In this study, we reported the updated chromosome and plasmid sequence of ZM4, and its two engineered xylose-utilizing strains derivatives (strains 2032 and 8b). The majority of plasmid genes have either homologs from other organisms or some conserved domains. Our bioinformatics analysis suggested that several plasmid genes may be essential genes of ZM4. To further validate the function of these plasmid genes, an RNA-Seq pipeline was developed for Z. mobilis and used to compare the gene expression under various conditions, including anaerobic and aerobic conditions, and in different biomass hydrolysates. Overall, these plasmids genes are more responsive to different concentrated hydrolysates than the different oxygen concentrations tested (such as anaerobic vs. aerobic conditions). Moreover, our results also indicate that the plasmid copy number is affected by growth conditions and foreign gene insertion while the chromosomal gene expression is relatively stable across different conditions in different strain backgrounds. Overall design: We combined and cross-compared the genome resequencing and RNA-Seq results of four independent research laboratories from three countries: USA, Greece, and China. This GEO submission contains 17 paired-end RNA-seq samples for Zymomonas mobilis subsp. mobilis strain 2032 with a minimum of two biological replicates per test condition. Cell samples were collected at different timepoints during batch fermentation culture.