Longnecker, K., E.B. Sherr, B.F. Sherr . Cell-specific rates of leucine incorporation by HNA, LNA, and CTC-positive bacterioplankton in an upwelling ecosystem.

There are several different methods used to assess the level of metabolic activity in marine bacterioplankton, however there is no consensus on which method is best. Furthermore, different methods used to assess the level of metabolic activity can result in different answers about the variation of metabolic activity.

Three methods used to assess metabolic activity are:

1. Measurement of the incorporation of radioactively-labeled metabolic precursors, such as thymidine or leucine (Kirchman 1993)
2. Use of a redox sensitive compund that fluoresceces when reduced by an active electron transport system (CTC, Rodriguez et al. 1992; Smith and McFeters 1997)
3. Research in the Mediterranean and Celtic Sea has raised the possibilty of using the amount of nucleic acids within a cell as a proxy for metabolic acitivity (Lebaron et al. 2001; Zubkov et al. 2001). Lebaron et al. (2001) have shown that cells with high cell-specific nucleic acid contents (HNA cells) are more metabolically active than cells with low cell-specific nucleic acid contents (LNA cells).

We set out on the RV Wecoma in 2002 to address the variability in marine bacterioplankton metabolic activity using samples collected from stations west of Newport, Oregon (Figure1). Immediately after water samples were collected, they were incubated with tritiated leucine and, for some of the samples, CTC. After our return to shore the samples were processed following the standard methods for bacterial production, or were sorted on a flow cytometer into four groups: HNA cells, LNA cells, total bacteria (sum of the HNA and LNA cell regions), and CTC-positive cells. Sorting the different populations on the flow cytometer has allowed us to determine the cell-specific leucine incorporation individually for each group.

Our preliminary results indicate the the cell-specific leucine incorporation rates for the HNA cells are higher than the rates for the LNA cells. Furthermore, on a cell-specific level the leucine incorporation rates for the CTC-positive cells are higher than for the HNA or LNA cells, however there are samples where the leucine incorporation rates for the CTC-positive cells can be less than the leucine incorporation rates for the heterotropic bacterioplankton. The cell-specific leucine incorporation rates decreased as the distance from shore increased, indicating the cells are, in general, more active in the near shore environment (Figure 2).

We defined the total leucine incorporation as the cell-specific leucine incorporation for a group multiplied by the abundance of the group. As volumetric bacterial production from the samples processed following standard methods increased, so did the total leucine incorporation rate for the total bacterial cells sorted on the flow cytometer (Figure 3). Examining the total leucine incorporation for each of the different groups sorted on the flow cytometer indicated that the HNA cells were responsible for most of the bacterial production in all three ecosystems (Table 1). However, the percent of the bacterial production attributable to the LNA cells and the CTC-positive cells increased at the offshore station. The increased role of the LNA cells at the offshore station may indicate a different population of cells inhabiting the offshore station, or may indicate the LNA cells are more active at that station due to the different environmental conditions present at the offshore station compared to the inshore stations.

Table 1	Total production	Mean percent attributable to:
Region	(pMol Leu L-1 hr-1)	HNA cells	LNA cells	CTC+ cells
Shelf	2-481	116%	25%	7%
Slope	2-159	72%	25%	7%
Offshore	0.2-29	82%	38%	14%

References cited:

Kirchman, D. L. 1993. Leucine incorporation as a measure of biomass production by heterotrophic bacteria, p. 509-512. In P. F. Kemp, B. F. Sherr, E. B. Sherr and J. J. Cole [eds.], Current Methods in Aquatic Microbial Ecology. Lewis Publishing.

Lebaron, P., P. Servais, H. Agogué, C. Courties, and F. Joux. 2001. Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems? Appl. Environ. Microbiol. 67: 1775-1782.

Rodriguez, G. G., D. Phipps, K. Ishiguro, and H. F. Ridgway. 1992. Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl. Environ. Microbiol. 58: 1801-1808.

Smith, J. J., and G. A. McFeters. 1997. Mechanisms of INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride), and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) reduction in Escherichia coli K-12. J. Microbiol. Methods 29: 161-175.

Zubkov, M. V., B. M. Fuchs, P. H. Burkill, and R. Amann. 2001. Comparison of cellular and biomass specific activities of dominant bacterioplankton groups in stratified waters of the Celtic Sea. Appl. Environ. Microbiol. 67: 5210-5218.