Proteomic analysis of Pseudomonas putida F1 during growth on toluene-phenol mixtures

Abstract

In both natural and engineered environments, microorganisms grow on mixtures of organics. However. little is known about microbial growth on mixtures, especially in the context of biodegradation. For example, previous research showed that the biodegradation kinetics of Pseudomonas putida F1 growing on mixtures of toluene and phenol were difficult to predict, despite the fact that the metabolic pathway for these substrates is the same. To investigate the physiological effects of the toluene-phenol mixtures on the growth of P. putida F1, a proteomics approach was utilized. An optimal proteome analysis procedure was developed and the proteomes of Pseudomonas putida F1 grown on either a single substrate (toluene or phenol) or a toluene-phenol mixture were analyzed by two-dimensional polyacrylamide gel electrophoresis. Three groups of proteins (Group T. P. and M). which were differentially produced when the bacterium grew on toluene, phenol, or the toluene-phenol mixture, respectively, were categorized by two strict criteria: reproducibility and uniqueness. Based on the proteome analysis, selected Group T. P. and M proteins were subjected to N-terminal sequencing, and mass spectrometry, and results of these analyses were used in searches of the protein identities. Two different mass spectrometers were utilized: a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer for mass fingerprinting and a tandem mass spectrometer (electrospray ionization mass spectrometer) for de novo peptide sequencing. Two proteins were identified: acyl carrier protein (ACP) and β-ketoadipate:succinyl CoA transferase. The function of ACP, an essential protein in the biosynthesis of fatty acids, triggered the investigation of the compositional changes in the phospholipid fatty acids of the cellular membrane. The phospholipid fatty acid composition was found to dramatically change depending on the growth substrate(s). These membrane compositional changes during the biodegradation of the toluene-phenol mixture were used to form the hypothesis that the presence of toluene altered the structure of cellular membrane in favor of toluene transport and inhibited the transport of phenol into the cell. Once the cell consumed toluene, the membrane structure changed to be favorable to phenol transport across the cell membrane. The production of β-ketoadipate:succinyI CoA transferase during the biodegradation of phenol might suggest either incidental gene induction or a basal level of phenol metabolism via the P-ketoadipate pathway metabolism. To consider more diverse contaminant mixtures, the growth of P. putida F1 on mixtures of toluene-phenol and two different heavy metals was also examined. The bacterium displayed better tolerance to cadmium than to chromate during growth on toluene, phenol, and their mixture. The metal tolerance of the bacterium remained at a similar level regardless of the substrate. Heavy metals substantially inhibited the cell growth rate on all substrates and the inhibitory effect was also reflected in decreased cell growth yields. Transmission electron microscopy was utilized to observe any alterations in the cellular membrane integrity. The microscopy images suggested that the presence of chromate, even at a low concentration (1.0 ppm), severely disturbed the cellular membrane and resulted in a loss of cell integrity. This research provides (1) physiological observations of Pseudomonas putida F1 in the biodegradation of toluene-phenol mixtures with proteomic analysis. (2) some fundamental evidence to better understand the relationships between biodegradation and the transmembrane transport of toluene-phenol mixtures, and (3) the effects of heavy metals on the biodegradation of toluene-phenol mixtures by P. putida F1.