The Molecular Mechanisms Of Alkane Degradation By Pseudomonas Aeruginosa Strain SJTD-1

The petroleum pollution thereof has brought great hazards to the ecological environment. Physical and chemical techniques have been used to deal with those issues, whereas they failed to meet the sustainable standards because of high cost and risk of secondary pollution. Recently, bioremediation as a newly emerging method is now showing its status on pollution control, for the safety of bio-metaboites, low cost and easy manipulation, however, it is also limited by low degradation efficiency, the potential of ecological hazard and great impact of enviromental facts such as temperature or p H. Currently, lack of knowledge on genetic background and metabolism of hydrocarbons has led to the slow development of promising microorganisms. Therefore, screening of alkane-degradation strains, studies on mechanisms, and further constructing engineering strains have become an important area for bioremediation.A gram-negative aliphatic hydrocarbon-degrading bacterium isolated from oil-contaminated soil of Daqing oil field was designated as SJTD-1. We have had systematic studies on its degradation curves of n-alkanes, whole genome analysis and proteomic research, identification of alkane monooxygenases and their regulation mechanisms.C16-C20 alkanes have been verified as optimal substrates for SJTD-1 and higher concentration below 2 g/L of n-hexadecane would promote better viabilities of SJTD-1. 500 mg/L of tetradecane, hexadecane, and octadecane were transformed completely in 36 h and more than 95% of alkanes with even higher concentration were still consumed by SJTD-1. Thus, strain SJTD-1 has high efficiency of n-alkane degradation. It was further identified as belonging to the species of Pseudomonas aeruginosa by comparative analyses of the 16 S r RNA sequence.The whole genome of strain SJTD-1 was sequenced via Roche 454 FLX+ and Illumina Miseq platform, which reveals strain SJTD-1 harbors a complement of alkane-degrading genes: alk B1（SJTD-1₄712） and alk B2（SJTD-1₃623）, two P450 genes（SJTD-1₅482 and SJTD-1₄609）, and a putative flavin-containing monooxygenase alm A（SJTD-1₃206）.In this study, the i TRAQ-LC–MS/MS strategy was applied for quantification of proteins in response to the stimuli of C18 alkane in this study. As a result, 476 proteins（account of 8.4% of the total gene encoding proteins） were identified with significant changes of expression. Most of those differently expressed proteins were functionally mapped into pathways of alkane degradation or metabolism, including alkane uptake and transport, energy transfer, secretion, environmental stress and regulation. Especially, the abundance of a series of proteins related to glyoxylate cycle, chenmotaxis, VI secretion system are larger, indicating that those physiological processes are more active. It provides a new research perspective on alkane deragadation pathway and construction of bioengineering strains. Above all, the expression of Alk B2 has been elevated by 4.05 fold, again highlighted for their outstanding contribution to alkane use. Also, the expression of another putative novel Alm A-like monooxygenase has increase by 2.35 fold and its role on long chain alkane hydroxylation were firstly discussed in this study.This paragraph then addressed a diverse contribution of five alkane hydroxylases to the hydrocarbon degradation by means of genetic deletion and transcription study. alk B1 and alk B2 genes were induced in the presence of medium- and short- chain alkanes（n ? 16）, besides, alk B2 gene was also induced by long chain alkanes（n? 18）. Inactivation of either Alk B would evidently decrease the biotransformation rate of alkanes and it went even worse when alk B2 was deleted. However, only Alk B2 and Alm A have been determined as two key factors involved in the degradation of long chain alkanes（≥ C18）. Transcriptional study revealed that one alk B would be induced when strain SJTD-1 was deprived of the other alk B gene. All these findings showcased the significant roles of Alk B family and particularly the contribution of Alk B2 to the degradation of medium- and longchain alkanes.To clarify the regulation mechanisms of alkane hydroxylases, ds DNA sequences upstream of alk B2 were used, and verified to be bound by one Gnt R regulator（encoded by SJTD-1₃624）. Binding relations were also found with alk B1 promoter regions, but weaker than Alk B2. DNA footprinting assay showed one palindromic sequence of 5‘-att-GT-cag-AC-aat-3‘ located 30 bp ahead of alk B2 ATG codon was the key motif for Gnt R binding. Further study confirmed Gnt R repressed the transcription of alk B2 and relieved in the presence of alkanes. Our findings were the first to report the roles of Gnt R on the regulation of alk B genes.Taken together, this study extensively addressed the issue of Pseudomonas aeruginosa SJTD-1 consuming linear alkanes, from the aspects of biotransformation capacities, key genes and their coding pathways, and microbial regulation systems. With the deeper knowledge into the inner mechanism, the days when microbial remediation of oil-polluted water and soil would come sooner and closer.