to gaseous Noxides, such as nitric oxide (NO), nitrous oxide (N 2 O), and nitrogen (N 2 ), concomitant with energy generation (61). The switch from aerobic respiration to denitrification is usually known to be regulated by a response to N-oxides and oxygen levels (2, 28, 31). These N-oxides and oxygen levels are sensed through the CRP/FNR (cAMP receptor protein/fumarate and nitrate reductase regulator) family, the members of which are global regulators that activate transcription of denitrifying genes (49). In Pseudomonas aeruginosa, a ubiquitous gramnegative environmental bacterium, denitrifying genes are also regulated by N-oxides and oxygen levels through a regulatory network requiring ANR, DNR regulatory proteins, and a nitrate-responding two-component regulator, NarXL (45).Although how respiration is regulated by physicochemical factors such as oxygen and N-oxide concentrations is...

The mouse calvarial osteoblast MC3T3-E1 cells released 92 kDa and 68 kDa of gelatinase activities into the conditioned media (CMs) from undifferentiated cells. When differentiation was induced by cultivating cells with ascorbate-2-phosphate (AscP), 68-kDa activity increased significantly in parallel with production of 60-kDa activity. These enzymes required Ca 2؉ and Zn 2؉ ions for their proteolytic activities. The 68-kDa activity was immunologically identified as latent matrix metalloproteinase 2 (MMP-2). The 92-kDa activity was deduced to be latent MMP-9 based on its molecular mass. The 60-kDa activity band was found to possess both gelatin and ␤-casein hydrolyzing activities, indicating that this activity band might comprise the active form of MMP-2 and latent MMP-13. MC3T3-E1 cells were found to express MMP-2, MMP-13, and membrane type (

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Chitin, a water insoluble β (1,4) linked polymer of N acetyl D glucosamine (GlcNAc), is one of the most abundant renewable forms of biomass. In the natural environment, chitin is decomposed mainly by microorganisms that can utilize it as a nutrient source. Various hydrolases are involved in chitin degradation [i.e., chitinase (EC 3.2.1.14), β N acetylhexosaminidase (EC 3.2.1.52), chitin deacetylase (EC 3.5.1.41) and chitin oligosaccharide deacetylase (COD, EC 3.1.1)]. Chitinivorous microorganisms produce water soluble mono or oligosaccharides from chitin using one or several of these chitinolytic enzymes, and then transport these soluble saccharides into the cell for further degradation. In a previous paper, we reported that Vibrio parahaemolyticus KN1699 produces the heterodisaccharide β N acetyl D glucosaminyl (1,4) D glucosamine (GlcNAc GlcN) as the primary chitin degradation product using following two types of extracellular enzymes: glycoside hydrolase family 18 chitinase, which produces N,N diacetylchitobiose, (GlcNAc)2, from chitin, and carbohydrate esterase (CE) family 4 COD, which hydrolyzes the N acetyl group at the reducing end GlcNAc residue of (GlcNAc)2. Recently, we clarified that GlcNAc GlcN is not only a nutrient for strain KN1699, but also functions as an inducer of chitinase production by this bacterium.4) Moreover, effect of this heterodisaccharide on the chitinase induction was confirmed also in other chitin decomposing Vibrio strains harboring CE family 4 COD genes. These findings suggest that CODs involved in the synthesis of this signal compound are key enzymes for chitin catabolism in some species of Vibrio.To date, aside from COD of strain KN1699, only two CODs, one from Vibrio alginolyticus H 8 5,6) and one from Vibrio cholerae EI Tor N16961, 7) have been purified and characterized. Although these three enzymes have high amino acid sequence homologies (80 99%), their specificities for chitin oligosaccharides are different, indicating the importance of characterizing other microbial CODs. We therefore screened natural sources for bacteria that secrete enzymes that can convert (GlcNAc)2 to GlcNAc GlcN. As a result, we isolated a target bacterium (strain SN184) from the bacteria that adhered on the surface of α chitin flakes (wrapped with tea bag filter paper) sunk in the sea near Tsumekizaki (Shimoda City, Shizuoka Prefecture, Japan). Gram stain results indicated that strain SN 184 is Gram negative. The genotype of strain SN184 was investigated by comparing the nucleotide sequence of its 16S rDNA (GenBank accession no. AB469367) to the sequence database BLASTN. The results confirmed that the isolate is most closely related to many strains of the genus Vibrio (about 97% identity). Although we tried to determine the species of this isolate by investigating its physiological characteristics, it was not possible to determine the species of this bacterium using the methodology outlined in Bergey s Manual of Systematic Bacteriology. 8)The bacterium was therefore named Vibrio sp. SN184.To clone ...