Nozomu Obana

Stabilization of Clostridium perfringens collagenase mRNA by VR‐RNA‐dependent cleavage in 5′ leader sequence

The small RNA (sRNA), VR‐RNA that is directly regulated by the VirR/VirS two‐component system, regulates many genes including toxin genes such as collagenase (colA) and phospholipase C (plc) in Clostridium perfringens. Although the VR‐RNA 3′ region is sufficient to regulate the colA and plc genes, the molecular mechanism of toxin gene regulation by VR‐RNA remains unclear. Here, we found that colA mRNA is cleaved at position −79 and −78 from the A of the first codon (ATG) in the presence of VR‐RNA. The processed transcripts were stable compared with longer intact transcripts. On the other hand, colA mRNA was labile in a VR‐RNA‐deficient strain, and processed transcripts were undetectable. The stability and processing of colA mRNA were restored by transformation of the 3′ region of VR‐RNA‐expression vector. The 3′ region of VR‐RNA and colA mRNA had significant complementation and interacted in vitro. These results show that VR‐RNA base pairs with colA mRNA and induces cleavage in the 5′ untranslated region (UTR) of colA mRNA, which leads to the stabilization of colA mRNA and the activation of colA expression.

Environmental factors that shape biofilm formation

Cells respond to the environment and alter gene expression. Recent studies have revealed the social aspects of bacterial life, such as biofilm formation. Biofilm formation is largely affected by the environment, and the mechanisms by which the gene expression of individual cells affects biofilm development have attracted interest. Environmental factors determine the cell’s decision to form or leave a biofilm. In addition, the biofilm structure largely depends on the environment, implying that biofilms are shaped to adapt to local conditions. Second messengers such as cAMP and c-di-GMP are key factors that link environmental factors with gene regulation. Cell-to-cell communication is also an important factor in shaping the biofilm. In this short review, we will introduce the basics of biofilm formation and further discuss environmental factors that shape biofilm formation. Finally, the state-of-the-art tools that allow us investigate biofilms under various conditions are discussed. Graphical abstract Environmental factors affect biofilm formation throughout their development.

A Sporulation Factor Is Involved in the Morphological Change of Clostridium perfringens Biofilms in Response to Temperature

Biofilm formation has been associated with bacterial pathogenesis, such as nosocomial and chronic infections, as the resistance of biofilms to environmental stresses has increased. Clostridium perfringens is a Gram-positive spore-forming anaerobic pathogen. This organism survives antibiotic treatment through the formation of biofilms or spores, but the environmental and regulatory factors involved in the biofilm formation remain unclear. Here, we observed that temperature regulates C. perfringens biofilm morphology. At 37°C, C. perfringens adhered to the substrate surface and formed a flat, thin biofilm, herein referred to as adhered biofilm. However, at 25°C, this bacterium did not adhere and produced a threadlike extracellular matrix, forming a viscous, thick biofilm, herein referred to as pellicle biofilm. Pellicle biofilm formation requires the sporulation master regulator, Spo0A, and the toxin regulator, CtrAB, and is enhanced in the absence of the global repressor, AbrB. These transcriptional regulator genes are regulated by each other and temperature. Adhered-biofilm formation requires AbrB and pilA2, which encodes a component of type IV pili (TFP). TFP expression was activated at 37°C and regulated through Spo0A, AbrB, and CtrAB. These results indicate that the morphology of C. perfringens biofilm is dependent on temperature through the differential production of extracellular matrix and the activity of TFP. Moreover, pellicle biofilm formation is involved in sporulation and toxin production. Here, we demonstrated that clostridial biofilm formation is closely associated with sporulation and that the morphological change of the biofilms could play an important role in the pathogenesis of this organism.

A Novel Toxin Regulator, the CPE1446-CPE1447 Protein Heteromeric Complex, Controls Toxin Genes in Clostridium perfringens

Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium that is widespread in environmental soil and sewage, as well as in animal intestines. It is also a causative agent of diseases in humans and other animals, and it produces numerous extracellular enzymes and toxins. Although these toxins have been characterized in detail, regulators of toxin genes are less well understood. The present study identified CPE1447 and CPE1446 as novel regulators of toxin gene expression. CPE1447 and CPE1446 are cotranscribed as an operon, and the encoded proteins have a helix-turn-helix (HTH) motif at the N termini of their amino acid sequences, suggesting that CPE1447 and CPE1446 control the target genes as transcriptional regulators. The expression of several genes encoding toxins was changed in both a CPE1446 mutant and a CPE1447-CPE1446 deletion mutant. Complementation of CPE1446 and CPE1447 revealed that CPE1447 and CPE1446 coordinately regulate their target genes. CPE1447 protein was coprecipitated with His-tagged CPE1446 protein, indicating that the CPE1447 and CPE1446 proteins form a stable complex in C. perfringens under their native conditions. Although the small RNA that regulates several genes under the VirR/VirS two-component system (VR-RNA) positively affected CPE1447-CPE1446 mRNA expression, it did not control expression of the CPE1447-CPE1446 regulon, demonstrating that CPE1447 and CPE1446 regulate a different set of toxin genes from the VirR/VirS-VR-RNA cascade.

Effects of Depletion of RNA-Binding Protein Tex on the Expression of Toxin Genes in Clostridium perfringens

Tex was originally identified in Bordetella pertussis, where it serves as a transcriptional regulator of toxin genes. However, the Tex of Streptococcus pneumoniae has no regulatory function in the expression of the pneumococcal major toxin pneumolysin. Here, we identified the CPE2168 gene as Tex in Clostridium perfringens, and examined the roles of Tex in toxin gene expression. We found that the deletion mutant for Tex does not affect growth, but the mRNA levels of three hyaluronidase genes (nagH, nagJ, and nagL) and an exo-sialidase (nanJ) were reduced to less than 50% as compared to the parent strain, C. perfringens strain 13. On the other hand, Tex did not affect the expression of proteases, enterotoxins, hemolysins, either of two hyaluronidase genes (nagI and nagK), an exo-sialidase (nanI), or adhesins. Moreover, purified Tex bound to the 5′-portion of target gene mRNAs. Based on these results, we propose that Tex positively regulates the gene expression of a set of toxin genes in C. perfringens.

A novel approach for purification and selective capture of membrane vesicles of the periodontopathic bacterium, Porphyromonas gingivalis: membrane vesicles bind to magnetic beads coated with epoxy groups in a noncovalent, species-specific manner.

Membrane vesicles (MVs) of Porphyromonas gingivalis are regarded as an offensive weapon of the bacterium, leading to tissue deterioration in periodontal disease. Therefore, isolation of highly purified MVs is indispensable to better understand the pathophysiological role of MVs in the progression of periodontitis. MVs are generally isolated by a conventional method based on ultracentrifugation of the bacterial culture supernatant. However, the resulting MVs are often contaminated with co-precipitating bacterial appendages sheared from the live bacteria. Here, we report an intriguing property of P. gingivalis MVs–their ability to bind superparamagnetic beads coated with epoxy groups (SB-Epoxy). Analysis of fractions collected during the purification revealed that all MVs of five tested P. gingivalis stains bound to SB-Epoxy. In contrast, free fimbriae in the crude MV preparation did not bind to the SB-Epoxy. The SB-Epoxy-bound MVs were easily dissociated from the SB-Epoxy using a mild denaturation buffer. These results suggest that the surface chemistry conferred by epoxy on the beads is responsible for the binding, which is mediated by noncovalent bonds. Both the structural integrity and purity of the isolated MVs were confirmed by electron microscopy. The isolated MVs also caused cell detachment from culture dishes at a physiologically relevant concentration. Assays of competitive binding between the SB-Epoxy and mixtures of MVs from five bacterial species demonstrated that only P. gingivalis MVs could be selectively eliminated from the mixtures. We suggest that this novel approach enables efficient purification and selective elimination of P. gingivalis MVs.

Biofilm formation by Paracoccus denitrificans requires a type I secretion system-dependent adhesin BapA

Abstract Paracoccus denitrificans is a non‐swimming Gram‐negative bacterium, with versatile respiration capability which has remarkable potentials for bioremediation, especially in water treatment. Although biofilms are important in water treatment systems, the genetic mechanisms underlying the cellular adherence and biofilm formation of this bacterium remain unknown. We show that P. denitrificans forms a thin biofilm on surfaces at the air‐liquid interface under static conditions. The initial step of biofilm formation requires a biofilm‐associated protein BapA, which we identified by transposon mutant screening. BapA contains a unique sequence of dipeptide repeats of aspartate and alanine. Our data indicate that BapA is translocated to the extracellular milieu by a type 1 secretion system, where it enables the cells to attach to the substratum. Furthermore, superresolution microscopy shows that BapA is localized on the cell surface, which alters the cell surface hydrophobicity. Our results show a crucial role of BapA that promotes the adhesion and biofilm formation of P. denitrificans.

SP10 Infectivity Is Aborted after Bacteriophage SP10 Infection Induces nonA Transcription on the Prophage SPβ Region of the Bacillus subtilis Genome

Bacteria have developed various strategies for phage resistance. Infection with phage induces the transcription of part of the phage resistance gene, but the regulatory mechanisms of such transcription remain largely unknown. The phage resistance gene nonA is located on the SPβ prophage region of the Bacillus subtilis Marburg strain genome. The nonA transcript was detected at the late stage of SP10 infection but is undetectable in noninfected cells. The nonA transcript was detected after the induction of the sigma factor Orf199-Orf200 (σ(Orf199-200)), when sigma factors encoded in the SP10 genome were expressed from a xylose-inducible plasmid. Thus, the SP10 sigma factor is an activator of a set of SP10 genes and nonA. The nonA gene encodes a 72-amino-acid protein with a transmembrane motif and has no significant homology with any protein in any database. NonA overexpression halted cell growth and reduced the efficiency of B. subtilis colony formation and respiration activity. In addition, SP10 virion protein synthesis was inhibited in the nonA(+) strain, and SP10 virion particles were scarce in it. These results indicate that NonA is a novel protein that can abort SP10 infection, and its transcription was regulated by SP10 sigma factor.

Immunoactive Clostridial Membrane Vesicle Production Is Regulated by a Sporulation Factor

ABSTRACT Recently, many Gram-positive bacteria as well as Gram-negative bacteria have been reported to produce membrane vesicles (MVs), but little is known regarding the regulators involved in MV formation. We found that a Gram-positive anaerobic pathogen, Clostridium perfringens, produces MVs predominantly containing membrane proteins and cell wall components. These MVs stimulated proinflammatory cytokine production in mouse macrophage-like cells. We suggested that MVs induced interleukin-6 production through the Toll-like receptor 2 (TLR2) signaling pathway. Thus, the MV could have a role in the bacterium-host interaction and bacterial infection pathogenesis. Moreover, we found that the sporulation master regulator gene spo0A was required for vesiculogenesis. A conserved, phosphorylated aspartate residue of Spo0A was indispensable for MV production, suggesting that the phosphorylation of Spo0A triggers MV production. Multiple orphan sensor kinases necessary for sporulation were also required to maximize MV production. These findings imply that C. perfringens actively produces immunoactive MVs in response to the environment changing, as recognized by membrane-spanning sensor kinases and by modulating the phosphorylation level of Spo0A.

A Versatile and Rapidly Deployable Device to Enable Spatiotemporal Observations of the Sessile Microbes and Environmental Surfaces

Although microbes typically associate with surfaces, detailed observations of surface-associated microbes on natural substrata are technically challenging. We herein introduce a flow channel device named the Stickable Flow Device, which is easily configurable and deployable on various surfaces for the microscopic imaging of environmental microbes. We demonstrated the utility of this device by creating a flow channel on different types of surfaces including live leaves. This device enables the real-time imaging of bacterial biofilms and their substrata. The Stickable Flow Device expands the limits of conventional real-time imaging systems, thereby contributing to a deeper understanding of microbe-surface interactions on various surfaces.