| Petroleum pollution is a severe environmental issue.Microbial remediation has become one of the most reliable methods for its remediation.Comprehensively revealing the genetic backgrounds of hydrocarbon-degrading microorganisms contributes to developing effective methods for remediation of petroleum pollution.Biosurfactants have wide applications.Revealing the genetic basis for its synthesis in microorganisms is crucial for realizing heterologous expression and production with a low cost.It has been shown that Achromobacter sp.HZ01 is capable of degrading petroleum hydrocarbons and producing biosurfactants.Its transcriptome(transcriptome_#1)regarding petroleum hydrocarbon degradation has been preliminarily analyzed.However,the pathways of hydrocarbon degradation and some crucial functional genes have not been revealed,and its genome has not been sequenced yet.Deep analysis of transcriptome_#1 and sequencing analyses of genome and transcriptome_#2(the transcriptome regarding biosurfactant synthesis)were performed in this study using strain HZ01 as the research object.The obtained major innovative results are as follows.(1)Transcription regulation,osmoregulation,stress response,DNA damage and repair,and significantly differentially expressed genes(DEGs)were analyzed in transcriptome_#1.Petroleum treatment for 16 h resulted in transcription changes of many genes in strain HZ01.YhbH(related to the reduction of translation efficiency)and CotE(related to sporulation)were significantly up-regulated,and some genes coding for important structural proteins were significantly down-regulated,suggesting that petroleum treatment for 16 h caused partial toxicity to strain HZ01.(2)The draft genome sequence(5,532,918 bp)of strain HZ01 was obtained,containing 5,162 predicted genes.Abundant genes related to secondary metabolites synthesis(including gene clusters terpene,phosphonate,siderophore,t1 pks,arylpolyene,resorcinol,and ectoine)were identified.The analysis of carbon source utilization suggests that strain HZ01 is not capable of using some common carbohydrates as the sole carbon source,which is due to that it contains less genes associated with carbohydrate transport and lacks some important enzymes related to glycometabolism.The Kirby-Bauer disk diffusion assay showed that strain HZ01 was resistant to cephalosporins,aminoglycosides,polypeptides,and carbapenems.Drug resistant genes,virulence genes and types II and VI secretion systems related to pathogenicity were identified from its genome.(3)The fermentation broth of strain HZ01 using citric acid as the sole carbon source exhibited a favourable emulsification activity,suggesting that biosurfactants are present in the fermentation broth.The emulsification of the biosurfactants may promote the degradation of petroleum hydrocarbons in strain HZ01.Strain HZ01 contains abundant proteins directly related to petroleum hydrocarbon degradation.AlkB hydroxylase and its homologs were not identified.It harbors a complete enzyme system of terminal oxidative pathway for n-alkane degradation,and the enzymes involved in the catechol pathway are relatively complete.This strain lacks several essential enzymes for methane oxidation,and Baeyer-Villiger monooxygenase involved in the subterminal oxidation pathway and cycloalkane degradation was not identified.These results suggest that strain HZ01 degrades n-alkanes via the terminal oxidative pathway,degrades aromatic compounds primarily via the catechol pathway and can not perform methane oxidation or cycloalkane degradation.After strain HZ01 was treated with petroleum for 16 h,the tricarboxylic acid(TCA)cycle was inhibited,and the following steps of terminal oxidative pathway were activated: the initial oxidation of n-alkanes;reactions catalyzed by dehydrogenases,fatty acid hydroxylases and acyl-CoA synthases;and fatty acid β-oxidation.(4)A total of 5,028 transcripts were identified from transcriptome_#2.Significantly differentially expressed genes,ABC transporters,two-component systems and TCA cycle were analyzed.Quantitative real-time PCR suggests that the results from the analysis of DEGs in transcriptome_#2 are correct and reliable.(5)Genes coding for the following proteins were identified at least from two sequencing analyses: LuxR transcriptional regulation factor,3-oxoacyl-ACP reductase,acyltransferase,OmpA,thioesterase,glycosyltransferase,and 3-oxoacyl-ACP synthase.These proteins are likely to be involved in the biosurfactant synthesis in strain HZ01.An intact glyoxylate cycle pathway may be related to the glycolipid synthesis in this bacterium.The genes related to biosurfactant synthesis are not existent in the form of gene clusters in strain HZ01. |
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