Influence of Osmotic and Nutritional Stress on Physiology ofPenicillium fellutanum in Removal of Phosphocholine and Modification of Phospho-1-O-[N-Peptidyl-(2-Aminoethanol)] Phosphodiesters of Peptidophosphogalactomannan Species

Influence of NaCl concentration on activity of AP and phosphodiesterases. P. fellutanum cultured in a medium (LPSG) containing 2 mM phosphate has high activities of AP, PC:PCH, and Bis-PDase compared with SG medium cultures (9). Nutrients and culture conditions for P. fellutanum have been previously described (5, 6). The substrates for AP, PC:PCH, and Bis-PDase activities were p-nitrophenyl-phosphate,p-nitro-phenylphosphoryl choline, andbis-(p-nitrophenyl)-phosphate, respectively. Sterile NaCl was added 72 h after inoculation of cultures ofP. fellutanum in LPSG media. The LPSG cultures contained 0.01 (control), 0.51, 1.01, 2.01, and 3.01 M NaCl. Two to three milliliters of culture was removed daily through day 15. After filtration through Whatman no. 4 paper, a mixture containing 100 μl of filtrate, 500 μl of 10 mM sodium citrate buffer (pH 5.0), 2 μmol of substrate, and double-distilled water (to 850 μl) was incubated at 22°C for 20 min. Reactions were stopped with 1 ml of 0.2 N NaOH, and the amount of p-nitrophenoxide produced was determined at 410 nm. The experimental results of the assays were reproducible within 10% or less. The concentration of NaCl, up to 0.40 M, seemed to have little effect on the measured in vitro activity of PC:PCH (data not shown).

Adjusting day 3 cultures to 0.51, 1.01, or 2.01 M NaCl had minimal effect on PC:PCH activity up to day 8 (Fig.2B), compared with PC:PCH activities of controls containing 10 mM NaCl (Fig. 2A). However, the activities of AP and Bis-PDase progressively decreased in cultures containing 0.51 to 3.01 M NaCl. The maximum activity of PC:PCH remained unchanged at 0.51, 1.01, and 2.01 M NaCl (Fig. 2B to D) and decreased by about 30% in the culture containing 3.01 M NaCl (Fig. 2E). We conclude that extracellular PC:PCH activity, which catalyzes the hydrolysis of phosphocholine phosphodiesters, remains relatively high in culture filtrates even in cultures adjusted up to 3.01 M NaCl. Extracellular AP or Bis-PDase activity is negligible after day 5 in cultures containing either 2.01 or 3.01 M NaCl.

• Open in new tab
• Download powerpoint

Fig. 2.

Influence of NaCl concentration on the production of phosphoesterases in LPSG medium. Sterile solid NaCl was added to 150 ml of P. fellutanum cultures 72 h after inoculation of spore suspensions into LPSG medium to the concentration shown in each panel. Samples of culture were removed, filtered, and assayed (9) for AP, PC:PCH, and Bis-PDase activities daily through day 15. All activities were reproducible within 5%. Salt concentration did not influence the activity of PC:PCH.

Ratio of pP_xGMⁱⁱ to pP_xGMⁱⁱⁱ from LPSG cultures.The culture filtrates from day 8 P. fellutanum cultured on LPSG medium and on LPSG medium adjusted to 3.01 M NaCl at 72 h after inoculation were exhaustively dialyzed against water in 14-kDa molecular weight cutoff membranes. The pP_xGMs were concentrated by precipitation with Cetrimide and fractionated on DEAE-cellulose (Fig. 3) (5). Carbohydrate content of fractions was determined by a modified microphenol-sulfuric acid assay (2). Eighty percent or more of the pP_xGM from the LPSG control medium was obtained as pP_xGMⁱⁱ (peak I) and the remainder as pP_xGMⁱⁱⁱ (peak II) (Fig. 3A). This ratio of pP_xGMⁱⁱ to pP_xGMⁱⁱⁱapproximates that found previously (2, 10). In contrast, fractionation of pP_xGM from filtrates of cultures adjusted to 3.01 M NaCl resulted in a pP_xGMⁱⁱ/pP_xGMⁱⁱⁱ ratio of about 1:2 (Fig. 3B). The total mass of pP_xGMs recovered was about the same as that in controls. The data suggest that cultures in 3 M NaCl either convert pP_xGMⁱⁱ to pP_xGMⁱⁱⁱ or de novo synthesize significantly more pP_xGMⁱⁱⁱ and less pP_xGMⁱⁱ than controls.

• Open in new tab
• Download powerpoint

Fig. 3.

Fractionation of pP_xGMs from LPSG medium (A) and LPSG medium adjusted to contain 3.01 M NaCl (B). The cultures were filtered through Whatman no. 4 paper, and the pP_xGMs were precipitated with Cetrimide as described previously (7) and fractionated on DEAE-cellulose (7). pP_xGMⁱⁱ (peaks I) was eluted with 0.01 N HCl–0.06 M LiCl, and pP_xGMⁱⁱⁱ (peaks II) was eluted with 0.01 N HCl–0.4 M LiCl.

³¹P NMR spectra of pP_xGMⁱⁱ and pP_xGMⁱⁱⁱ from LPSG cultures containing 3.01 M NaCl.The pP_xGMⁱⁱ (150 mg) from the control LPSG culture (Fig. 3A, peak I) and both pP_xGMⁱⁱ (Fig. 3B, peak I) and pP_xGMⁱⁱⁱ (Fig. 3B, peak II) from a culture containing 3.01 M NaCl were analyzed separately by proton-decoupled³¹P nuclear magnetic resonance (NMR) spectroscopy. The spectrum of pP_xGMⁱⁱ from the control (Fig.4A) is similar to that of LPSG cultures previously reported (2, 5, 9) with³¹P signals at 0.22, 1.15, 1.33, and 1.47 ppm and minor signals of phospho-1-O-[2-aminethanol] (0.9 ppm), phospho-1-O-[N-methyl-2-aminoethanol] (0.8 ppm), and phospho-[N,N′-dimethyl-2-aminoethanol] (0.7 ppm). The relative heights of peaks b to d were comparable in pP_xGMⁱⁱ from both LPSG and SG medium, indicating little change in relative quantities of these phospho-1-O-[N-peptidyl-(2-aminoethanol)] phosphodiester species (1, 9).

• Open in new tab
• Download powerpoint

Fig. 4.

³¹P NMR spectra of pP_xGMⁱⁱ and pP_xGMⁱⁱⁱisolated from day 8 P. fellutanum cultured in LPSG medium containing 3.01 M NaCl. Proton-decoupled ³¹P NMR spectroscopy of 150 mg of pP_xGMⁱⁱ from LPSG (A) and pP_xGMⁱⁱ (B) and pP_xGMⁱⁱⁱ (C) from LPSG cultures amended to contain 3.01 M NaCl at 72 h after inoculation was carried out. Data acquisition parameters for panel A are as follows: spectral width, 2,403.8 Hz; pulse width, 21 μs; acquisition time, 1.70 s. Spectrometer frequency was 121.4685 MHz; temperature was 23°C. Chemical shifts were assigned previously (1, 12): peak a, phosphocholine phosphodiester, 0.22 ppm; peaks b through d, phospho-1-O-[N-peptidyl-(2-aminoethanol)] phosphodiesters, 1.15, 1.33, and 1.47 ppm, respectively, attached to the mannopyranosyl-6-O- and galactofuranosyl-6-O- positions of pP_xGMs.

In contrast to the spectrum of pP_xGMⁱⁱ from the control LPSG medium, that of pP_xGMⁱⁱ from LPSG medium amended to 3.01 M NaCl has quite different relative signal heights and areas with respect to chemical shifts of phospho-1-O-[N-peptidyl-(2-aminoethanol)] phosphodiester signals. The signal at 1.47 ppm is decreased significantly and that at 1.15 ppm is increased in magnitude, compared with the control. Considering that both are from 150-mg samples and that the signal-to-noise ratios are similar, the spectral data suggest that no significant change in phosphocholine phosphodiester (signal a) residues (Fig. 4A and B) occurred in either sample. However, the relative size of signals at 1.15, 1.33, and 1.44 ppm in Fig. 4B is inverted compared with those of Fig. 4A, although there appears to be little or no loss of total phosphodiester residues. If a significant reduction in phosphodiester content had occurred, then the signal-to-noise ratio would have decreased, creating a noisier and wider baseline for spectrum 4B than for spectrum 4A. Considering that the extracellular enzymes are mostly hydrolases, it is possible that a loss in aminoacyl residues from the species represented by the signal at 1.47 ppm resulted in formation of a species with a signal at 1.15 ppm.

The proton-decoupled ³¹P NMR spectrum (Fig. 4C) of pP_xGMⁱⁱⁱ, the major species isolated from the LPSG medium in 3.01 M NaCl, shows that pP_xGMⁱⁱⁱhas negligible phosphocholine phosphodiester residues (0.22 ppm) and no 1-O-phosphodiesters of ethanolamine,N-methyl-2-aminoethanol andN,N′-dimethyl-2-aminoethanol species based on a lack of signals in the region of 0.9 to 0.6 ppm. The major phosphodiester signal in pP_xGMⁱⁱⁱ is that represented by the signal at 1.15 ppm; a small signal at 1.33 ppm is observed.

Previous studies show that the effect of adding NaCl to day 3 P. fellutanum cultures is to increase the levels of cytoplasmic COS and GB (5-7). This suggests that P. fellutanumin 3 M NaCl are osmotically stressed and synthesize COS and GB as a source of osmoprotectants. The available known potential osmolyte sources are phosphocholine esters and amino acyl residues from pP_xGM. Glycine, serine and threonine from the 3-kDa mannopeptide and/or the phospho-1-O-[N-peptidyl-(2-aminoethanol)] phosphodiesters of pP_xGM may be responsible for a decrease in pP_xGMⁱⁱ from 71 to 37 mg and the increase in pP_xGMⁱⁱⁱ from 29 to 63 mg. The ³¹P NMR data show nearly a complete loss of ³¹P NMR signal at 1.47 ppm and a severe reduction in that at 1.33 ppm with an increase in signal at 1.15 ppm and with no significant change in total phosphodiester content of the pP_xGM species. Removal of amino acyl residues from theseN-peptidyl-(2-aminoethanol)-containing phosphodiesters with a resulting loss of delocalization of the electron cloud about the phosphorus atom could alter the chemical shift of the ³¹P species from 1.47 and 1.33 ppm to 1.15 ppm.

Amino acid composition of peptidophosphomannan from cultures in LPSG medium containing 5 M NaCl.A peptidophosphomannan, pP_xM, was isolated from filtrates of P. fellutanum cultured for more than 20 days in LPSG medium containing 5 M NaCl. pP₂M eluted from DE-52 in 0.01 N HCl–0.06 M LiCl. This polymer was treated with 6 N HCl at 110°C for 24 h, and amino acid analyses were performed. Serine was the only amino acid found in the neutralized hydrolysate. Similar acid treatment of pP₃₀GMⁱⁱ isolated from day 5 P. fellutanum, cultured in SG medium containing 20 mM phosphate, showed that serine, threonine, and glycine, potential precursors of COS and GB, represent 46 mol% of the total amino acyl residues. pP₃₀GMⁱⁱ was treated with 0.01 N HCl at 100°C for 90 min to release galactofuranosyl residues. The hydrolysate was neutralized and fractionated by dialysis in a 1-kDa molecular weight cutoff membrane. The mole percent of glycine, serine, and threonine in the high-molecular-weight peptidophosphomannan in the retentate fraction was 47, compared with 42 in the low-molecular-weight galactofuranosyl-containing anionic fraction. This shows that both the peptidophosphomannan-region and galactofuranosyl-6-O-phospho-1′-O-[N-peptidyl-(2′-aminoethanol)]-containing galactofuran chains are potential sources of glycine, serine, and threonine.

These data, considered with studies (6, 7) on the effect of NaCl on the levels of COS and GB that showed essentially complete removal of phosphocholine phosphodiester residues from pP_xGMⁱⁱ, indicate that adjusting 72-h cultures of P. fellutanum in LPSG medium to 3.01 M NaCl most likely resulted in the formation of pP_xGMⁱⁱⁱ from pP_xGMⁱⁱ, and possibly other sources, in day 8 cultures and altered the distribution of galactofuranosyl-containing phosphodiesters. The lack of detectable galactofuranosyl residues, the presence of only two phosphoesters, and the loss of all amino acids except serine in a “peptidophosphomannan” isolated from day 20 cultures in LPSG medium in 5 M NaCl show that P. fellutanumcan remove all of the amino acids except a serine from the peptidophosphomannan region of pP_xGM. Considering the amino acid composition of pP_xGMⁱⁱ, the data suggest that glycine, threonine, and serine in the 3-kDa peptides andN-peptidyl-(2-aminoethanol)-containing phosphodiesters may be sources of precursors required for synthesis of COS and GB in osmotically stressed P. fellutanum.