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

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. mobilisIMPORTANCE 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.

Increased salt tolerance in Zymomonas mobilis strain generated by adaptative evolution.

BACKGROUND: Ethanologenic alphaproteobacterium Zymomonas mobilis has been acknowledged as a promising biofuel producer. There have been numerous efforts to engineer this species applicable for an industrial-scale bioethanol production. Although Z. mobilis is robustly resilient to certain abiotic stress such as ethanol, the species is known to be sensitive to saline stress at a mild concentration, which hampers its industrial use as an efficient biocatalyst. To overcome this issue, we implemented a laboratory adaptive evolution approach to obtain salt tolerant Z. mobilis strain. RESULTS: During an adaptive evolution, we biased selection by cell morphology to exclude stressed cells. The evolved strains significantly improved growth and ethanol production in the medium supplemented with 0.225 M NaCl. Furthermore, comparative metabolomics revealed that the evolved strains did not accumulate prototypical osmolytes, such as proline, to counter the stress during their growth. The sequenced genomes of the studied strains suggest that the disruption of ZZ6_1149 encoding carboxyl-terminal protease was likely responsible for the improved phenotype. CONCLUSIONS: The present work successfully generated strains able to grow and ferment glucose under the saline condition that severely perturbs parental strain physiology. Our approach to generate strains, cell shape-based diagnosis and selection, might be applicable to other kinds of strain engineering in Z. mobilis.

Cell-Biological Studies of Osmotic Shock Response in Streptomyces spp.

Most bacteria are likely to face osmotic challenges, but there is yet much to learn about how such environmental changes affect the architecture of bacterial cells. Here, we report a cell-biological study in model organisms of the genus Streptomyces, which are actinobacteria that grow in a highly polarized fashion to form branching hyphae. The characteristic apical growth of Streptomyces hyphae is orchestrated by protein assemblies, called polarisomes, which contain coiled-coil proteins DivIVA and Scy, and recruit cell wall synthesis complexes and the stress-bearing cytoskeleton of FilP to the tip regions of the hyphae. We monitored cell growth and cell-architectural changes by time-lapse microscopy in osmotic upshift experiments. Hyperosmotic shock caused arrest of growth, loss of turgor, and hypercondensation of chromosomes. The recovery period was protracted, presumably due to the dehydrated state of the cytoplasm, before hyphae could restore their turgor and start to grow again. In most hyphae, this regrowth did not take place at the original hyphal tips. Instead, cell polarity was reprogrammed, and polarisomes were redistributed to new sites, leading to the emergence of multiple lateral branches from which growth occurred. Factors known to regulate the branching pattern of Streptomyces hyphae, such as the serine/threonine kinase AfsK and Scy, were not involved in reprogramming of cell polarity, indicating that different mechanisms may act under different environmental conditions to control hyphal branching. Our observations of hyphal morphology during the stress response indicate that turgor and sufficient hydration of cytoplasm are required for Streptomyces tip growth. IMPORTANCE: Polar growth is an intricate manner of growth for accomplishing a complicated morphology, employed by a wide range of organisms across the kingdoms of life. The tip extension of Streptomyces hyphae is one of the most pronounced examples of polar growth among bacteria. The expansion of the cell wall by tip extension is thought to be facilitated by the turgor pressure, but it was unknown how external osmotic change influences Streptomyces tip growth. We report here that severe hyperosmotic stress causes cessation of growth, followed by reprogramming of cell polarity and rearrangement of growth zones to promote lateral hyphal branching. This phenomenon may represent a strategy of hyphal organisms to avoid osmotic stress encountered by the growing hyphal tip.

Dynamic gradients of an intermediate filament-like cytoskeleton are recruited by a polarity landmark during apical growth.

Intermediate filament (IF)-like cytoskeleton emerges as a versatile tool for cellular organization in all kingdoms of life, underscoring the importance of mechanistically understanding its diverse manifestations. We showed previously that, in Streptomyces (a bacterium with a mycelial lifestyle similar to that of filamentous fungi, including extreme cell and growth polarity), the IF protein FilP confers rigidity to the hyphae by an unknown mechanism. Here, we provide a possible explanation for the IF-like function of FilP by demonstrating its ability to self-assemble into a cis-interconnected regular network in vitro and its localization into structures consistent with a cytoskeletal network in vivo. Furthermore, we reveal that a spatially restricted interaction between FilP and DivIVA, the main component of the Streptomyces polarisome complex, leads to formation of apical gradients of FilP in hyphae undergoing active tip extension. We propose that the coupling between the mechanism driving polar growth and the assembly of an IF cytoskeleton provides each new hypha with an additional stress-bearing structure at its tip, where the nascent cell wall is inevitably more flexible and compliant while it is being assembled and matured. Our data suggest that recruitment of cytoskeleton around a cell polarity landmark is a broadly conserved strategy in tip-growing cells.

Genome Copy Number Quantification Revealed That the Ethanologenic Alpha-Proteobacterium Zymomonas mobilis Is Polyploid.

The alpha-proteobacterium Zymomonas mobilis is a promising biofuel producer, based on its native metabolism that efficiently converts sugars to ethanol. Therefore, it has a high potential for industrial-scale biofuel production. Two previous studies suggested that Z. mobilis strain Zm4 might not be monoploid. However, a systematic analysis of the genome copy number is still missing, in spite of the high potential importance of Z. mobilis. To get a deep insight into the ploidy level of Z. mobilis and its regulation, the genome copy numbers of three strains were quantified. The analyses revealed that, during anaerobic growth, the lab strain Zm6, the Zm6 type strain obtained from DSMZ (German Collection of Microorganisms), and the lab strain Zm4, have copy numbers of 18.9, 22.3 and 16.2, respectively, of an origin-adjacent region. The copy numbers of a terminus-adjacent region were somewhat lower with 9.3, 15.8, and 12.9, respectively. The values were similar throughout the growth curves, and they were only slightly downregulated in late stationary phase. During aerobic growth, the copy numbers of the lab strain Zm6 were much higher with around 40 origin-adjacent copies and 17 terminus-adjacent copies. However, the cells were larger during aerobic growth, and the copy numbers per µm3 cell volume were rather similar. Taken together, this first systematic analysis revealed that Z. mobilis is polyploid under regular laboratory growth conditions. The copy number is constant during growth, in contrast to many other polyploid bacteria. This knowledge should be considered in further engineering of the strain for industrial applications.

An assessment of serial co-cultivation approach for generating novel Zymomonas mobilis strains.

OBJECTIVE: The alphaproteobacterium Zymomonas mobilis is an efficient ethanol producer, and Z. mobilis-based biorefinery shows great potential for biofuel production. Serial co-cultivation is an emerging approach that promotes inter-species interactions which can improve or rewire the metabolic features in industrially useful microorganisms by inducing frequent mutations. We applied this method to assess if it improves or rewires the desirable physiological features of Z. mobilis, especially ethanol production. RESULTS: We performed serial co-culture of Z. mobilis with the baker's yeast, Saccharomyces cerevisiae. We observed filamentation of Z. mobilis cells in the co-culture, indicating that the Z. mobilis cells were exposed to stress due to the presence of a competitor. After 50 times of serial transfers, we characterized the generated Z. mobilis strains, showing that long term co-culture did not drive significant changes in either the growth or profile of excreted metabolites in the generated strains. In line with this, whole genome sequencing of the generated Z. mobilis strains revealed only minor genetic variations from the parental strain. 50 generations of Z. mobilis monoculture did not induce morphological changes or any significant genetic variations. The result indicates that the method needs to be carefully optimized for Z. mobilis strain improvement.

Metabolic Profiling of Glucose-Fed Metabolically Active Resting Zymomonas mobilis Strains.

Zymomonas mobilis is the most efficient bacterial ethanol producer and its physiology is potentially applicable to industrial-scale bioethanol production. However, compared to other industrially important microorganisms, the Z. mobilis metabolome and adaptation to various nutritional and genetic perturbations have been poorly characterized. For rational metabolic engineering, it is essential to understand how central metabolism and intracellular redox balance are maintained in Z. mobilis under various conditions. In this study, we applied quantitative mass spectrometry-based metabolomics to explore how glucose-fed non-growing Z. mobilis Zm6 cells metabolically adapt to change of oxygen availability. Mutants partially impaired in ethanol synthesis (Zm6 adhB) or oxidative stress response (Zm6 cat) were also examined. Distinct patterns of adaptation of central metabolite pools due to the change in cultivation condition and between the mutants and Zm6 reference strain were observed. Decreased NADH/NAD ratio under aerobic incubation corresponded to higher concentrations of the phosphorylated glycolytic intermediates, in accordance with predictions of the kinetic model of Entner-Doudoroff pathway. The effects on the metabolite pools of aerobic to anaerobic transition were similar in the mutants, yet less pronounced. The present data on metabolic plasticity of non-growing Z. mobilis cells will facilitate the further metabolic engineering of the respective strains and their application as biocatalysts.

Zwitterionic HILIC tandem mass spectrometry with isotope dilution for rapid, sensitive and robust quantification of pyridine nucleotides in biological extracts.

The pyridine nucleotides nicotineamide adenine dinucleotide (NAD) and nicotineamide adenine dinucleotide phosphate (NADP) are conserved coenzymes across all domains of life, and are involved in more than 200 different hydride transfer reactions supporting essential catabolic and anabolic functions. The intracellular levels of these metabolites, and the ratio of their oxidized to reduced forms regulate an extensive network of reactions ranging beyond metabolism. Hence, monitoring their intracellular levels provides information about, but not limited to, the metabolic state of a cell or tissue. Interconversion between oxidized and reduced forms, varying pH liability and varying intracellular concentrations of the different species leaves absolute quantification of the pyridine nucleotides analytically challenging. These polar metabolites are poorly retained on conventional reverseed-phase stationary phases without ion-pair reagents that contaminates the LC-system. Herein we demonstrate that zwitterionic HILIC-tandem mass spectroemtry can be applied to successfully resolve the pyridine nucleotides in biological extracts in a fast, robust and highly sensitive way. The presented method applies isotope dilution to compensate potential loss of these labile metabolites and is validated for low, medium and high biomass samples of two popular biological model systems; Escherichia coli and the human cell line JJN-3. High stability and rapid sample preparation without solvent removal allows for long sequence runs, making this method ideal for high-throughput analysis of biological extracts.

Absolute Quantification of the Central Carbon Metabolome in Eight Commonly Applied Prokaryotic and Eukaryotic Model Systems.

Absolute quantification of intracellular metabolite pools is a prerequisite for modeling and in-depth biological interpretation of metabolomics data. It is the final step of an elaborate metabolomics workflow, with challenges associated with all steps-from sampling to quantifying the physicochemically diverse metabolite pool. Chromatographic separation combined with mass spectrometric (MS) detection is the superior platform for high coverage, selective, and sensitive detection of metabolites. Herein, we apply our quantitative MS-metabolomics workflow to measure and present the central carbon metabolome of a panel of commonly applied biological model systems. The workflow includes three chromatographic methods combined with isotope dilution tandem mass spectrometry to allow for absolute quantification of 68 metabolites of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and the amino acid and (deoxy) nucleoside pools. The biological model systems; Bacillus subtilis, Saccharomyces cerevisiae, two microalgal species, and four human cell lines were all cultured in commonly applied culture media and sampled in exponential growth phase. Both literature and databases are scarce with comprehensive metabolite datasets, and existing entries range over several orders of magnitude. The workflow and metabolite panel presented herein can be employed to expand the list of reference metabolomes, as encouraged by the metabolomics community, in a continued effort to develop and refine high-quality quantitative metabolomics workflows.

Improvement of Acetaldehyde Production in Zymomonas mobilis by Engineering of Its Aerobic Metabolism.

Acetaldehyde is a valuable product of microbial biosynthesis, which can be used by the chemical industry as the entry point for production of various commodity chemicals. In ethanologenic microorganisms, like yeast or the bacterium Zymomonas mobilis, this compound is the immediate metabolic precursor of ethanol. In aerobic cultures of Z. mobilis, it accumulates as a volatile, inhibitory byproduct, due to the withdrawal of reducing equivalents from the alcohol dehydrogenase reaction by respiration. The active respiratory chain of Z. mobilis with its low energy-coupling efficiency is well-suited for regeneration of NAD+ under conditions when acetaldehyde, but not ethanol, is the desired catabolic product. In the present work, we sought to improve the capacity Z. mobilis to synthesize acetaldehyde, based on predictions of a stoichiometric model of its central metabolism developed herein. According to the model analysis, the main objectives in the course of engineering acetaldehyde producer strains were determined to be: (i) reducing ethanol synthesis via reducing the activity of alcohol dehydrogenase (ADH), and (ii) enhancing the respiratory capacity, either by overexpression of the respiratory NADH dehydrogenase (NDH), or by mutation of other components of respiratory metabolism. Several mutants with elevated respiration rate, decreased alcohol dehydrogenase activity, or a combination of both, were obtained. They were extensively characterized by determining their growth rates, product yields, oxygen consumption rates, ADH, and NDH activities, transcription levels of key catabolic genes, as well as concentrations of central metabolites under aerobic culture conditions. Two mutant strains were selected, with acetaldehyde yield close to 70% of the theoretical maximum value, almost twice the previously published yield for Z. mobilis. These strains can serve as a basis for further development of industrial acetaldehyde producers.