Exoproteolytic activity in an Atlantic pond (France): estimates of in situ activity

Abstract

In order to estlrnate In n t u bactenal exoproteolysis and its contribution to bactenal secondary production (BSP), we studied microbial activities (BSP and kinetlcs of exoproteolytic activity) in relation to d~ssolved comblned amino acids (DCAA) dunng the survey of a n Atlantic marine pond In thls pond, high concentrations of DCAA (mean = 4 27 pM), probably generated by zooplankton grazing, allowed large bacterial biomass development and high production (max = 10" cells I-' and 18 8 X log cells 1-' d-' respectively) Exoproteolytic enzyme activity (mean V, = 55 9 pM d-l) was tightly coupled to bactenal production, suggesting that protein utlllzation was essential for bacterial growth Calculation, according to the Michaelis-Menten equation of the dissolved protein In sltu hydrolysis rates using the lunetic parameters (V, and K,,,) and the ambient concentratlon of DCAA proved to be a better est~rnation of the actual rate of hydrolytic activlty than the potential activlty based on V,,, only The rates found with thls calculat~on agreed wlth those obtalned from different environments with other methods (DCAA and labelled protelns uptake) Furthermore our estimates of In sltu proteolys~s were consistent with BSP (nitrogen fluxes onglnating from dissolved protein hydrolysis averaging 56% of the N bactenal production), and as a result, these estimates seem to provide a realistic estimation of the actual dissolved protein hydrolysis rates KEY WORDS: Bacterioplankton . In situ exoproteolytic hydrolysis rate . Kinet~c parameters . DCAA Manne pond

Cell-surface proteolytic activity of photosynthetic dinoflagellates

“…LAP activity of non-axenic dinoflagellate cultures, 20°C temperate coastal and estuarine waters (Martinez & Azam 1993a, Crottereau & Delmas 1998. During dense blooms, dinoflagellates are expected to elevate LAP in the nano-and microplankton size fractions, resulting in an increase in total activity.…”

“…For assays, 20 µl of the substrate stock was added to 1 ml of culture, natural sample, or filtrate in semi-UV cuvettes (Fisherbrand); this resulted in a final substrate concen- The samples were incubated in the cuvettes in the dark at 15 or 20°C, as appropriate, for 30 min. At the end of the incubation the reactions were stopped by adding 100 µl of 10% sodium dodecyl (lauryl) sulfate to each vial and either stored frozen (-20°C) until fluorescence was determined (Crottereau & Delmas 1998) or readings were made immediately with a Shimadzu Rf-5301PC spectrofluorometer (excitation 380 nm with a 10 nm slit, emission 440 nm with a 1.5 nm slit width). A standard curve was prepared using concentrations of the product, 7-amino-4-methyl-coumarin (AMC) (Sigma Chemical), ranging from 0 to 10 µM in sterile seawater media.…”

“…In contrast, ecto-enzymes are bound to the cell surface or are found in the periplasmic space (Chróst 1991). Leucine aminopepidase (LAP) activity is commonly used as a measure of exo-and ecto-proteolytic activity in the plankton (Rosso & Azam 1987, Martinez et al 1996, Crottereau & Delmas 1998. LAP degrades polypeptides sequentially from the N-terminus.…”

Extracellular enzyme activity and uptake of carbon and nitrogen along an estuarine salinity and nutrient gradient

“…Commercially available dipeptide-like substrates, such as L-leucine 7-amido-4-methyl-coumarin (leu-MCA), have been used to assess leucine aminopeptidase activity (e.g. Rosso & Azam 1987, Crottereau & Delmas 1998, Stoecker & Gustafson 2003. In this study of the Pocomoke River, LYA-dipeptides were the primary products of hydrolysis; further hydrolysis of dipeptides to free amino acids was very slow.…”

Virus-like particle analysis in a seston-rich coastal pond using transmission electron microscopy

“…These ponds exhibit high concentrations of suspended particulate matter, nutrients and DOM (Robert et al 1982). Together, they support bacterial abundances 10-fold higher than in the open sea (Frikha et al 1987, Crottereau & Delmas 1998, as well as high concentrations of phytoplankton (1 to 17 µg chl a l -1; Delmas et al 1992) and protozoa (0.1 to 20 × 10 6 flagellates and 0.5 to 8 × 10 4 ciliates l -1 ; Dupuy et al 2000). In order to assess virus abundance in these seston-rich waters, we needed to develop a method to separate seston (particles ≥0.2 µm) from virus particles.…”