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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.

Ovalbumin (OVA), one of the members of the serpin-superfamily, is the major protein in chicken eggs. Many studies have demonstrated the polymerization ability of OVA but the detailed molecular mechanisms demonstrating its conformational changes accompanying fibril formation are still unclear. This study revealed nitric oxide (NO) induced conformational changes and oligomerization of egg white OVA, resulting in polymerized fibrils. Electron microscopic analysis showed that NO treatment to OVA under mild acidic condition resulted in morphological changes, producing structures similar to the long protein fibrils found in egg white. Spectroscopic analysis and mass spectrometry found that NO-treated OVA contains increased number of β-sheet, indicating transition from α-helixes to β-sheets, and S-nitrosylation of OVA cysteine residue 367. Structural modeling showed that S-nitrosocysteine, Cys367 NO , is located in the amyloidogenic core region of the C-terminal region, nearby the N-terminal core region where the α-to-β transition is induced. Such results provide a potential mechanism for non-disease-associated fibril formation of OVA. [ABSTRACT FROM AUTHOR]

Alkyl hydroperoxide reductase E (AhpE) is a member of the peroxidase family of enzymes that catalyse the reduction of peroxides, however its structural and functional roles are still unclear in details. In this study, we used the Thermococcus kodakarensis AhpE-like protein as a model to investigate structure–function relationships including the molecular properties of DNA binding activity. Multiple sequence alignment, structural comparison and biochemical analyses revealed that TkAhpE includes conserved peroxidase residues in the active site, and exhibits peroxidase activity with structure-dependent holdase chaperone function. Following electrophoretic mobility shift assays and electron microscopy analysis demonstrated distinctive binding features of TkAhpE to the DNA showing that their dimeric conformer can bind to the double-stranded DNA, but not to the single-stranded DNA, indicating its striking molecular features to double-stranded DNA-specific interactions. Based on our results, we provided that TkAhpE is a multifunctional peroxidase displaying structure-dependent molecular chaperone and DNA binding activities. [ABSTRACT FROM AUTHOR]