Abstract
BackgroundZymomonas mobilis is a natural ethanologen being developed and deployed as an industrial biofuel producer. To date, eight Z. mobilis strains have been completely sequenced and found to contain 2–8 native plasmids. However, systematic verification of predicted Z. mobilis plasmid genes and their contribution to cell fitness has not been hitherto addressed. Moreover, the precise number and identities of plasmids in Z. mobilis model strain ZM4 have been unclear. The lack of functional information about plasmid genes in ZM4 impedes ongoing studies for this model biofuel-producing strain.ResultsIn this study, we determined the complete chromosome and plasmid sequences of ZM4 and its engineered xylose-utilizing derivatives 2032 and 8b. Compared to previously published and revised ZM4 chromosome sequences, the ZM4 chromosome sequence reported here contains 65 nucleotide sequence variations as well as a 2400-bp insertion. Four plasmids were identified in all three strains, with 150 plasmid genes predicted in strain ZM4 and 2032, and 153 plasmid genes predicted in strain 8b due to the insertion of heterologous DNA for expanded substrate utilization. Plasmid genes were then annotated using Blast2GO, InterProScan, and systems biology data analyses, and most genes were found to have apparent orthologs in other organisms or identifiable conserved domains. To verify plasmid gene prediction, RNA-Seq was used to map transcripts and also compare relative gene expression under various growth conditions, including anaerobic and aerobic conditions, or growth in different concentrations of biomass hydrolysates. Overall, plasmid genes were more responsive to varying hydrolysate concentrations than to oxygen availability. Additionally, our results indicated that although all plasmids were present in low copy number (about 1–2 per cell), the copy number of some plasmids varied under specific growth conditions or due to heterologous gene insertion.ConclusionsThe complete genome of ZM4 and two xylose-utilizing derivatives is reported in this study, with an emphasis on identifying and characterizing plasmid genes. Plasmid gene annotation, validation, expression levels at growth conditions of interest, and contribution to host fitness are reported for the first time.
Highlights
Zymomonas mobilis is a natural ethanologen being developed and deployed as an industrial biofuel producer
The results from each sequencing endeavor were assembled independently using different bioinformatics pipelines. These parallel sequencing efforts consistently identified a total of four plasmids in strain ZM4, which were named according to their sequence sizes: pZM32 (32,791 bp), pZM33 (33,006 bp), pZM36 (36,494 bp), and pZM39 (39,266 bp) in ZM4 and 2032 (Fig. 1)
Final plasmid sequence assembles were confirmed by primer walking (Additional file 1: Figure S1A), and our results were consistent with previously published gel electrophoresis results in which three plasmids, approximately 32.5, 34, and 40.5 kb in size, were observed in ZM4 [25]
Summary
Zymomonas mobilis is a natural ethanologen being developed and deployed as an industrial biofuel producer. Eight Z. mobilis strains have been completely sequenced and found to contain 2–8 native plasmids. The precise number and identities of plasmids in Z. mobilis model strain ZM4 have been unclear. The lack of functional information about plasmid genes in ZM4 impedes ongoing studies for this model biofuel-producing strain. Zymomonas mobilis is a natural ethanologen being developed for industrial conversion of biomass feedstocks into biofuels [1, 2]. Z. mobilis has recently been engineered to produce 2,3-butanediol, which can serve as a precursor for the generation of advanced biofuels and bioplastics [8]
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