ReportProkaryotic aminopeptidase activity along a continuous salinity gradient in a hypersaline coastal lagoon ( the Coorong , South Australia )

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 cellassociated enzymes) and (ii) obligate the prokaryotic communities to modulate rapidly their aminopeptidase activity according to the nutritive conditions available along the gradient. Findings 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 [3], proteolytic activity in natural aquatic systems has been assessed by measuring the activity of leucine-aminopeptidase as a model enzyme [4]. 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) [5]. 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% [6] and the effect of higher salinity conditions was mainly investigated in highly saline ponds from solar salterns [7]. 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. The Coorong is a South Australian shallow coastal lagoon characterized by a strong salinity gradient with salinity continuously ranging from brackish (1.8%) to hypersaline (15.5%). Constrained between the last interglacial dune and the modern dune that has been established from the mid-holocene, this lagoon receives inputs from the ocean through the Murray Mouth and from underground and freshwater inputs from Lake Alexandrina and Lake Albert, which are the terminal system of the River Murray (Fig. 1). If freshwater inputs lead to lower salinities in the northwest part of the Coorong, the excess in evapora* Correspondence: thomas.pollet@thonon.inra. fr 1 School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide SA 5001, Australia Full list of author information is available at the end of the article © 2010 Pollet et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pollet et al. Saline Systems 2010, 6:5 http://www.salinesystems. org/content/6/1/5 Page 2 of 6 tion over precipitation increases salinity along the northsouth axis, especially during the summer period. 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 [8]. Water samples were collected at each station using acid-washed 1-liter borosilicate bottles. Water temperature ranged between 25.2 and 27.7°C. DO concentrations decreased from 3.46 mg l-1 in S1 to 1.48 mg l-1 in S4 (Table 1). Concentration of suspended particular matter (SPM), determined following Hewson et al [9], increased from 38 mg l-1 at S1 to 540 mg l-1 at S4 (Table 1). Nutrient concentrations were determined in the field using a portable LF 2400 photometer according to standard colorimetric methods for (Indophenol blue), (Naphtylethylene diamine), (Naphtylethylene diamine after zinc reduction) and (Ascorbic acid reduction). Ammonium was the most abundant form of nitrogen with concentrations consistently increasing from 2.5 μM in S1 to more than 110 μM in S3 and S4. Phosphate concentrations were low at S1, S2 and S3 (i.e. < 8.5 μM) whereas S4 was characterized by high phosphate concentrations (> 50 μM). Chl a (μg l-1), determined following Strickland and Parson [10] using a Turner 450 fluorometer after extraction in methanol of the samples collected on glass-fiber filters, were low at S1, S2 and S4 (i.e. < 3 μg l-1). In contrast, S3 was characterized by relatively high Chl a concentrations (13.5 μg l-1; Table 1). Prokaryotic populations were identified and enumerated by flow cytometry (FCM) using a FACScanto flow cytometer. Samples were fixed and prepared following Brussaard [11]. Sub-populations were discriminated based on the differences in SYBR-I Green fluorescence and rightangle 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). In accordance with previous observations from solar salterns [12], the abundance of prokaryotes showed a significant increase with salinity (p < 0.05), with values ranging from 2.1 × 106 ml-1 at S1 to 1.7 × 108 ml-1 at S4 (Fig. 2). The high SPM and phosphate concentrations observed at the hypersaline station (S4) are favourable to high microbial abundance [13-17]. In addition, the decrease in viral lysis and bacterivory as well as the rapid growth of bacteria under high salinity conditions (i.e. > 15%) [12,18], might also have contributed to the high prokaryotic abundance observed in the hypersaline part of the lagoon. The prokaryotic cytometric richness also varied along the gradient (Fig. 3). Four to seven discrete sub-populations of prokaryotes were identified along the salinity gradient. Four distinct sub-populations were observed at S1 (S = 2.6%) and S4 (S = 15.4%) (Fig. 3A) whereas 7 and 6 subpopulations were identified at S2 (S = 8.3%) and S3 (S = 13.4%), respectively (Fig. 3B). The variability of the prokaryotic cytometric richness observed along the salinity gradient (Fig. 3C) could reflect a modification of both bacterial populations and their activity level [19]. It is now well known that salinity represents one of the main factors structuring the distribution of prokaryotic assemblages, favouring the dominance of some groups adapted NH4 +