Genome-encoded ABCF factors implicated in intrinsic antibiotic resistance in Gram-positive bacteria: VmlR2, Ard1 and CplR.

Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by uORF4u, our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge.

Colony-Based Electrochemistry Reveals Electron Conduction Mechanisms Mediated by Cytochromes and Flavins in Shewanella oneidensis.

Bacteria utilize electron conduction in their communities to drive their metabolism, which has led to the development of various environmental technologies, such as electrochemical microbial systems and anaerobic digestion. It is challenging to measure the conductivity among bacterial cells when they hardly form stable biofilms on electrodes. This makes it difficult to identify the biomolecules involved in electron conduction. In the present study, we aimed to identify c-type cytochromes involved in electron conduction in Shewanella oneidensis MR-1 and examine the molecular mechanisms. We established a colony-based bioelectronic system that quantifies bacterial electrical conductivity, without the need for biofilm formation on electrodes. This system enabled the quantification of the conductivity of gene deletion mutants that scarcely form biofilms on electrodes, demonstrating that c-type cytochromes, MtrC and OmcA, are involved in electron conduction. Furthermore, the use of colonies of gene deletion mutants demonstrated that flavins participate in electron conduction by binding to OmcA, providing insight into the electron conduction pathways at the molecular level. Furthermore, phenazine-based electron transfer in Pseudomonas aeruginosa PAO1 and flavin-based electron transfer in Bacillus subtilis 3610 were confirmed, indicating that this colony-based system can be used for various bacteria, including weak electricigens.

Identification of lthB, a Gene Encoding a Putative Glycosyltransferase Family 8 Protein Required for Leptothrix Sheath Formation.

The bacterium Leptothrix cholodnii generates cell chains encased in sheaths that are composed of woven nanofibrils. The nanofibrils are mainly composed of glycoconjugate repeats, and several glycosyltransferases (GTs) are required for its biosynthesis. However, only one GT (LthA) has been identified to date. In this study, we screened spontaneous variants of L. cholodnii SP6 to find those that form smooth colonies, which is one of the characteristics of sheathless variants. Genomic DNA sequencing of an isolated variant revealed an insertion in the locus Lcho_0972, which encodes a putative GT family 8 protein. We thus designated this protein LthB and characterized it using deletion mutants and antibodies. LthB localized adjacent to the cell envelope. ΔlthB cell chains were nanofibril free and thus sheathless, indicating that LthB is involved in nanofibril biosynthesis. Unlike the ΔlthA mutant and the wild-type strain, which often generate planktonic cells, most ΔlthB organisms presented as long cell chains under static conditions, resulting in deficient pellicle formation, which requires motile planktonic cells. These results imply that sheaths are not required for elongation of cell chains. Finally, calcium depletion, which induces cell chain breakage due to sheath loss, abrogated the expression of LthA, but not LthB, suggesting that these GTs cooperatively participate in glycoconjugate biosynthesis under different signaling controls. IMPORTANCE In recent years, the regulation of cell chain elongation of filamentous bacteria via extracellular signals has attracted attention as a potential strategy to prevent clogging of water distribution systems and filamentous bulking of activated sludge in industrial settings. However, a fundamental understanding of the ecology of filamentous bacteria remains elusive. Since sheath formation is associated with cell chain elongation in most of these bacteria, the molecular mechanisms underlying nanofibril sheath formation, including the intracellular signaling cascade in response to extracellular stimuli, must be elucidated. Here, we isolated a sheathless variant of L. cholodnii SP6 and thus identified a novel glycosyltransferase, LthB. Although mutants with deletions of lthA, encoding another GT, and lthB were both defective for nanofibril formation, they exhibited different phenotypes of cell chain elongation and pellicle formation. Moreover, LthA expression, but not LthB expression, was influenced by extracellular calcium, which is known to affect nanofibril formation, indicating the functional diversities of LthA and LthB. Such molecular insights are critical for a better understanding of ecology of filamentous bacteria, which, in turn, can be used to improve strategies to control filamentous bacteria in industrial facilities.

Porous Pellicle Formation of a Filamentous Bacterium, Leptothrix.

The bacterium Leptothrix cholodnii generates filaments encased in a sheath comprised of woven nanofibrils. In static liquid culture, L. cholodnii moves toward the air-liquid interface, where it forms porous pellicles. Observations of aggregation at the interface reveal that clusters consisting of only a few bacteria primarily grow by netting free cells. These growing clusters hierarchically enlarge through the random docking of other small clusters. We find that the bacteria swim using their polar flagellum toward the interface, where their sheath assists them in intertwining with others and thereby promotes the formation of small clusters. In contrast, sheathless hydrophobic mutant cells get stuck to the interface. We find that the nanofibril sheath is vital for robust pellicle formation as it lowers cell surface hydrophobicity by 60%, thereby reducing their adsorption and enabling cells to move toward and stick together at the air-liquid interface. IMPORTANCE Efficient and sustainable management of water resources is becoming a fundamental issue for supporting growing populations and for developing economic activity. Fundamental to this management is the treatment of wastewater. Microorganisms are the active component of activated sludge that is employed in the biodegradation process of many wastewater treatment facilities. However, uncontrolled growth of filamentous bacteria such as Sphaerotilus often results in filamentous bulking, lowering the efficiency of water treatment systems. To prevent this undesirable condition, strategies based on a fundamental understanding of the ecology of filamentous bacteria are required. Although the filamentous bacterium Leptothrix cholodnii, which is closely related to Sphaerotilus, is a minor inhabitant of activated sludge, its complete genome sequence is known, making gene manipulation relatively easy. Moreover, L. cholodnii generates porous pellicles under static conditions, which may be a characteristic of filamentous bulking. We show that both swimming motility and nanofibril-mediated air-liquid interface attachment are required for porous pellicle formation. These insights are critical for a better understanding of the characteristics of filamentous bulking and might improve strategies to control activated sludge.

Autolysis-mediated membrane vesicle formation in Bacillus subtilis.

It is known that Bacillus subtilis releases membrane vesicles (MVs) during the SOS response, which is associated with cell lysis triggered by the PBSX prophage-encoded cell-lytic enzymes XhlAB and XlyA. In this study, we demonstrate that MVs are released under various stress conditions: sucrose fatty acid ester (SFE; surfactant) treatment, cold shock, starvation, and oxygen deficiency. B. subtilis possesses four major host-encoded cell wall-lytic enzymes (autolysins; LytC, LytD, LytE, and LytF). Deletions of the autolysin genes abolished autolysis and the consequent MV production under these stress conditions. In contrast, deletions of xhlAB and xlyA had no effect on autolysis-triggered MV biogenesis, indicating that autolysis is a novel and prophage-independent pathway for MV production in B. subtilis. Moreover, we found that the cell lysis induced by the surfactant treatment was effectively neutralized by the addition of exogenous purified MVs. This result suggests that the MVs can serve as a decoy for the cellular membrane to protect the living cells in the culture from membrane damage by the surfactant. Our results indicate a positive effect of B. subtilis MVs on cell viability and provide new insight into the biological importance of the autolysis phenomenon in B. subtilis.

Roadmap on emerging concepts in the physical biology of bacterial biofilms: from surface sensing to community formation.

Bacterial biofilms are communities of bacteria that exist as aggregates that can adhere to surfaces or be free-standing. This complex, social mode of cellular organization is fundamental to the physiology of microbes and often exhibits surprising behavior. Bacterial biofilms are more than the sum of their parts: single-cell behavior has a complex relation to collective community behavior, in a manner perhaps cognate to the complex relation between atomic physics and condensed matter physics. Biofilm microbiology is a relatively young field by biology standards, but it has already attracted intense attention from physicists. Sometimes, this attention takes the form of seeing biofilms as inspiration for new physics. In this roadmap, we highlight the work of those who have taken the opposite strategy: we highlight the work of physicists and physical scientists who use physics to engage fundamental concepts in bacterial biofilm microbiology, including adhesion, sensing, motility, signaling, memory, energy flow, community formation and cooperativity. These contributions are juxtaposed with microbiologists who have made recent important discoveries on bacterial biofilms using state-of-the-art physical methods. The contributions to this roadmap exemplify how well physics and biology can be combined to achieve a new synthesis, rather than just a division of labor.

Competence-Stimulating-Peptide-Dependent Localized Cell Death and Extracellular DNA Production in Streptococcus mutans Biofilms.

Extracellular DNA (eDNA) is a biofilm component that contributes to the formation and structural stability of biofilms. Streptococcus mutans, a major cariogenic bacterium, induces eDNA-dependent biofilm formation under specific conditions. Since cell death can result in the release and accumulation of DNA, the dead cells in biofilms are a source of eDNA. However, it remains unknown how eDNA is released from dead cells and is localized within S. mutans biofilms. We focused on cell death induced by the extracellular signaling peptide called competence-stimulating peptide (CSP). We demonstrate that nucleic acid release into the extracellular environment occurs in a subpopulation of dead cells. eDNA production induced by CSP was highly dependent on the lytF gene, which encodes an autolysin. Although lytF expression was induced bimodally by CSP, lytF-expressing cells further divided into surviving cells and eDNA-producing dead cells. Moreover, we found that lytF-expressing cells were abundant near the bottom of the biofilm, even when all cells in the biofilm received the CSP signal. Dead cells and eDNA were also abundantly present near the bottom of the biofilm. The number of lytF-expressing cells in biofilms was significantly higher than that in planktonic cultures, which suggests that adhesion to the substratum surface is important for the induction of lytF expression. The deletion of lytF resulted in reduced adherence to a polystyrene surface. These results suggest that lytF expression and eDNA production induced near the bottom of the biofilm contribute to a firmly attached and structurally stable biofilm.IMPORTANCE Bacterial communities encased by self-produced extracellular polymeric substances (EPSs), known as biofilms, have a wide influence on human health and environmental problems. The importance of biofilm research has increased, as biofilms are the preferred bacterial lifestyle in nature. Furthermore, in recent years it has been noted that the contribution of phenotypic heterogeneity within biofilms requires analysis at the single-cell or subpopulation level to understand bacterial life strategies. In Streptococcus mutans, a cariogenic bacterium, extracellular DNA (eDNA) contributes to biofilm formation. However, it remains unclear how and where the cells produce eDNA within the biofilm. We focused on LytF, an autolysin that is induced by extracellular peptide signals. We used single-cell level imaging techniques to analyze lytF expression in the biofilm population. Here, we show that S. mutans generates eDNA by inducing lytF expression near the bottom of the biofilm, thereby enhancing biofilm adhesion and structural stability.

Potential Risk of Spreading Resistance Genes within Extracellular-DNA-Dependent Biofilms of Streptococcus mutans in Response to Cell Envelope Stress Induced by Sub-MICs of Bacitracin.

Antibiotics are used to treat or prevent some types of bacterial infection. The inappropriate use of antibiotics unnecessarily promotes antibiotic resistance and increases resistant bacteria, and controlling these bacteria is difficult. While the emergence of drug-resistant bacteria is a serious problem, the behavior of drug-resistant bacteria is not fully understood. In this study, we investigated the behavior of Streptococcus mutans, a major etiological agent of dental caries that is resistant to bacitracin, which is a cell wall-targeting antibiotic, and focused on biofilm formation in the presence of bacitracin. S. mutans UA159 most strongly induced extracellular DNA (eDNA)-dependent biofilm formation in the presence of bacitracin at 1/8× MIC. The ΔmbrC and ΔmbrD mutant strains, which lack bacitracin resistance, also formed biofilms in the presence of bacitracin at 1/2× MIC. This difference between the wild type and the mutants was caused by the induction of atlA expression in the mid-log phase. We also revealed that certain rgp genes involved in the synthesis of rhamnose-glucose polysaccharide related to cell wall synthesis were downregulated by bacitracin. In addition, glucosyltransferase-I was also involved in eDNA-dependent biofilm formation. The biofilm led to increased transformation efficiencies and promoted horizontal gene transfer. Biofilms were also induced by ampicillin and vancomycin, antibiotics targeting cell wall synthesis, suggesting that cell envelope stress triggers biofilm formation. Therefore, the expression of the atlA and rgp genes is regulated by S. mutans, which forms eDNA-dependent biofilms, promoting horizontal gene transfer in response to cell envelope stress induced by sub-MICs of antibiotics.IMPORTANCE Antibiotics have been reported to induce biofilm formation in many bacteria at subinhibitory concentrations. Accordingly, it is conceivable that the MIC against drug-sensitive bacteria may promote biofilm formation of resistant bacteria. Since drug-resistant bacteria have spread, it is important to understand the behavior of resistant bacteria. Streptococcus mutans is bacitracin resistant, and the 1/8× MIC of bacitracin, which is a cell wall-targeted antibiotic, induced eDNA-dependent biofilm formation. The ΔmbrC and ΔmbrD strains, which are not resistant to bacitracin, also formed biofilms in the presence of bacitracin at 1/2× MIC, and biofilms of both the wild type and mutants promoted horizontal gene transfer. Another cell wall-targeted antibiotic, vancomycin, showed effects on biofilms and gene transfer similar to those of bacitracin. Thus, treatment with cell wall-targeted antibiotics may promote the spread of drug-resistant genes in biofilms. Therefore, the behavior of resistant bacteria in the presence of antibiotics at sub-MICs should be investigated when using antibiotics.

Synergy between Sophorolipid Biosurfactant and SDS Increases the Efficiency of P. aeruginosa Biofilm Disruption.

Biofilms are communities of bacteria encased in self-secreted extracellular polymeric substances (EPS) that adhere stubbornly to submerged surfaces. Once established, these communities can cause serious chronic illnesses in medical settings, while they can promote corrosion and biofouling in industrial settings. Due to the difficulty of their removal, strongly oxidizing chemicals and detergents can be used to degrade and remove biofilms by killing the cells and degrading the matrix; however, the choice of compounds is limited in delicate environments due to the potential damage they may cause. In the case of detergents, most are synthesized from nonrenewable petrochemicals that have a degree of aquatic toxicity. There is a growing need to identify and characterize alternatives to synthetic surfactants. Biosurfactants, which are surfactants produced by microorganisms, are a promising alternative since they can be synthesized from renewable resources, have low environmental toxicity, and have been shown to have higher degrees of specificity in the mechanism of action. Sophorolipids are a class of glycolipid surfactants produced by yeast that have demonstrated great promise due to large yields from renewable feedstocks and for antimicrobial properties; however, the effect of the application of sophorolipids to Gram-negative bacterial biofilms has not been well studied. We investigate the antibiofilm properties of sophorolipids by demonstrating its ability to cause the catastrophic disruption of Pseudomonas aeruginosa PAO1 biofilms in microfluidic channels. We show that while sophorolipids inflict little damage to the bacteria, they weaken the EPS biofilm matrix, leading to surface-detachment and breakup of the biofilm. Furthermore, we find that sophorolipids act cooperatively with the widely used surfactant, sodium dodecyl sulfate. When combined, concentrations ∼100-fold lower than the minimum effective concentration, when used independently, recover potency. Biosurfactants are typically expensive to produce, thus our work demonstrates a means to improve efficacy while simultaneously reducing both cost and the amount of environmentally harmful substances used.

Process Description of an Unconventional Biofilm Formation by Bacterial Cells Autoagglutinating through Sticky, Long, and Peritrichate Nanofibers.

In this study, we elucidated the formation process of an unconventional biofilm formed by a bacterium autoagglutinating through sticky, long, and peritrichate nanofibers. Understanding the mechanisms of biofilm formation is essential to control microbial behavior and improve environmental biotechnologies. Acinetobacter sp. Tol 5 autoagglutinate through the interaction of the long, peritrichate nanofiber protein AtaA, a trimeric autotransporter adhesin. Using AtaA, without cell growth or extracellular polymeric substances production, Tol 5 cells quickly form an unconventional biofilm. The process forming this unconventional biofilm started with cell-cell interactions, proceeded to cell clumping, and led to the formation of large cell aggregates. The cell-cell interaction was described by Derjaguin-Landau-Verwey-Overbeek (DLVO) theory based on a new concept, which considers two independent interactions between two cell bodies and between two AtaA fiber tips forming a discontinuous surface. If cell bodies cannot collide owing to an energy barrier at low ionic strengths but approach within the interactive distance of AtaA fibers, cells can agglutinate through their contact. Cell clumping proceeds following the cluster-cluster aggregation model, and an unconventional biofilm containing void spaces and a fractal nature develops. Understanding its formation process would extend the utilization of various types of biofilms, enhancing environmental biotechnologies.

Peculiarities of biofilm formation by Paracoccus denitrificans.

Most bacteria form biofilms, which are thick multicellular communities covered in extracellular matrix. Biofilms can become thick enough to be even observed by the naked eye, and biofilm formation is a tightly regulated process. Paracoccus denitrificans is a non-motile, Gram-negative bacterium that forms a very thin, unique biofilm. A key factor in the biofilm formed by this bacterium is a large surface protein named biofilm-associated protein A (BapA), which was recently reported to be regulated by cyclic diguanosine monophosphate (cyclic-di-GMP or c-di-GMP). Cyclic-di-GMP is a major second messenger involved in biofilm formation in many bacteria. Though cyclic-di-GMP is generally reported as a positive regulatory factor in biofilm formation, it represses biofilm formation in P. denitrificans. Furthermore, quorum sensing (QS) represses biofilm formation in this bacterium, which is also reported as a positive regulator of biofilm formation in most bacteria. The QS signal used in P. denitrificans is hydrophobic and is delivered through membrane vesicles. Studies on QS show that P. denitrificans can potentially form a thick biofilm but maintains a thin biofilm under normal growth conditions. In this review, we discuss the peculiarities of biofilm formation by P. denitrificans with the aim of deepening the overall understanding of bacterial biofilm formation and functions.

Intra- and Interspecies Variability of Single-Cell Innate Fluorescence Signature of Microbial Cell.

Here we analyzed the innate fluorescence signature of the single microbial cell, within both clonal and mixed populations of microorganisms. We found that even very similarly shaped cells differ noticeably in their autofluorescence features and that the innate fluorescence signatures change dynamically with growth phases. We demonstrated that machine learning models can be trained with a data set of single-cell innate fluorescence signatures to annotate cells according to their phenotypes and physiological status, for example, distinguishing a wild-type Aspergillus nidulans cell from its nitrogen metabolism mutant counterpart and log-phase cells from stationary-phase cells of Pseudomonas putida We developed a minimally invasive method (confocal reflection microscopy-assisted single-cell innate fluorescence [CRIF] analysis) to optically extract and catalog the innate cellular fluorescence signatures of each of the individual live microbial cells in a three-dimensional space. This technique represents a step forward from traditional techniques which analyze the innate fluorescence signatures at the population level and necessitate a clonal culture. Since the fluorescence signature is an innate property of a cell, our technique allows the prediction of the types or physiological status of intact and tag-free single cells, within a cell population distributed in a three-dimensional space. Our study presents a blueprint for a streamlined cell analysis where one can directly assess the potential phenotype of each single cell in a heterogenous population by its autofluorescence signature under a microscope, without cell tagging.IMPORTANCE A cell's innate fluorescence signature is an assemblage of fluorescence signals emitted by diverse biomolecules within a cell. It is known that the innate fluoresce signature reflects various cellular properties and physiological statuses; thus, they can serve as a rich source of information in cell characterization as well as cell identification. However, conventional techniques focus on the analysis of the innate fluorescence signatures at the population level but not at the single-cell level and thus necessitate a clonal culture. In the present study, we developed a technique to analyze the innate fluorescence signature of a single microbial cell. Using this novel method, we found that even very similarly shaped cells differ noticeably in their autofluorescence features, and the innate fluorescence signature changes dynamically with growth phases. We also demonstrated that the different cell types can be classified accurately within a mixed population under a microscope at the resolution of a single cell, depending solely on the innate fluorescence signature information. We suggest that single-cell autofluoresce signature analysis is a promising tool to directly assess the taxonomic or physiological heterogeneity within a microbial population, without cell tagging.

Antifungal susceptibility trend and analysis of resistance mechanism for Candida species isolated from bloodstream at a Japanese university hospital.

We compared the susceptibility of six commercially available antifungal agents (fluconazole, itraconazole, voriconazole, caspofungin, micafungin, and amphotericin B) against 133 Candida bloodstream isolates between 2008 and 2013 at Aichi Medical University Hospital. C. albicans was the most common isolate, followed by C. parapsilosis, C. glabrata, and C. tropicalis. MIC90s of voriconazole against C. albicans, C. parapsilosis, and C. tropicalis were the lowest and that of micafungin against C. glabrata was the lowest among the agents tested. Of the 133 isolates, two strains were identified as drug-resistant. One was a fluconazole-resistant C. glabrata strain, in which the ATP-binding cassette (ABC) transporter gene expression was upregulated. The other was a micafungin-resistant C. glabrata strain, that had 13 amino acid substitutions in FKS1 and FKS2, including a novel substitution V1342I in FKS1 hotspot 2. We also evaluated the susceptibility of T-2307, a novel class of antifungal agents used in clinical trials, against the fluconazole- and micafungin-resistant C. glabrata strain; the MICs of T-2307 were 0.0039 and 0.0078 µg/mL, respectively. In conclusion, the incidence of bloodstream infection caused by drug-resistant Candida spp. was rare from 2008 to 2013 at our hospital. Of 133 isolates, only two strains of C. glabrata were resistant to azoles or echinocandins, that upregulated the ABC transporter genes or had novel FKS mutations, respectively.

Salicylic Acid and Jasmonic Acid Pathways are Activated in Spatially Different Domains Around the Infection Site During Effector-Triggered Immunity in Arabidopsis thaliana.

The innate immune response is, in the first place, elicited at the site of infection. Thus, the host response can be different among the infected cells and the cells surrounding them. Effector-triggered immunity (ETI), a form of innate immunity in plants, is triggered by specific recognition between pathogen effectors and their corresponding plant cytosolic immune receptors, resulting in rapid localized cell death known as hypersensitive response (HR). HR cell death is usually limited to a few cells at the infection site, and is surrounded by a few layers of cells massively expressing defense genes such as Pathogenesis-Related Gene 1 (PR1). This virtually concentric pattern of the cellular responses in ETI is proposed to be regulated by a concentration gradient of salicylic acid (SA), a phytohormone accumulated around the infection site. Recent studies demonstrated that jasmonic acid (JA), another phytohormone known to be mutually antagonistic to SA in many cases, is also accumulated in and required for ETI, suggesting that ETI is a unique case. However, the molecular basis for this uniqueness remained largely to be solved. Here, we found that, using intravital time-lapse imaging, the JA signaling pathway is activated in the cells surrounding the central SA-active cells around the infection sites in Arabidopsis thaliana. This distinct spatial organization explains how these two phythormone pathways in a mutually antagonistic relationship can be activated simultaneously during ETI. Our results re-emphasize that the spatial consideration is a key strategy to gain mechanistic insights into the apparently complex signaling cross-talk in immunity.

Role of RNase Y in Clostridium perfringens mRNA Decay and Processing.

RNase Y is a major endoribonuclease that plays a crucial role in mRNA degradation and processing. We study the role of RNase Y in the Gram-positive anaerobic pathogen Clostridium perfringens, which until now has not been well understood. Our study implies an important role for RNase Y-mediated RNA degradation and processing in virulence gene expression and the physiological development of the organism. We began by constructing an RNase Y conditional knockdown strain in order to observe the importance of RNase Y on growth and virulence. Our resulting transcriptome analysis shows that RNase Y affects the expression of many genes, including toxin-producing genes. We provide data to show that RNase Y depletion repressed several toxin genes in C. perfringens and involved the virR-virS two-component system. We also observe evidence that RNase Y is indispensable for processing and stabilizing the transcripts of colA (encoding a major toxin collagenase) and pilA2 (encoding a major pilin component of the type IV pili). Posttranscriptional regulation of colA is known to be mediated by cleavage in the 5' untranslated region (5'UTR), and we observe that RNase Y depletion diminishes colA 5'UTR processing. We show that RNase Y is also involved in the posttranscriptional stabilization of pilA2 mRNA, which is thought to be important for host cell adherence and biofilm formation. IMPORTANCE: RNases have important roles in RNA degradation and turnover in all organisms. C. perfringens is a Gram-positive anaerobic spore-forming bacterial pathogen that produces numerous extracellular enzymes and toxins, and it is linked to digestive disorders and disease. A highly conserved endoribonuclease, RNase Y, affects the expression of hundreds of genes, including toxin genes, and studying these effects is useful for understanding C. perfringens specifically and RNases generally. Moreover, RNase Y is involved in processing specific transcripts, and we observed that this processing in C. perfringens results in the stabilization of mRNAs encoding a toxin and bacterial extracellular apparatus pili. Our study shows that RNase activity is associated with gene expression, helping to determine the growth, proliferation, and virulence of C. perfringens.

Immunoactive Clostridial Membrane Vesicle Production Is Regulated by a Sporulation Factor.

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.

Impact of the pneumococcal conjugate vaccine on serotype distribution of adult non-invasive Streptococcus pneumoniae isolates in Tokai region, Japan, 2008-2016.

The introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) in February 2010 markedly reduced the burden of invasive pneumococcal disease and changed serotype distribution in Japan. In November 2013, PCV7 was replaced by the 13-valent pneumococcal conjugate vaccine (PCV13). We investigated the serotype distribution and susceptibility trends of non-invasive Streptococcus pneumoniae isolates collected from adult patients. A total of 504 pneumococcal isolates were collected during 4 periods between 2008 and 2016 (Period 1; between June 2008 and April 2009, Period 2; between September 2010 and March 2011, Period 3; between October 2011 and March 2012, Period 4; between August 2015 and January 2016). The coverage of PCV7 and PCV13 significantly decreased from 38.6% and 60.5% in Period 1 to 6.6% and 31.1% in Period 4. This change was mainly due to a large decrease in the frequency of serotype 19F, 6B, and 14. Serotype 3 was the most frequently isolated, and gradually increased. Additionally, non-PCV13 serotypes 11A, 33F, and 35B significantly increased. Most of the PCV7 serotypes 19F, 23F, 6B, and 14 had mutations of penicillin-binding protein genes and macrolide resistance genes, and these serotypes showed low susceptibilities to cefdinir and clarithromycin. On the other hand, a significant change in susceptibility to various antimicrobial agents was not observed throughout the study period, except for decreased susceptibility to carbapenems. Continuous surveillance studies of pneumococcal serotype changes and drug susceptibility are necessary in future.

T-2307, a novel arylamidine, is transported into Candida albicans by a high-affinity spermine and spermidine carrier regulated by Agp2.

OBJECTIVES: T-2307, a novel arylamidine, exhibits potent broad-spectrum activities against pathogenic fungi, particularly Candida albicans. We previously reported that T-2307 uptake was mainly mediated by a saturable high-affinity carrier at the MIC for C. albicans. Since we hypothesized that the potent anticandidal activity arose from accumulation via the high-affinity carrier, we characterized the specificity and kinetic features of the carrier. METHODS: The MICs of T-2307 for C. albicans strains were evaluated in the presence and absence of potential competitive substrates. The cells were exposed to [(14)C]T-2307, [(14)C]spermine or [(14)C]spermidine in the presence of unlabelled T-2307, pentamidine, propamidine, or competitive substrates if necessary, and the radioactivity in the cells was measured. C. albicans gene deletion was performed using a one-step PCR-based technique. RESULTS: Coapplication with exogenous spermine or spermidine decreased the antifungal activity and uptake of T-2307 in C. albicans strains. T-2307 competitively inhibited spermine and spermidine uptake with inhibition constants similar to its Km for the high-affinity carrier. The comparison of MICs and kinetic values between T-2307 and other diamidine compounds suggested that the different antifungal properties could be partially attributable to the variations in their affinity with the carrier. Studies of gene deletion mutants revealed that T-2307 was transported into C. albicans by a high-affinity spermine and spermidine carrier regulated by Agp2. CONCLUSIONS: Uptake of T-2307 via the high-affinity spermine and spermidine carrier regulated by Agp2 could contribute to its potent antifungal activity. Further investigation is required to identify the high-affinity carrier for potential targeting with novel therapies.

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.

Inhibition of Pseudomonas aeruginosa swarming motility by 1-naphthol and other bicyclic compounds bearing hydroxyl groups.

Many bacteria convert bicyclic compounds, such as indole and naphthalene, to oxidized compounds, including hydroxyindoles and naphthols. Pseudomonas aeruginosa, a ubiquitous bacterium that inhabits diverse environments, shows pathogenicity against animals, plants, and other microorganisms, and increasing evidence has shown that several bicyclic compounds alter the virulence-related phenotypes of P. aeruginosa. Here, we revealed that hydroxyindoles (4- and 5-hydroxyindoles) and naphthalene derivatives bearing hydroxyl groups specifically inhibit swarming motility but have minor effects on other motilities, including swimming and twitching, in P. aeruginosa. Further analyses using 1-naphthol showed that this effect is also associated with clinically isolated hyperswarming P. aeruginosa cells. Swarming motility is associated with the dispersion of cells from biofilms, and the addition of 1-naphthol maintained biofilm biomass without cell dispersion. We showed that this 1-naphthol-dependent swarming inhibition is independent of changes of rhamnolipid production and the intracellular level of signaling molecule cyclic-di-GMP (c-di-GMP). Transcriptome analyses revealed that 1-naphthol increases gene expression associated with multidrug efflux and represses gene expression associated with aerotaxis and with pyochelin, flagellar, and pilus synthesis. In the present study, we showed that several bicyclic compounds bearing hydroxyl groups inhibit the swarming motility of P. aeruginosa, and these results provide new insight into the chemical structures that inhibit the specific phenotypes of P. aeruginosa.