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Fusobacterium nucleatum is a key member of the human oral biofilm. It is also implicated in preterm birth and colorectal cancer. To facilitate basic studies of fusobacterial virulence, we describe here a versatile transposon mutagenesis procedure and a pilot screen for mutants defective in biofilm formation. Out of 10 independent biofilm-defective mutants isolated, the affected genes included the homologs of the Escherichia coli cell division proteins FtsX and EnvC, the electron transport protein RnfA, and four proteins with unknown functions. Next, a facile new gene deletion method demonstrated that nonpolar, in-frame deletion of ftsX or envC produces viable bacteria that are highly filamentous due to defective cell division. Transmission electron and cryo-electron microscopy revealed that the ΔftsX and ΔenvC mutant cells remain joined with apparent constriction, and scanning electron microscopy (EM) uncovered a smooth cell surface without the microfolds present in wild-type cells. FtsX and EnvC proteins interact with each other as well as a common set of interacting partners, many with unknown function. Last, biofilm development is altered when cell division is blocked by MinC overproduction; however, unlike the phenotypes of ΔftsX and ΔenvC mutants, a weakly adherent biofilm is formed, and the wild-type rugged cell surface is maintained. Therefore, FtsX and EnvC may perform novel functions in Fusobacterium cell biology. This is the first report of an unbiased approach to uncover genetic determinants of fusobacterial biofilm development. It points to an intriguing link among cytokinesis, cell surface dynamics, and biofilm formation, whose molecular underpinnings remain to be elucidated.IMPORTANCE Little is known about the virulence mechanisms and associated factors in F. nucleatum, due mainly to the lack of convenient genetic tools for this organism. We employed two efficient genetic strategies to identify F. nucleatum biofilm-defective mutants, revealing FtsX and EnvC among seven biofilm-associated factors. Electron microscopy established cell division defects of the ΔftsX and ΔenvC mutants, accompanied with a smooth cell surface, unlike the microfold, rugged appearance of wild-type bacteria. Proteomic studies demonstrated that FtsX and EnvC interact with each other as well as a set of common and unique interacting proteins, many with unknown functions. Importantly, blocking cell division by MinC overproduction led to formation of a weakly adherent biofilm, without alteration of the wild-type cell surface. Thus, this work links cell division and surface dynamics to biofilm development and lays a foundation for future genetic and biochemical investigations of basic cellular processes in this clinically significant pathogen.
Pubmed ID: 29691334
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View all literature mentionsA collection of Pathway/Genome Databases which describes the genome and metabolic pathways of a single organism. The BioCyc collection of Pathway/Genome Databases (PGDBs) provides an electronic reference source on the genomes and metabolic pathways of sequenced organisms. BioCyc PGDBs are generated by software that predicts the metabolic pathway complements of completely sequenced organisms from their genome sequences. They also include the results of a number of other computational inference procedures applied to these genomes, including predictions of which genes code for missing enzymes in metabolic pathways, and predicted operons. The BioCyc Web site provides a suite of software tools for database searching and visualization, for omics data analysis, and for comparative genomics and comparative pathway questions. The databases within the BioCyc collection are organized into tiers according to the amount of manual review and updating they have received. Tier 1 PGDBs have been created through intensive manual efforts, and receive continuous updating. Tier 2 PGDBs were computationally generated by the PathoLogic program, and have undergone moderate amounts of review and updating. Tier 3 PGDBs were computationally generated by the PathoLogic program, and have undergone no review and updating. There are 967 DBs in Tier 3. The downloadable version of BioCyc that includes the Pathway Tools software provides more speed and power than the BioCyc Web site.
View all literature mentionsA free, cross-platform set of image processing, modeling and display programs used for tomographic reconstruction and for 3D reconstruction of EM serial sections and optical sections. The package contains tools for assembling and aligning data within multiple types and sizes of image stacks, viewing 3-D data from any orientation, and modeling and display of the image files. IMOD 4.1.8 Is Now Available for Linux, Windows, and Mac OS X
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