Proteolytic enzymes in the marine bacterium Pseudoalteromonas atlantica: Post-secretional activation and effects of environmental conditions

Abstract

Production and activities of cellular and extracellular proteolytic enzymes associated with the marine bacterium Pseudoalteromonas atlantica were examined in response to physiological state and changing environmental variables under laboratory conditions. Enzymes evidenced by zymography were metalloproteases, as defined by substrate preference and susceptibility to inhibitors (EDTA and 1,10-phenanthroline). Distinct bands (i.e., molecular-weight [MW] size classes) of enzymes were isolated from within cellular and extracellular compartments. Susceptibility of extracellular enzymes to environmental stressors was related, in part, to the MW size classes of the enzymes. While all bands of enzymes showed some degree of inhibition when pH was decreased, the very high MW (103 to 93 kDa) and the very low MW (34 to 31 kDa) bands of extracellular enzymes were most susceptible. Some mid-sized enzymes (75 to 69 kDa) were most resilient, and remained active at pH as low as 4.4 and as high as 10.5-values which may occur in seawater micro-environments during high levels of heterotrophic respiration and photosynthetic activities, respectively. Extracellular enzymes retained activity in seawater (25 ppt) as long as 15 d after secretion. Finally, our data strongly suggest that extracellular enzymes, which were recently secreted by cells, may be modified post-secretion, as evidenced by changes in MW banding patterns over time (in presence of chloramphenicol). This study showed that P. atlantica produces a flexible array of extracellular enzymes. In ocean systems the adaptive value of flexibility in enzyme activities may relate to changing microenvironments which bacteria may encounter over microspatial scales.