Dataset Information

Development of a Gene Replacement Method for the Filamentous BacteriumLeptothrix cholodniiSP-6.

ABSTRACT: Genetic strategies such as gene disruption and fluorescent protein tagging largely contribute to understanding the molecular mechanisms of biological functions in bacteria. However, the methods for gene replacement remain underdeveloped for the filamentous bacteriaLeptothrix cholodniiSP-6. Their cell chains are encased in sheath composed of entangled nanofibrils, which may prevent the conjugation for gene transfer. Here, we describe a protocol optimized for gene disruption through gene transfer mediated by conjugation withEscherichia coliS17-1 with details on cell ratio, sheath removal, and loci validation. The obtained deletion mutants for specific genes can be used to clarify the biological functions of the proteins encoded by the target genes. Graphical overview.

SUBMITTER: Kunoh T

PROVIDER: S-EPMC10127049 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Development of a Gene Replacement Method for the Filamentous Bacterium<i>Leptothrix cholodnii</i>SP-6.

Kunoh Tatsuki T Ono Erika E Yamamoto Tatsuya T Suzuki Ichiro I Takeda Minoru M Nomura Nobuhiko N

Bio-protocol 20230420 8

Genetic strategies such as gene disruption and fluorescent protein tagging largely contribute to understanding the molecular mechanisms of biological functions in bacteria. However, the methods for gene replacement remain underdeveloped for the filamentous bacteria<i>Leptothrix cholodnii</i>SP-6. Their cell chains are encased in sheath composed of entangled nanofibrils, which may prevent the conjugation for gene transfer. Here, we describe a protocol optimized for gene disruption through gene tr ...[more]

PMID: 37113333

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Project description:Species belonging to the genus Leptothrix are widely distributed in the environment and in activated sludge (AS) wastewater treatment plants (WWTPs). They are commonly found in iron-rich environments and reported to cause filamentous bulking in WWTPs. In this study, the diversity, distribution, and metabolic potential of the most prevalent Leptothrix spp. found in AS worldwide were studied. Our 16S rRNA amplicon survey showed that Leptothrix belongs to the general core community of AS worldwide, comprising 32 species with four species being most commonly found. Their taxonomic classification was re-evaluated based on both 16S rRNA gene and genome-based phylogenetic analysis showing that three of the most abundant "Leptothrix" species represented species in three other genera, Rubrivivax, Ideonella, and the novel genus, Ca. Intricatilinea. New fluorescence in situ hybridization (FISH) probes revealed rod-shaped morphology for the novel Ca. Rubrivivax defluviihabitans and Ca. Ideonella esbjergensis, while filamentous morphology was found only for Ca. Intricatilinea gracilis. Analysis of high-quality metagenome-assembled genomes revealed metabolic potential for aerobic growth, fermentation, storage of intracellular polymers, partial denitrification, photosynthesis, and iron reduction. FISH in combination with Raman microspectroscopy confirmed the in situ presence of chlorophyll and carotenoids in Ca. Rubrivivax defluviihabitans and Ca. Intricatilinea gracilis. This study resolves the taxonomy of abundant but poorly classified "Leptothrix" species, providing important insights into their diversity, morphology, and function in global AS wastewater treatment systems.IMPORTANCEThe genus Leptothrix has been extensively studied and described since the 1880s, with six species currently described but with the majority uncultured and undescribed. Some species are assumed to have a filamentous morphology and can cause settling problems in wastewater treatment plants (WWTPs). Here, we revised the classification of the most abundant Leptothrix spp. present in WWTPs across the world, showing that most belong to other genera, such as Rubrivivax and Ideonella. Furthermore, most do not have a filamentous morphology and are not problematic in WWTPs as previously believed. Metabolic reconstruction, including some traits validated in situ by the application of new fluorescence in situ hybridization probes and Raman microspectroscopy, provided additional insights into their metabolism. The study has contributed to a better understanding of the diversity, morphology, and function of "Leptothrix," which belong to the abundant core community across global activated sludge WWTPs.

Project description:Leptothrix ochracea is known for producing large volumes of iron oxyhydroxide sheaths that alter wetland biogeochemistry. For over a century, these delicate structures have fascinated microbiologists and geoscientists. Because L. ochracea still resists long-term in vitro culture, the debate regarding its metabolic classification dates back to 1885. We developed a novel culturing technique for L. ochracea using in situ natural waters and coupled this with single-cell genomics and nanoscale secondary-ion mass spectrophotometry (nanoSIMS) to probe L. ochracea's physiology. In microslide cultures L. ochracea doubled every 5.7 h and had an absolute growth requirement for ferrous iron, the genomic capacity for iron oxidation, and a branched electron transport chain with cytochromes putatively involved in lithotrophic iron oxidation. Additionally, its genome encoded several electron transport chain proteins, including a molybdopterin alternative complex III (ACIII), a cytochrome bd oxidase reductase, and several terminal oxidase genes. L. ochracea contained two key autotrophic proteins in the Calvin-Benson-Bassham cycle, a form II ribulose bisphosphate carboxylase, and a phosphoribulose kinase. L. ochracea also assimilated bicarbonate, although calculations suggest that bicarbonate assimilation is a small fraction of its total carbon assimilation. Finally, L. ochracea's fundamental physiology is a hybrid of those of the chemolithotrophic Gallionella-type iron-oxidizing bacteria and the sheathed, heterotrophic filamentous metal-oxidizing bacteria of the Leptothrix-Sphaerotilus genera. This allows L. ochracea to inhabit a unique niche within the neutrophilic iron seeps.IMPORTANCELeptothrix ochracea was one of three groups of organisms that Sergei Winogradsky used in the 1880s to develop his hypothesis on chemolithotrophy. L. ochracea continues to resist cultivation and appears to have an absolute requirement for organic-rich waters, suggesting that its true physiology remains unknown. Further, L. ochracea is an ecological engineer; a few L. ochracea cells can generate prodigious volumes of iron oxyhydroxides, changing the ecosystem's geochemistry and ecology. Therefore, to determine L. ochracea's basic physiology, we employed new single-cell techniques to demonstrate that L. ochracea oxidizes iron to generate energy and, despite having predicted genes for autotrophic growth, assimilates a fraction of the total CO2 that autotrophs do. Although not a true chemolithoautotroph, L. ochracea's physiological strategy allows it to be flexible and to extensively colonize iron-rich wetlands.

Dataset Information

Development of a Gene Replacement Method for the Filamentous BacteriumLeptothrix cholodniiSP-6.

Publications

Development of a Gene Replacement Method for the Filamentous Bacterium<i>Leptothrix cholodnii</i>SP-6.

Similar Datasets

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