The portal protein plays essential roles at different steps of the SPP1 DNA packaging process

AbstractA large number of viruses use a specialized portal for entry of DNA to the viral capsid and for its polarized exit at the beginning of infection. These families of viruses assemble an icosahedral procapsid containing a portal protein oligomer in one of its 12 vertices. The viral ATPase (terminase) interacts with the portal vertex to form a powerful molecular motor that translocates DNA to the procapsid interior against a steep concentration gradient. The portal protein is an essential component of this DNA packaging machine. Characterization of single amino acid substitutions in the portal protein gp6 of bacteriophage SPP1 that block DNA packaging identified sequential steps in the packaging mechanism that require its action. Gp6 is essential at early steps of DNA packaging and for DNA translocation to the capsid interior, it affects the efficiency of DNA packaging, it is a central component of the headful sensor that determines the size of the packaged DNA molecule, and is essential for closure of the portal pore by the head completion proteins to prevent exit of the DNA encapsidated. Functional regions of gp6 necessary at each step are identified within its primary structure. The similarity between the architecture of portal oligomers and between the DNA packaging strategies of viruses using portals strongly suggests that the portal protein plays the same roles in a large number of viruses

Overview of Clostridium difficile Infection: Life Cycle, Epidemiology, Antimicrobial Resistance and Treatment

The use of antimicrobial agents and acquired resistances explains in part the emergence and spreading of epidemic strains of Clostridium difficile. Continued use of antimicrobial therapy still represents an acute danger in triggering the emergence and spreading of new resistant and multiresistant strains including against first-line antibiotics. We examine the pathway of peptidoglycan synthesis in this organism and associated resistances, as well as resistance to other classes of antibiotics. The life cycle of C. difficile involves growth, spore formation and germination. Spores endow the organism with a formidable capacity of persistence in the environment and in the host, resistance, dissemination and infectious potential. Highly resistant spores produced by antibiotic-resistant/multiresistant strains may be one of the most serious challenges we face in what concerns the containment of C. difficile. Finally, we review recent developments in the treatment and prevention of C. difficile infection

Cellular and population strategies underpinning neurotoxin production and sporulation in Clostridium botulinum type E cultures

Funding Information: The work was funded by the European Research Council (ERC-CoG 683099), Academy of Finland (299700), Marie Skłodowska Curie Innovative Training Network (CLOSPORE 642068), the Doctoral Programmes in Microbiology and Biotechnology and in Food Chain and Health of the University of Helsinki, Fundação para a Ciência e a Tecnologia, Portugal (PEst-OE/EQB/LA0004/2011), FEDER funds through COMPETE2020 “Programa Operacional Competitividade e Internacionalização” (LISBOA-01-0145-FEDER-007660), and the national funds from OE-"Orçamento de Estado" and European funds from FEDER "Fundo Europeu de Desenvolvimento Regional" (PPBI-POCI-01-0145-FEDER-022122). Funding Information: We warmly thank Nigel Minton, University of Nottingham, for the generous provision of vectors and bacterial strains required in mutagenesis. Mikael Niku is thanked for technical advice with fluorescence microscopy, and Hanna Korpunen is thanked for technical assistance. The work was funded by the European Research Council (ERC-CoG 683099), Academy of Finland (299700), Marie Skłodowska Curie Innovative Training Network (CLOSPORE 642068), the Doctoral Programmes in Microbiology and Biotechnology and in Food Chain and Health of the University of Helsinki, Fundação para a Ciência e a Tecnologia, Portugal (PEst-OE/EQB/LA0004/2011), FEDER funds through COMPETE2020 “Programa Operacional Competitividade e Internacionalização” (LISBOA-01-0145-FEDER-007660), and the national funds from OE-"Orçamento de Estado" and European funds from FEDER "Fundo Europeu de Desenvolvimento Regional" (PPBI-POCI-01-0145-FEDER-022122). A.M., G.M., A.O.H., H.K., and M.L. conceived and designed the study; A.M., G.M., and M.N. performed the laboratory experiments; A.M., G.M., and A.O.H. performed the image analysis; A.M. and M.L. analyzed the data and wrote the manuscript; and all authors contributed to the final manuscript. Funder EC | European Research Council (ERC) Academy of Finland (AKA) Marie Sk&łodowska-Curie Innovative Training Network CLOSPORE University of Helsinki, Doctoral Programme in Food Chain and Health University of Helsinki, Doctoral Programme in Microbiology and Biotechnology Fundação para a Ciência e a Tecnologia, Portugal Orcamento de Estado/Fundo Europeu de Desenvolvimento Regional Fundo Europeu de Desenvolvimento Regional Grant(s) 683099 299700 642068 PEst-OE/EQB/ LA0004/2011 PPBI-POCI-01-0145-FEDER-022122 PPBI-POCI-01-0145-FEDER-022122 Author(s) Miia Lindstrom Miia Lindstrom Miia Lindstrom Anna Mertaoja Maria B. Nowakowska Anna Mertaoja Adriano O. Henriques Adriano O. Henriques Adriano O. Henriques Publisher Copyright: Copyright © 2023 Mertaoja et al.Toxin production and sporulation are key determinants of pathogenesis in Clostridia. Clostridium botulinum produces the most potent toxin known, the botulinum neurotoxin (BoNT), which blocks neurotransmission and causes a life-threatening paralysis called botulism. BoNT production and sporulation share a common regulator Spo0A, which suggests coordination of the two traits. Describing the relationship between toxin production and sporulation is fundamental toward understanding the evolutionary and mechanistic logic and further control of clostridial pathogenesis. Here, we provide the first single-cell resolution analysis of BoNT production and sporulation in C. botulinum type E cultures by using a fluorescent reporter to follow the activation of the BoNT gene promoter. BoNT was expressed by a subpopulation of cells and was released through Spo0A-mediated autolysis of vegetative cells or upon release of mature spores. All possible combinations of toxin production and sporulation resided in wild-type C. botulinum type E cultures, indicating neither tight co-regulation nor strict independence of the two traits. The population structure and the degree of overall heterogeneity were affected by growth phase and environmental conditions, with cold temperature inducing large diversity and cultural stability, in line with adaptation to fluctuating temperatures that C. botulinum type E strains likely encounter in nature. We also observed Spo0A-independent BoNT production by a small cell subpopulation of the spo0A-null strain. Our observation of toxin gene activation in the forespore invites speculation on possible alternative biological roles for toxin production by vegetative and sporulating cells and reflection on the evolutionary rationale of toxin production with respect to the ecology of spore-forming pathogens.publishersversionpublishe

Characterization of Clostridium difficile 027 strains from an outbreak in a Portuguese hospital

C. difficile infection (CDI) is the cause of an intestinal disease mediated by two potent cytotoxins, TcdA and TcdB. Symptoms of CDI can range from asymptomatic colonization or mild diarrhea, to life-threatening inflammatory lesions such as pseudomembraneous colitis, toxic megacolon or bowel perforation. In part because of the recent emergence of so-called hypervirulent strains, especially (but not exclusively) those belonging to ribotype 027, C. difficile is now considered a main nosocomial enteric pathogen. Hypervirulent epidemic strains have been associated with more severe disease conditions, with higher relapse rates and increased mortality. Health care-associated CDI develops in hospitalized patients undergoing antibiotic treatment because C. difficile can colonize the gut if the normal intestinal microbiota is disturbed. However, C. difficile is also emerging as an important pathogen in the community, as well as in animal husbandry. The organism is an obligate anaerobe, and has the ability to form spores. Spores are extremely resilient and can accumulate and remain viable in the environment or in the host for long periods of time. Spores that remain latent in the gut are responsible for the recurrence of C. difficile-associated disease (CDAD) when antibiotic therapy is stopped. At least some of the hypervirulent epidemic strains show a greater sporulation capacity in vitro, as well as robust toxin production. The first detection of C. difficile 027 hypervirulent epidemic strains implicated in a hospital outbreak in Portugal dates from January 2012, involving 12 patients, with a crude mortality rate of 50%. Here we report on the genetic characterization of those strains as well as the antibiotic resistance profile, toxin production, and rate and efficiency of spore formation. In parallel, C. difficile 027 non-outbreak strains isolated from other Portuguese health care facilities are also investigated

Rethinking the Niche of Upper-Atmosphere Bacteria: Draft Genome Sequences of Bacillus aryabhattai C765 and Bacillus aerophilus C772, Isolated from Rice Fields

Here, we report two genome sequences of endospore-forming bacteria isolated from the rice fields of Comporta, Portugal, identified as Bacillus aryabhattai C765 and Bacillus aerophilus C772. Both species were previously identified in air samples from the upper atmosphere, but our findings suggest their presence in a wider range of environmental niches.FCT grant: (PEST-E/EQB/LA0004/2011), FCT contracts: (IF/00268/2013/CP1173/CT00061, SFRH/BPD/89907/2012)

Genetic Competence Drives Genome Diversity in Bacillus subtilis

This deposit is composed by the main article plus the supplementary materials of the publication.Prokaryote genomes are the result of a dynamic flux of genes, with increases achieved via horizontal gene transfer and reductions occurring through gene loss. The ecological and selective forces that drive this genomic flexibility vary across species. Bacillus subtilis is a naturally competent bacterium that occupies various environments, including plant-associated, soil, and marine niches, and the gut of both invertebrates and vertebrates. Here, we quantify the genomic diversity of B. subtilis and infer the genome dynamics that explain the high genetic and phenotypic diversity observed. Phylogenomic and comparative genomic analyses of 42 B. subtilis genomes uncover a remarkable genome diversity that translates into a core genome of 1,659 genes and an asymptotic pangenome growth rate of 57 new genes per new genome added. This diversity is due to a large proportion of low-frequency genes that are acquired from closely related species. We find no gene-loss bias among wild isolates, which explains why the cloud genome, 43% of the species pangenome, represents only a small proportion of each genome. We show that B. subtilis can acquire xenologous copies of core genes that propagate laterally among strains within a niche. While not excluding the contributions of other mechanisms, our results strongly suggest a process of gene acquisition that is largely driven by competence, where the long-term maintenance of acquired genes depends on local and global fitness effects. This competence-driven genomic diversity provides B. subtilis with its generalist character, enabling it to occupy a wide range of ecological niches and cycle through them.Fundação para a Ciência e a Tecnologia grants: (PTDC/EBB-BIO/119006/2010, PEst-OE/EQB/LA0004/2011, SFRH/BPD/89907/2012, SFRH/BD/29397/06); FEDER grant: (LISBOA-01-0145-FEDER-007660).info:eu-repo/semantics/publishedVersio

Novel Secretion Apparatus Maintains Spore Integrity and Developmental Gene Expression in Bacillus subtilis

Sporulation in Bacillus subtilis involves two cells that follow separate but coordinately regulated developmental programs. Late in sporulation, the developing spore (the forespore) resides within a mother cell. The regulation of the forespore transcription factor σG that acts at this stage has remained enigmatic. σG activity requires eight mother-cell proteins encoded in the spoIIIA operon and the forespore protein SpoIIQ. Several of the SpoIIIA proteins share similarity with components of specialized secretion systems. One of them resembles a secretion ATPase and we demonstrate that the ATPase motifs are required for σG activity. We further show that the SpoIIIA proteins and SpoIIQ reside in a multimeric complex that spans the two membranes surrounding the forespore. Finally, we have discovered that these proteins are all required to maintain forespore integrity. In their absence, the forespore develops large invaginations and collapses. Importantly, maintenance of forespore integrity does not require σG. These results support a model in which the SpoIIIA-SpoIIQ proteins form a novel secretion apparatus that allows the mother cell to nurture the forespore, thereby maintaining forespore physiology and σG activity during spore maturation

A new fluorescence-based approach for direct visualization of coat formation during sporulation in Bacillus cereus

Funding Information: The Ph.D. thesis of A.L. was funded by INRAE and the PACA Region and was partly supported by a grant of the MICA division and a Perdiguier grant of Avignon University. Part of this work was supported by the microscopy facilities of the Platform 3A, funded by the European Regional Development Fund, the French Ministry of Research, Higher Education and Innovation, the Provence-Alpes-Côte d’Azur region, the Departmental Council of Vaucluse and the Urban Community of Avignon. This work was also funded through grants PEst-OE/EQB/LA0004/2011 to AOH, by project LISBOA-01-0145-FEDER-007660 (“Microbiologia Molecular, Estrutural e Celular”) funded by FEDER funds through COMPETE2020 – “Programa Operacional Competitividade e Internacionalização”, and by project PPBI—Portuguese Platform of BioImaging (PPBI-POCI-01-0145-FEDER-022122) co-funded by national funds from OE—"Orçamento de Estado" and by European funds from FEDER—"Fundo Europeu de Desenvolvimento Regional". Work and Lattice SIM imaging in the R.C-L. lab was supported by funding from the European Research Council (ERC) under the Horizon 2020 research and innovation program (grant agreement No 772178, ERC Consolidator grant to R.C.-L.). Publisher Copyright: © 2023, Springer Nature Limited.The human pathogenic bacteria Bacillus cereus, Bacillus anthracis and the entomopathogenic Bacillus thuringiensis form spores encased in a protein coat surrounded by a balloon-like exosporium. These structures mediate spore interactions with its environment, including the host immune system, control the transit of molecules that trigger germination and thus are essential for the spore life cycle. Formation of the coat and exosporium has been traditionally visualized by transmission electronic microscopy on fixed cells. Recently, we showed that assembly of the exosporium can be directly observed in live B. cereus cells by super resolution-structured illumination microscopy (SR-SIM) using the membrane MitoTrackerGreen (MTG) dye. Here, we demonstrate that the different steps of coat formation can also be visualized by SR-SIM using MTG and SNAP-cell TMR-star dyes during B. cereus sporulation. We used these markers to characterize a subpopulation of engulfment-defective B. cereus cells that develops at a suboptimal sporulation temperature. Importantly, we predicted and confirmed that synthesis and accumulation of coat material, as well as synthesis of the σK-dependent protein BxpB, occur in cells arrested during engulfment. These results suggest that, unlike the well-studied model organism Bacillus subtilis, the activity of σK is not strictly linked to the state of forespore development in B. cereus.publishersversionpublishe

Clostridioides difficile para-Cresol Production Is Induced by the Precursor para-Hydroxyphenylacetate.

Clostridioides difficile is an etiological agent for antibiotic-associated diarrheal disease. C. difficile produces a phenolic compound, para-cresol, which selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. C. difficile decarboxylates para-hydroxyphenylacetate (p-HPA) to produce p-cresol by the action of the HpdBCA decarboxylase encoded by the hpdBCA operon. Here, we investigate regulation of the hpdBCA operon and directly compare three independent reporter systems; SNAP-tag, glucuronidase gusA, and alkaline phosphatase phoZ reporters to detect basal and inducible expression. We show that expression of hpdBCA is upregulated in response to elevated p-HPA. In silico analysis identified three putative promoters upstream of hpdBCA operon-P1, P2, and P?54; only the P1 promoter was responsible for both basal and p-HPA-inducible expression of hpdBCA We demonstrated that turnover of tyrosine, a precursor for p-HPA, is insufficient to induce expression of the hpdBCA operon above basal levels because it is inefficiently converted to p-HPA in minimal media. We show that induction of the hpdBCA operon in response to p-HPA occurs in a dose-dependent manner. We also identified an inverted palindromic repeat (AAAAAG-N13-CTTTTT) upstream of the hpdBCA start codon (ATG) that is essential for inducing transcription of the hpdBCA operon in response to p-HPA, which drives the production of p-cresol. This provides insights into the regulatory control of p-cresol production, which affords a competitive advantage for C. difficile over other intestinal bacteria, promoting dysbiosis.IMPORTANCE Clostridioides difficile infection results from antibiotic-associated dysbiosis. para-Cresol, a phenolic compound produced by C. difficile, selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. Here, we demonstrate that expression of the hpdBCA operon, encoding the HpdBCA decarboxylase which converts p-HPA to p-cresol, is upregulated in response to elevated exogenous p-HPA, with induction occurring between >0.1 and ?0.25?mg/ml. We determined a single promoter and an inverted palindromic repeat responsible for basal and p-HPA-inducible hpdBCA expression. We identified turnover of tyrosine, a p-HPA precursor, does not induce hpdBCA expression above basal level, indicating that exogenous p-HPA was required for p-cresol production. Identifying regulatory controls of p-cresol production will provide novel therapeutic targets to prevent p-cresol production, reducing C. difficile's competitive advantage

Resistance of Clostridium difficile from ribotype 017 to imipenem: contribution of the whole genome sequencing

A infeção por Clostridium difficile é a principal causa de diarreia infeciosa associada aos cuidados de saúde. Neste estudo, caracterizámos um conjunto de estirpes clínicas de Clostridium difficile, provenientes de diversos hospitais portugueses, com o objetivo de estudar a resistência aos carbapenemos neste agente patogénico. Um total de 191 estirpes clínicas, isoladas entre 2012 e 2015 de 15 hospitais em Portugal, foram incluídas no estudo; a suscetibilidade ao imipenemo foi determinada por um método de gradiente de difusão em agar. Foram selecionadas estirpes sensíveis e resistentes ao imipenemo, para estudos fenotípicos adicionais e para contributo da sequenciação do genoma completo. A resistência ao imipenemo foi detetada em 24 (12,6%) das estirpes, 22 das quais pertencentes ao ribotipo (RT) 017 (apenas toxina B positivo), todas provenientes do mesmo hospital, durante o período em estudo, e com perfil de multiresistência. Pela análise dos dados de sequenciação dos genomas, foram identificadas duas substituições de aminoácidos (Ala555Thr e Tyr721Ser) nos domínios funcionais de duas enzimas envolvidas na síntese do peptidoglicano (penicillin-binding proteins - PBP). Uma PBP adicional foi também identificada nas estirpes RT017. Este estudo descreve pela primeira vez alterações em PBPs como base genética provável da resistência ao imipenemo em C. difficile.Clostridium dif ficile is a major cause of healthcare-associated infections. Here, we characterized C. dif ficile strains isolated in Por tuguese hospitals, in order to search for impenem resistance and the underlying genetic determinants. Imipenem susceptibility testing by agar gradient dif fusion was per formed on 191 C. dif ficile strains, isolated from 15 portuguese hospitals, between 2012-2015. Some of the imipenem-resistant and imipenem-susceptible strains were selected for downstream phenotypic analyses and for whole genome sequencing (WGS). Resistance to imipenem was detected in 24 (12.6 %) strains, 22 of which were ribotype (RT) 017 strains, only positive for toxin B, isolated in the same hospital, and presenting resistance to several other antibiotics. Through analysis of WGS data, two amino acid changes (Ala555Thr and Tyr721Ser) targeting the transpeptidase domain of two penicillin-binding proteins (PBP) were identified. An additional PBP was also identified in this ribotype. We describe, for the first time, mutations in PBP-encoding genes as the probable genetic basis for C. dif ficile imipenem resistance.Este trabalho foi financiado pelo INSA (projeto 2016DDI1284) e pela Fundação para a Ciência e Tecnologia (bolsa de investigação no âmbito do projeto Pest-C/EQB/LA0006/2011; programa IF IF/00268/2013/CP1173/CT0006, a MS; bolsa de doutoramento PD/BD/105738/2014, a ALM).info:eu-repo/semantics/publishedVersio