Biology subjects

Fuchino, K.

Publications and source records attributed to Fuchino, K..

5 recordsLinked to original sources

bioRxiv2026

PopZ is dispensable for cell fitness but required for polar localization of ParB in Zymomonas mobilis.

Chromosome segregation is an essential process in monoploid bacteria, in which faithful inheritance of the genetic material is required for viability of daughter cell. In several alpha-proteobacteria, the polar organizing protein PopZ plays a crucial role in chromosome segregation by anchoring the chromosome partitioning machinery at a cell pole. While this process has been studied in monoploid species, it remains unclear whether this type of segregation mechanisms play a significant role in bacterial cells that carry multiple copies of chromosome. The alpha-proteobacterium Zymomonas mobilis exhibits ethanologenic physiology, and thus, is a promising chassis for biofuel production. Recent studies showed that Z. mobilis may serve as a non-model system for studying bacterial cell biology, owing to its relatively simple physiology and reduced genome size. Z. mobilis has previously been shown to be polyploid, and possesses a homolog of PopZ, a key regulator of chromosome organisation and cell polarity in alpha-proteobacteria. This raises the question of whether PopZ is involved in the organization of multiple chromosome copies. Here, we investigated the function of PopZ in Z. mobilis. Unlike in previously studied alpha-proteobacterial species, PopZ is dispensable for growth and cell morphology in Z. mobilis. However, the loss of PopZ altered the polar accumulation of chromosome segregation protein ParB, indicating an involvement in chromosome organization in Z. mobilis. These findings indicate that Z. mobilis might possess a chromosome segregation machinery that is dispensable under favourable growth conditions but might become important under certain conditions that reduce chomorome copy numbers.

microbiology↗

bioRxiv2026

Hypothetical lytic transglycosylase SleB is important for cell fitness in Zymomonas mobilis

Bacterial peptidoglycan (PG) undergoes a variety of chemical modifications. O-acetylation at the C6 hydroxyl group of N-acetylmuramic acid is a widespread PG-modification found across diverse bacterial phyla. It contributes to virulence in pathogenic bacteria because the O-acetyl group reduces the activity of the PG-degrading host defense enzyme, lysozyme. Beyond its role in host defense evasion, recent studies suggest that PG O-acetylation also regulates the activity of endogenous lytic transglycosylase (LT) autolysins. The ethanologenic alpha-proteobacterium Zymomonas mobilis O-acetylates its PG, which is associated with tolerance to environmental stresses, including salt. To better understand how PG O-acetylation contributes to stress tolerance, we investigated the predicted lytic transglycosylase SleB. Intriguingly, the sleB gene is located adjacent to the pat operon, which encodes the proteins responsible for PG O-acetylation. We showed that loss of SleB caused impaired growth and morphology, and a significant reduction of crosslinks in the PG of Z. mobilis. Furthermore, the sleB mutant was sensitive to environmental stress resembling the sensitivity of the patA mutant. Collectively, our findings unravelled an important role of SleB in PG remodelling and stress resilience.

microbiology↗

bioRxiv2026

Periplasmic SacB as a robust counter-selection tool for genome engineering in the polyploid bacterium Zymomonas mobilis

The alpha-proteobacterium Zymomonas mobilis exhibits exceptional ethanologenic physiology, which makes it a traditional alcoholic beverage producer and a promising chassis for biofuel production. Although genetic tools for this organism have expanded in recent years, a fundamental aspect of its chromosome organization remains to be understood. In particular, Z. mobilis has been suggested to exhibit polyploidy, but this feature is not fully confirmed because of discrepancies among studies reporting the copy number of chromosomes. Here, we tagged the chromosome-partitioning protein ParB with a fluorescent marker to visualise its cellular localisation and estimate chromosome copy number in individual cells. Imaging showed that Z. mobilis exhibits several distinctive ParB foci throughout the cytoplasm and an accumulated focus at the pole, demonstrating that a single Z. mobilis cell contains at >5 copies of the chromosome at the oriC regions. After verifying its polyploidy, we sought to establish an efficient counter-selection system, which is crucial for engineering multiple copies of the chromosome. We assessed the efficacy of levan-sucrase (SacB) toxicity in Z. mobilis. We found that, despite Z. mobilis secreting a native extra-cellular sucrase SacB, heterologous periplasmically-localised Bacillus subtilis SacB rendered Z. mobilis cells sensitive to sucrose. We successfully used this effect for counter-selection when deleting and inserting targeted DNA sequences into the Z. mobilis genome. Together, this work provides important insights and tools for advancing Z. mobilis genetics and its biotechnological applications. ImportanceZymomonas mobilis is a promising industrial bacterium with capacity to convert sugars into ethanol at nearly maximum theoretical yield. With its expanding use in industrial applications, it is crucial to clarify if individual Z. mobilis cells carry multiple copies of chromosome as this has important implications for genome engineering. Two previous studies have used quantitative PCR to address this question, but their reported chromosome copy numbers varied widely from 20 to 100. Here, we used a cell biological approach to estimate the copy number and confirmed that single Z. mobilis cell possesses multiple copies. In addition, we show that a SacB-based counter-selection works in Z. mobilis, enabling efficient and complete mutation of all chromosome copies.

microbiology↗

bioRxiv2025

Peptidoglycan remodelling improves salt resilience of Zymomonas mobilis

The alpha-proteobacterium Zymomonas mobilis is one of the most efficient microbial producers of ethanol and has the potential to be stablished as biofuel producer at industrial scale. However, a bottleneck hindering the full use of Z. mobilis in biorefinery is its sensitivity to environmental stresses, including sodium chloride (NaCl), a common component present in biomass from various sources. To address this limitation, we need to deepen our understanding of the cell envelope, the crucial barrier between bacteria and external stressors. To date, the cell envelope of Z. mobilis has remained largely uncharacterized. Here we show that the deletion of ctpA, which encodes a periplasmic protease, increases the salt resilience. Salt resilience is mediated by an increased level of the peptidoglycan endopeptidase MepM, which we identified as a substrate of CtpA through comparative proteomics. Supporting this, the overexpression of MepM in the wild-type enhanced salt resilience. We also discovered that the peptidoglycan of Z. mobilis is O-acetylated at MurNAc residues, a modification usually associated with virulence in pathogenic bacteria. Interestingly, O-acetylation was crucial for salt resilience, supporting a role in PG growth regulation under the stress condition. Overall, this study highlights the importance of investigating cell envelope biology in Z. mobilis as a foundation for engineering strains with improved resilience to environmental stress and, more general, studying the cell envelopes of non-model bacteria to expand our fundamental understanding of cell function ImportanceFossil fuels have negative impacts on the environment and will become limited in the next decades. Hence, alternative, sustainable energy sources need to be urgently established. Microbial fermentation of biomass for biofuel production presents a promising avenue. The Gram-negative alpha-proteobacterium Z. mobilis exhibits a superior capacity to convert sugars into ethanol, a clean, renewable and widely-used fuel. However, Z. mobilis has not been used as a first choice as a bio-fuel producer. The ethanol producer, bakers yeast Saccharomyces cerevisiae serves as a model species in cell biology, but we lack fundamental understanding of the cell envelope biology of Z. mobilis, which would be critical to engineer strain with increased resilience. Here, we demonstrate that knowledge about cell envelope biogenesis factors in Z. mobilis can help engineering optimised strains that grow under conditions of bio-fuel production.

microbiology↗

bioRxiv2020

Cell biological studies of ethanologenic bacterium Zymomonas mobilis

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 behaviour of Z. mobilis strain Zm6 at a single cell level. We observed that growing Z. mobilis cells often divided at non mid-cell position, which contributed to variant cell size at birth. Yet, 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 contents among individual cells, which might have been caused by asynchronous replication of chromosome that was not coordinated to cell growth. Furthermore, slightly angled divisions might have rendered temporary curvatures of attached Z. mobilis cells. Overall, the presented study uncovered a novel bacterial cell organization in Z. mobilis, the metabolism of which is not favoured for biosynthesis to build biomass. ImportanceWith increasing environmental concerns about the exhausting use of fossil fuels, a development of sustainable biofuel production platform has been attracting significant public attention. Ethanologenic Z. mobilis species are endowed with an efficient ethanol-fermentation capacity that surpass, in several aspects, that of the bakers yeast Saccharomyces cerevisiae, the most used microorganism for ethanol productions. For a development of 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 highlights that polyploid bacterium Z. mobilis exhibits a distinctive mode of bacterial cell organization, reflecting its unique metabolism that do not prioritize incorporation of nutrients to cell growth. Thus, another significance of presented work is to advance our general understanding in the diversity of bacterial cell architecture.

microbiology↗