Properties of the Inducible Hydroxyproline Transport System of Pseudomonas putida

Abstract

Features of the transport system for hydroxyproline in a strain of Pseudomonas putida were studied. A mutant, lacking hydroxyproline-2 epimerase and unable to metabolize hydroxy-L-proline, was shown to transport and accumulate this compound after induction. Both entry and exit rates were examined, and kinetic constants for the reaction were determined. Increasing the induction time from 0.5 to 3 hr increased the entry rate threeto fourfold but had only a small and variable effect on the exit rate. Entry followed saturation kinetics. For hydroxy-L-proline, the Km and Vmax values were found to be 3 X 10-5 M and 6 jAmoles per g (dry weight) per min, respectively. The Km and Vmax for the epimer allohydroxy-D-proline were 10-J M and 0.1 jbmole per g (dry weight) per min. Entry rates into "loaded" and "unloaded" cells were found to be the same. Exit was shown to be first order over the range of internal substrate concentrations measured. Exit rates were measured by several different methods and found to be independent of external substrate concentration. The first-order exit rate constant was computed to be 0.23 min-'. Several metabolic inhibitors were examined for their effect on transport. The inhibitory action of N-ethyl maleimide was shown to be greatly reduced if cells were allowed to accumulate hydroxy-L-proline before exposure to the inhibitor. A number of other amino acids interfered with the transport of hydroxy-L-proline; the greatest effect was produced by L-alanine and L-proline.

d -Lysine Catabolic Pathway in Pseudomonas putida: Interrelations with l -Lysine Catabolism

“…Experimentally, uptake studies by wild-type or mutant (M-7) cells showed differences no greater than twofold in the rate of L-lysine uptake compared with D-lysine, whether or not cells were induced by growth in the presence of DL-lysine (Table 2). Rates were similar in magnitude to saturating rates measured for the uptake of hydroxyproline by the same strain of cells after full induction (16). In contrast to the status of hydroxyproline uptake by our strain and to lysine uptake by another strain of P. putida (27), exposure to lysine produced no stimulation of uptake rate; in contrast, growth in lysine media led to consistently lower initial uptake rates of L-or D-lysine.…”

“…Transport measurements. Estimates of the initial rate of uptake by cells of Lor D-lysine were made as described previously for other substrates (16). Cells were washed and suspended in 0.033 M potassium phosphate, pH 7, to a final density of 100 Klett units.…”

Transport of Octanoate by Pseudomonas oleovorans

“…The wide variety of substrates supporting growth of pseudomonads is well documented, and many of the catabolic pathways are totally or partially dilineated (15,17). Knowledge of transport by a representative gram-negative organism, however, comes mainly from Escherichia coli, with investigations of transport by pseudomonads limited, with few exceptions, to some amino acids (3,7,13,14). Degradation of fatty acids by Pseudomonas oleovorans, for example, has been studied in some detail (8,9,11,12,18), while transport of these compounds is limited to a single study with E. coli (5).…”

Properties of α-Aminoisobutyric Acid Transport in a Thermophilic Microorganism

“…Regulation of AIB transport: induction. The transport of certain amino acids was found to be inducible (5,10,15,20). A two-to threefold increase in AIB uptake was obtained when -alanine ( For conditions of leucine starvation and AIB uptake see Materials and Methods.'…”