Prokaryotic aminopeptidase activity along a continuous salinity gradient in a hypersaline coastal lagoon (the Coorong, South Australia)
© Pollet et al; licensee BioMed Central Ltd. 2010
Received: 23 December 2009
Accepted: 30 April 2010
Published: 30 April 2010
The distribution and aminopeptidase activity of prokaryotes were investigated along a natural continuous salinity gradient in a hypersaline coastal lagoon, the Coorong, South Australia. The abundance of prokaryotes significantly increased from brackish to hypersaline waters and different sub-populations, defined by flow cytometry, were observed along the salinity gradient. While four sub-populations were found at each station, three additional ones were observed for 8.3% and 13.4%, suggesting a potential modification in the composition of the prokaryotic communities and/or a variation of their activity level along the salinity gradient. The aminopeptidase activity highly increased along the gradient and salinity appeared as the main factor favouring this enzymatic activity. However, while the aminopeptidase activity was dominated by free enzymes for salinities ranging from 2.6% to 13.4%, cell-attached aminopeptidase activity was predominant in more saline waters (i.e. 15.4%). Changes in substrate structure and availability, strongly related to salinity, might (i) modify patterns of both aminopeptidase activities (free and cell-associated enzymes) and (ii) obligate the prokaryotic communities to modulate rapidly their aminopeptidase activity according to the nutritive conditions available along the gradient.
Dissolved proteins and peptides are important sources of energy and nitrogen in aquatic systems [1, 2], but they must be hydrolysed to amino acids and oligopeptides to be useable by prokaryotes. Following the development of sensitive methods using fluorogenic substrates , proteolytic activity in natural aquatic systems has been assessed by measuring the activity of leucine-aminopeptidase as a model enzyme . However, microbial cells living in aquatic systems are influenced by a variety of environmental factors which affect the molecular control of their enzyme synthesis. Among these variables, salinity has been identified as a major driving force in both the composition of bacterioplankton and their efficiency in degrading dissolved organic carbon (DOC) . Previous studies focusing on the effect of salinity on the composition and metabolic activity of bacterial communities were mainly conducted in estuaries where salinity typically did not exceed 5%  and the effect of higher salinity conditions was mainly investigated in highly saline ponds from solar salterns . To our knowledge, little is still known about the dynamic of prokaryotic aminopeptidase activity along natural continuous hypersaline gradients. The objective of this study was to investigate the changes in aminopeptidase activity of prokaryotic communities identified using flow cytometry from brackish to hypersaline waters.
Sub-surface samples were collected at 4 stations (S1, S2, S3 and S4; Fig. 1) characterized by increasing salinities, i.e. 2.6%, 8.3%, 13.4% and 15.4%. Temperature (°C), conductivity (mS cm-1) and dissolved oxygen concentrations (DO; mg l-1) were recorded using a YSI 85 (Fondriest) multiparameter probe. Salinity (%) was calculated from temperature and conductivity following Fofonoff and Millard . Water samples were collected at each station using acid-washed 1-liter borosilicate bottles.
Physical and chemical parameters measured along the salinity gradient.
DO (mg l-1)
[NO3-] + [NO2-] (μM)
[Chl a] (μg l-1)
SPM (mg l-1)
Prokaryotic populations were identified and enumerated by flow cytometry (FCM) using a FACScanto flow cytometer. Samples were fixed and prepared following Brussaard . Sub-populations were discriminated based on the differences in SYBR-I Green fluorescence and right-angle light scatter (SSC). Fluorescent beads 1 μm in diameter were added to all samples as an internal standard. Working bead concentrations were estimated after each FCM session under epifluorescent microscopy to ensure reliability of the bead concentration and all FCM parameters were normalized to bead concentration and fluorescence. Finally, populations were identified and enumerated using WinMDI 2.9 (©Joseph Trotter) flow cytometry analysis software. No significant differences were found between FCM counts and epifluorescence microscopy (EM) counts conducted at each station (Wilcoxon-Mann-Whitney U-test, n = 5, p > 0.05).
Aminopeptidase activity was estimated using the fluorogenic substrate analog, L-leucine-4-methyl-coumarinyl-7amide (Leu-AMC). AMC fluorescence was determined at 340 nm (excitation) and 440 nm (emission), with a spectrofluorometer (Hitachi Fluorescence Spectrophotometer, Model F-3000) previously calibrated. Total enzymatic activity (i.e. free enzymes dissolved in water and cell surface bound enzymes) and free extracellular enzymatic activity were estimated for each sampling site. For free enzymatic activity, water samples were previously gravity filtered through 0.2 μm pore size filters. Before each spectrophotometry analysis, subsamples without substrates were used as blanks to determine the background fluorescence of the samples at each sampling station. Aminopeptidase activity was quantified through Michaelis-Menten kinetic parameters: the highest rate of substrate hydrolysis V max (μM h-1) and the half-saturation constant for the enzyme K m (μM), which indicates the enzyme affinity to the substrate.
Free and cell-associated aminopeptidase activities exhibit different patterns in relation to salinity. From 2.6% to 13.4%, free aminopeptidase activity seems to be favoured; for higher salinity (15.4%), cell-associated aminopeptidase activity is preferred (Fig. 4A). Under the assumption that protein substrates were likely more available at the hypersaline station (S4), this observation is congruent with Hollibaugh and Azam 's conclusions. Free enzymes could be less important in the protein degradation and close physical association between prokaryotes and proteins would be necessary for efficient protein degradation. This may explain the dominance of cell-associated aminopeptidase activity observed at the hypersaline station. In addition, the changes in ionic strength related to salinity might affect the structure of substrate molecules and consequently the activity of extracellular enzymes . Indeed, the solubility of proteins is known to be profoundly affected by the ionic strength and particularly by the presence of divalent cations . This low solubility of proteins under hypersaline conditions might favour the prokaryote/protein association and might thus explained the higher cell-associated aminopeptidase activity observed at the hypersaline station. Moreover, the increase in SPM concentration along the salinity gradient may also favour the creation of microscale environments leading to local hotspots of prokaryotes attached to particles . This may also explained the observed transition from free to cell-attached aminopeptidase activity along the gradient (Fig. 4A) and the increase in substrate affinity observed at station S4 (Fig. 4B) for high salinity values. It is finally stressed that free-aminopeptidase activity is unlikely to have been contaminated by cell lysis, hence over-estimated, because of the non-destructive gravity filtration conducted here. Note, however, that at the highest salinity, free enzymes might aggregate with particulate matter, leading to an overestimation of the cell-associated enzymatic activity. While this is beyond the aims of the present work, further work is needed to assess the contribution of salinity in bounding free enzymes to particulate material.
In accordance with previous reports, this first study performed along a continuous salinity gradient has shown that the increase in salinity appeared as the main factor favouring aminopeptidase activity. However, both aminopeptidase activities (free and cell-associated enzymes) are also influenced by the availability and structure of suspended materials that is susceptible of strong changes along the salinity gradient. Prokaryotic communities have then to rapidly modulate their aminopeptidase activities to optimize their fitness in response to the variability of the nutritive conditions along the salinity gradient.
Given the key role played by microbial communities in the functioning of aquatic systems, these results stress the need to extend our knowledge concerning the effect of salinity on the dynamics and activity of microbial communities in natural systems particularly in the context of global change which particularly affects local ecosystems, such as the Coorong, through changes in salinity related to modifications of freshwater discharge and evaporation. Further work is thus needed to assess the interplay between salinity and the global enzymatic activity of prokaryotic communities.
The authors wish to thank Dr. S. Bailey from the Flow Cytometry Unit of the Flinders Medical Centre, for providing technical support during the flow cytometry work. This work was supported financially and infrastructurally by the Australian Research Council (Discovery Projects DP0664681 and DP0666420) and Flinders University. Professor Seuront is the recipient of an Australian Professorial Fellowship (project number DP0988554).
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