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The Ethanologenic Bacterium Zymomonas mobilis Divides Asymmetrically and Exhibits Heterogeneity in DNA Content.

ABSTRACT: The alphaproteobacterium Zymomonas mobilis exhibits extreme ethanologenic physiology, making this species a promising biofuel producer. Numerous studies have investigated its biology relevant to industrial applications and mostly at the population level. However, the organization of single cells in this industrially important polyploid species has been largely uncharacterized. In the present study, we characterized basic cellular behavior of Z. mobilis strain Zm6 under anaerobic conditions at the single-cell level. We observed that growing Z. mobilis cells often divided at a nonmidcell position, which contributed to variant cell size at birth. However, the cell size variance was regulated by a modulation of cell cycle span, mediated by a correlation of bacterial tubulin homologue FtsZ ring accumulation with cell growth. The Z. mobilis culture also exhibited heterogeneous cellular DNA content among individual cells, which might have been caused by asynchronous replication of chromosome that was not coordinated with cell growth. Furthermore, slightly angled divisions might have resulted in temporary curvatures of attached Z. mobilis cells. Overall, the present study uncovers a novel bacterial cell organization in Z. mobilis IMPORTANCE With increasing environmental concerns about the use of fossil fuels, development of a sustainable biofuel production platform has been attracting significant public attention. Ethanologenic Z. mobilis species are endowed with an efficient ethanol fermentation capacity that surpasses, in several respects, that of baker's yeast (Saccharomyces cerevisiae), the most-used microorganism for ethanol production. For development of a Z. mobilis culture-based biorefinery, an investigation of its uncharacterized cell biology is important, because bacterial cellular organization and metabolism are closely associated with each other in a single cell compartment. In addition, the current work demonstrates that the polyploid bacterium Z. mobilis exhibits a distinctive mode of bacterial cell organization, likely reflecting its unique metabolism that does not prioritize incorporation of nutrients for cell growth. Thus, another significant result of this work is to advance our general understanding in the diversity of bacterial cell architecture.

SUBMITTER: Fuchino K

PROVIDER: S-EPMC8105015 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The excessive consumption of sugars can cause health issues. Different strategies have been developed to reduce sugars in the diets. However, sugars in fruits and commercial products may be difficult to reduce, limiting their usage among certain populations of people. Zymomonas mobilis is a generally recognized as safe (GRAS) probiotic bacterium with the capability to produce levan-type prebiotics, and thrives in high-sugar environments with unique characteristics to be developed for lignocellulosic biofuel and biochemical production. In this study, the sugar reduction capabilities of Z. mobilis ZM4 were examined using two fruits of pear and persimmon and three high-sugar-content commercial products of two pear pastes (PPs) and one Chinese traditional wine (CTW). Our results demonstrated that Z. mobilis ZM4 can utilize sugars in fruits with about 20 g/L ethanol and less than 5 g/L sorbitol produced within 22 h using pears, and about 45 g/L ethanol and 30 g/L sorbitol produced within 34 h using persimmons. When PPs made from pears were used, Z. mobilis can utilize nearly all glucose (ca. 60 g/L) and most fructose (110 g/L) within 100 h with 40 ~ 60 g/L ethanol and more than 20 g/L sorbitol produced resulting in a final sorbitol concentration above 80 g/L. In the high-sugar-content alcoholic Chinese traditional wine, which contains mostly glucose and ethanol, Z. mobilis can reduce nearly all sugars with about 30 g/L ethanol produced, resulting in a final ethanol above 90 g/L. The ethanol yield and percentage yield of Z. mobilis in 50 ~ 60% CTW were 0.44 ~ 0.50 g/g and 86 ~ 97%, respectively, which are close to its theoretical yields-especially in 60% CTW. Although the ethanol yield and percentage yield in PPs were lower than those in CTW, they were similar to those in fruits of pears and persimmons with an ethanol yield around 0.30 ~ 0.37 g/g and ethanol percentage yield around 60 ~ 72%, which could be due to the formation of sorbitol and/or levan in the presence of both glucose and fructose. Our study also compared the fermentation performance of the classical ethanologenic yeast Saccharomyces cerevisiae BY4743 to Z. mobilis, with results suggesting that Z. mobilis ZM4 had better performance than that of yeast S. cerevisiae BY4743 given a higher sugar conversion rate and ethanol yield for sugar reduction. This work thus laid a foundation for utilizing the advantages of Z. mobilis in the food industry to reduce sugar concentrations or potentially produce alcoholic prebiotic beverages.Supplementary informationThe online version contains supplementary material available at 10.1186/s40643-021-00467-2.

Project description:BACKGROUND: The ethanol-producing bacterium Zymomonas mobilis has attracted considerable scientific and commercial interest due to its exceptional physiological properties. Shuttle vectors derived from native plasmids have previously been successfully used for heterologous gene expression in this bacterium for a variety of purposes, most notably for metabolic engineering applications. RESULTS: A quantitative PCR (qPCR) approach was used to determine the copy numbers of two endogenous double stranded DNA plasmids: pZMO1A (1,647 bp) and pZMO7 (pZA1003; 4,551 bp) within the NCIMB 11163 strain of Z. mobilis. Data indicated pZMO1A and pZMO7 were present at ca. 3-5 and ca. 1-2 copies per cell, respectively. A ca. 1,900 bp fragment from plasmid pZMO7 was used to construct two Escherichia coli - Z. mobilis shuttle vectors (pZ7C and pZ7-184). The intracellular stabilities and copy numbers of pZ7C and pZ7-184 were characterized within the NCIMB 11163, ATCC 29191 and (ATCC 10988-derived) CU1 Rif2 strains of Z. mobilis. Both shuttle vectors could be stably maintained within the ATCC 29191 strain (ca. 20-40 copies per cell), and the CU1 Rif2 strain (ca. 2-3 copies per cell), for more than 50 generations in the absence of an antibiotic selectable marker. A selectable marker was required for shuttle vector maintenance in the parental NCIMB 11163 strain; most probably due to competition for replication with the endogenous pZMO7 plasmid molecules. N-terminal glutathione S-transferase (GST)-fusions of four endogenous proteins, namely the acyl-carrier protein (AcpP); 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA); DNA polymerase III chi subunit (HolC); and the RNA chaperone protein Hfq; were successfully expressed from pZ7C-derived shuttle vectors, and their protein-protein binding interactions were analyzed in Z. mobilis ATCC 29191. Using this approach, proteins that co-purified with AcpP and KdsA were identified. CONCLUSIONS: We show that a shuttle vector-based protein affinity 'pull-down' approach can be used to probe protein interaction networks in Z. mobilis cells. Our results demonstrate that protein expression plasmids derived from pZMO7 have significant potential for use in future biological or biotechnological applications within Z. mobilis.

Project description:UNLABELLED: BACKGROUND:High tolerance to ethanol is a desirable characteristics for ethanologenic strains used in industrial ethanol fermentation. A deeper understanding of the molecular mechanisms underlying ethanologenic strains tolerance of ethanol stress may guide the design of rational strategies to increase process performance in industrial alcoholic production. Many extensive studies have been performed in Saccharomyces cerevisiae and Escherichia coli. However, the physiological basis and genetic mechanisms involved in ethanol tolerance for Zymomonas mobilis are poorly understood on genomic level. To identify the genes required for tolerance to ethanol, microarray technology was used to investigate the transcriptome profiling of the ethanologenic Z. mobilis in response to ethanol stress. RESULTS:We successfully identified 127 genes which were differentially expressed in response to ethanol. Ethanol up- or down-regulated genes related to cell wall/membrane biogenesis, metabolism, and transcription. These genes were classified as being involved in a wide range of cellular processes including carbohydrate metabolism, cell wall/membrane biogenesis, respiratory chain, terpenoid biosynthesis, DNA replication, DNA recombination, DNA repair, transport, transcriptional regulation, some universal stress response, etc. CONCLUSION:In this study, genome-wide transcriptional responses to ethanol were investigated for the first time in Z. mobilis using microarray analysis.Our results revealed that ethanol had effects on multiple aspects of cellular metabolism at the transcriptional level and that membrane might play important roles in response to ethanol. Although the molecular mechanism involved in tolerance and adaptation of ethanologenic strains to ethanol is still unclear, this research has provided insights into molecular response to ethanol in Z. mobilis. These data will also be helpful to construct more ethanol resistant strains for cellulosic ethanol production in the future.

Project description:Zymomonas mobilis is an ethanologenic bacterium currently being developed for production of advanced biofuels. Recent studies have shown that Z. mobilis can fix dinitrogen gas (N2) as a sole nitrogen source. During N2 fixation, Z. mobilis exhibits increased biomass-specific rates of ethanol production. In order to better understand the physiology of Z. mobilis during N2 fixation and during changes in ammonium (NH4+) availability, we performed liquid chromatography-mass spectrometry (LC-MS)-based targeted metabolomics and shotgun proteomics under three regimes of nitrogen availability: continuous N2 fixation, gradual NH4+ depletion, and acute NH4+ addition to N2-fixing cells. We report dynamic changes in abundance of proteins and metabolites related to nitrogen fixation, motility, ammonium assimilation, amino acid biosynthesis, nucleotide biosynthesis, isoprenoid biosynthesis, and Entner-Doudoroff (ED) glycolysis, providing insight into the regulatory mechanisms that control these processes in Z. mobilis. Our analysis identified potential physiological mechanisms that may contribute to increased specific ethanol production during N2 fixation, including decreased activity of biosynthetic pathways, increased protein abundance of alcohol dehydrogenase (ADHI), and increased thermodynamic favorability of the ED pathway. Of particular relevance to advanced biofuel production, we found that intermediates in the methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis were depleted during N2 fixation, coinciding with decreased protein abundance of deoxyxylulose 5-phosphate synthase (DXS), the first enzyme in the pathway. This implies that DXS protein abundance serves as a native control point in regulating MEP pathway activity in Z. mobilis. The results of this study will inform metabolic engineering to further develop Z. mobilis as a platform organism for biofuel production. IMPORTANCE Biofuels and bioproducts have the potential to serve as environmentally sustainable replacements for petroleum-derived fuels and commodity molecules. Advanced fuels such as higher alcohols and isoprenoids are more suitable gasoline replacements than bioethanol. Developing microbial systems to generate advanced biofuels requires metabolic engineering to reroute carbon away from ethanol and other native products and toward desired pathways, such as the MEP pathway for isoprenoid biosynthesis. However, rational engineering of microbial metabolism relies on understanding metabolic control points, in terms of both enzyme activity and thermodynamic favorability. In Z. mobilis, the factors that control glycolytic rates, ethanol production, and isoprenoid production are still not fully understood. In this study, we performed metabolomic, proteomic, and thermodynamic analysis of Z. mobilis during N2 fixation. This analysis identified key changes in metabolite levels, enzyme abundance, and glycolytic thermodynamic favorability that occurred during changes in NH4+ availability, helping to inform future efforts in metabolic engineering.

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The Ethanologenic Bacterium Zymomonas mobilis Divides Asymmetrically and Exhibits Heterogeneity in DNA Content.

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