Proteomic Insights into Metabolic Adaptations inAlcanivorax borkumensisInduced by Alkane Utilization

Abstract

Alcanivorax borkumensis is a ubiquitous marine petroleum oil-degrading bacterium with an unusual physiology specialized for alkane metabolism. This "hydrocarbonoclastic" bacterium degrades an exceptionally broad range of alkane hydrocarbons but few other substrates. The proteomic analysis presented here reveals metabolic features of the hydrocarbonoclastic lifestyle. Specifically, hexadecane-grown and pyruvate-grown cells differed in the expression of 97 cytoplasmic and membrane-associated proteins whose genes appeared to be components of 46 putative operon structures. Membrane proteins up-regulated in alkane-grown cells included three enzyme systems able to convert alkanes via terminal oxidation to fatty acids, namely, enzymes encoded by the well-known alkB1 gene cluster and two new alkane hydroxylating systems, a P450 cytochrome monooxygenase and a putative flavin-binding monooxygenase, and enzymes mediating ␤-oxidation of fatty acids. Cytoplasmic proteins up-regulated in hexadecane-grown cells reflect a central metabolism based on a fatty acid diet, namely, enzymes of the glyoxylate bypass and of the gluconeogenesis pathway, able to provide key metabolic intermediates, like phosphoenolpyruvate, from fatty acids. They also include enzymes for synthesis of riboflavin and of unsaturated fatty acids and cardiolipin, which presumably reflect membrane restructuring required for membranes to adapt to perturbations induced by the massive influx of alkane oxidation enzymes. Ancillary functions up-regulated included the lipoprotein releasing system (Lol), presumably associated with biosurfactant release, and polyhydroxyalkanoate synthesis enzymes associated with carbon storage under conditions of carbon surfeit. The existence of three different alkane-oxidizing systems is consistent with the broad range of oil hydrocarbons degraded by A. borkumensis and its ecological success in oil-contaminated marine habitats.Alcanivorax borkumensis is a key marine oil-degrading bacterium that can dramatically increase in numbers after an oil spill and become the most abundant microbe in oil-polluted waters (21,26,27,43). The list of sites where it has been isolated and shown to be involved in oil degradation grows with microbiological investigations of oil spills (9, 37; M. M. Yakimov, personal communication). The physiology of A. borkumensis is characterized by oligotrophy and a highly restricted growth substrate profile, namely, petroleum hydrocarbons plus a few organic acids, though the spectrum of hydrocarbons degraded is exceptionally broad. Its unusual metabolic features, presumed global importance in the natural biological removal of oil entering marine systems, and biotechnological potential for mitigation of the ecological damage caused by oil spills stimulated recent and current functional genomic studies of this organism. A key question concerning the ubiquity and competitive success of A. borkumensis in many marine locations is the genomic and biochemical basis of its physiological specialization and broad hydrocarbon su...

Aerobic Degradation of Petroleum Components by Microbial Consortia

“…Since the cultivation of Alcanivorax borkumensis [88], functional genomic, biochemical and physiological analyses have revealed the underlying basis of its success [52,[89][90][91]. While it lacks catabolic versatility, utilising alkanes almost exclusively as carbon and energy sources, it has multiple alkane-catabolism pathways, with key enzymes including alkane hydoxylases (a non-haem diiron monooxygenase; AlkB1 and AlkB2) and three cytochrome P450-dependent alkane monooxygenases [89].…”

Proteomics of marine bacteria

“…Varying levels of light intensity iTRAQ and LC-M/MS [43] Heterotrophy/photoautotrophy 2-D PAGE, MALDI-MS and LC-MS/MS [50] Nutrient availability/different C-and energy sources [68,74,75] Growth phase/cell cycle…”

“…While A. borkumensis is hardly detectable in clean water, it can be one of the most abundant microorganisms found in oil-polluted waters [72,73]. Details of alkane degradation pathways used by A. borkumensis could recently be elucidated in a comprehensive proteomic study [74]. Comparing protein patterns from cells grown on pyruvate and hexadecane, respectively, a number of cytoplasmic as well as many membrane-associated proteins could be identified that are specifically induced during growth on alkanes.…”

Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene

“…For instance, AlkB-like monooxygenase typically oxidizes liquid alkanes but, lately, few members of this family of enzymes were described to be also induced by propane [66,67]. This underlines the diversity that can exist the proteomic analysis of its cytoplasmic and membrane proteins after the growth on nhexadecane showed the presence of multiple alkane hydroxylase systems belonging to different families [75] involved in the primary oxidation of a broad range of alkanes.A review of the molecular genetics and regulations of the categories of bacterial monooxygenases involved in the n-alkanes metabolism reported in literature is summarized below. …”

“…The expression of both alkB1 and alkB2 is very low when cells grow using pyruvate as the carbon source, but is strongly induced when C 10 -C 16 alkanes are metabolized; expression decreases considerably upon entry into stationary phase [70,75,213]. A gene coding for a protein showing similarity to the P. …”