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Zymomonas mobilis is an ethanologenic alphaproteobacterium with promise for the industrial conversion of renewable plant biomass into fuels and chemical bioproducts. Limited functional annotation of the Z. mobilis genome is a current barrier to both fundamental studies of Z. mobilis and its development as a synthetic biology chassis. To gain insight, we collected sample-matched multiomics data, including RNA sequencing (RNA-seq), transcription start site (TSS) sequencing (TSS-seq), termination sequencing (term-seq), ribosome profiling, and label-free shotgun proteomic mass spectrometry, across different growth conditions and used these data to improve annotation and assign functional sites in the Z. mobilis genome. Proteomics and ribosome profiling informed revisions of protein-coding genes, which included 44 start codon changes and 42 added proteins. We developed statistical methods for annotating transcript 5′ and 3′ ends, enabling the identification of 3,940 TSSs and their corresponding promoters and 2,091 transcription termination sites, which were distinguished from RNA processing sites by the lack of an adjacent RNA 5′ end. Our results revealed that Z. mobilis σA −35 and −10 promoter elements closely resemble canonical Escherichia coli −35 and −10 elements, with one notable exception: the Z. mobilis −10 element lacks the highly conserved −7 thymine observed in E. coli and other previously characterized σA promoters. The σA promoters of another alphaproteobacterium, Caulobacter crescentus, similarly lack the conservation of −7 thymine in their −10 elements. Our results anchor the development of Z. mobilis as a platform for synthetic biology and establish strategies for empirical genome annotation that can complement purely computational methods. IMPORTANCE Efforts to rationally engineer synthetic pathways in Zymomonas mobilis are impeded by a lack of knowledge and tools for predictable and quantitative programming of gene regulation at the transcriptional, posttranscriptional, and posttranslational levels. With the detailed functional characterization of the Z. mobilis genome presented in this work, we provide crucial knowledge for the development of synthetic genetic parts tailored to Z. mobilis. This information is vital as researchers continue to develop Z. mobilis for synthetic biology applications. Our methods and statistical analyses also provide ways to rapidly advance the understanding of poorly characterized bacteria via empirical data that enable the experimental validation of sequence-based prediction for genome characterization and annotation.

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