Characterization of vB_SalS_PSa2, a T5-like Demerecviridae bacteriophage, and its potential use in food matrices.

This study characterizes a newly isolated Demerecviridae phage, named vB_SalS_PSa2, belonging to the phage T5 group. The main variations between vB_SalS_PSa2 and T5 concern structural proteins related to morphology and host recognition. vB_SalS_PSa2 is infective to 19 out of the 25 tested Salmonella enterica (including the rare "Sendai" and "Equine" serotypes) and Escherichia coli isolates, most of them being multidrug resistant. vB_SalS_PSa2 displayed good thermal stability (4-60 °C) and broad pH stability (4.0-12.0). It also exhibited antibacterial activity against S. enterica sv. Paratyphi A Enb50 at 4 °C in milk during the whole tested period (5 d), and for 3-6 h at both 25 and 37 °C. Furthermore, vB_SalS_PSa2 was able to inhibit biofilm formation and to show degradation activity on mature biofilms of E. coli K12 and S. enterica sv. Paratyphi Enb50 in both LB and milk. Altogether, these results indicate that phage vB_SalS_PSa2 is a valuable candidate for controlling foodborne S. enterica and E. coli pathogens.

Bacillus cereus sensu lato antimicrobial arsenal: An overview.

The Bacillus cereus group contains genetically closed bacteria displaying a variety of phenotypic features and lifestyles. The group is mainly known through the properties of three major species: the entomopathogen Bacillus thuringiensis, the animal and human pathogen Bacillus anthracis and the foodborne opportunistic strains of B. cereus sensu stricto. Yet, the actual diversity of the group is far broader and includes multiple lifestyles. Another less-appreciated aspect of B. cereus members lies within their antimicrobial potential which deserves consideration in the context of growing emergence of resistance to antibiotics and pesticides, and makes it crucial to find new sources of antimicrobial molecules. This review presents the state of knowledge on the known antimicrobial compounds of the B. cereus group members, which are grouped according to their chemical features and biosynthetic pathways. The objective is to provide a comprehensive review of the antimicrobial range exhibited by this group of bacteria, underscoring the interest in its potent biocontrol arsenal and encouraging further research in this regard.

Design of a Porous Silicon Biosensor: Characterization, Modeling, and Application to the Indirect Detection of Bacteria.

The design of a porous silicon (PSi) biosensor is not often documented, but is of the upmost importance to optimize its performance. In this work, the motivation behind the design choices of a PSi-based optical biosensor for the indirect detection of bacteria via their lysis is detailed. The transducer, based on a PSi membrane, was characterized and models were built to simulate the analyte diffusion, depending on the porous nanostructures, and to optimize the optical properties. Once all performances and properties were analyzed and optimized, a theoretical response was calculated. The theoretical limit of detection was computed as 104 CFU/mL, based on the noise levels of the optical setup. The experimental response was measured using 106 CFU/mL of Bacillus cereus as model strain, lysed by bacteriophage-coded endolysins PlyB221. The obtained signal matched the expected response, demonstrating the validity of our design and models.

Comparative analyses of the bacterial communities present in the spontaneously fermented milk products of Northeast India and West Africa.

Introduction: Spontaneous fermentation of raw cow milk without backslopping is in practice worldwide as part of the traditional food culture, including "Doi" preparation in earthen pots in Northeast India, "Kindouri" of Niger and "Fanire" of Benin prepared in calabash vessels in West Africa. Very few reports are available about the differences in bacterial communities that evolved during the spontaneous mesophilic fermentation of cow milk in diverse geographical regions. Methods: In this study, we used high throughput amplicon sequencing of bacterial 16S rRNA gene to investigate 44 samples of naturally fermented homemade milk products and compared the bacterial community structure of these foods, which are widely consumed in Northeast India and Western Africa. Results and discussion: The spontaneous milk fermentation shared the lactic acid bacteria, mainly belonging to Lactobacillaceae (Lactobacillus) and Streptococcaceae (Lactococcus) in these two geographically isolated regions. Indian samples showed a high bacterial diversity with the predominance of Acetobacteraceae (Gluconobacter and Acetobacter) and Leuconostoc, whereas Staphylococcaceae (Macrococcus) was abundant in the West African samples. However, the Wagashi cheese of Benin, prepared by curdling the milk with proteolytic leaf extract of Calotrophis procera followed by natural fermentation, contained Streptococcaceae (Streptococcus spp.) as the dominant bacteria. Our analysis also detected several potential pathogens, like Streptococcus infantarius an emerging infectious foodborne pathogen in Wagashi samples, an uncultured bacterium of Enterobacteriaceae in Kindouri and Fanire samples, and Clostridium spp. in the Doi samples of Northeast India. These findings will allow us to develop strategies to address the safety issues related to spontaneous milk fermentation and implement technological interventions for controlled milk fermentation by designing starter culture consortiums for the sustainable production of uniform quality products with desirable functional and organoleptic properties.

Indirect Detection of Bacteria on Optically Enhanced Porous Silicon Membrane-Based Biosensors Using Selective Lytic Enzymes.

In this work, we developed a biosensor for the indirect detection of bacteria via their lysate. The developed sensor is based on porous silicon membranes, which are known for their many attractive optical and physical properties. Unlike traditional porous silicon biosensors, the selectivity of the bioassay presented in this work does not rely on bio-probes attached to the sensor surface; the selectivity is added to the analyte itself, by the addition of lytic enzymes that target only the desired bacteria. The resulting bacterial lysate is then able to penetrate into the porous silicon membrane and affects its optical properties, while intact bacteria accumulate on top of the sensor. The porous silicon sensors, fabricated using standard microfabrication techniques, are coated with TiO2 layers using atomic layer deposition. These layers serve as passivation but also enhance the optical properties. The performance of the TiO2-coated biosensor is tested for the detection of Bacillus cereus, using the bacteriophage-encoded PlyB221 endolysin as the lytic agent. The sensitivity of the biosensor is much improved compared to previous works, reaching 103 CFU/mL, with a total assay time of 1 h 30 min. The selectivity and versatility of the detection platform are also demonstrated, as is the detection of B. cereus in a complex analyte.

On the use of antibiotics to control plant pathogenic bacteria: a genetic and genomic perspective.

Despite growing attention, antibiotics (such as streptomycin, oxytetracycline or kasugamycin) are still used worldwide for the control of major bacterial plant diseases. This raises concerns on their potential, yet unknown impact on antibiotic and multidrug resistances and the spread of their genetic determinants among bacterial pathogens. Antibiotic resistance genes (ARGs) have been identified in plant pathogenic bacteria (PPB), with streptomycin resistance genes being the most commonly reported. Therefore, the contribution of mobile genetic elements (MGEs) to their spread among PPB, as well as their ability to transfer to other bacteria, need to be further explored. The only well-documented example of ARGs vector in PPB, Tn5393 and its highly similar variants (carrying streptomycin resistance genes), is concerning because of its presence outside PPB, in Salmonella enterica and Klebsiella pneumoniae, two major human pathogens. Although its structure among PPB is still relatively simple, in human- and animal-associated bacteria, Tn5393 has evolved into complex associations with other MGEs and ARGs. This review sheds light on ARGs and MGEs associated with PPB, but also investigates the potential role of antibiotic use in resistance selection in plant-associated bacteria.

Interspecies Horizontal Transfer and Specific Integration of the Mosquitocidal Toxin-Encoding Plasmid pTAND672-2 from Bacillus thuringiensis subsp. israelensis to Lysinibacillus sphaericus.

pTAND672-2, a 144-kb resident plasmid of Bacillus thuringiensis serovar israelensis strain TAND672, was sequenced and characterized. This extrachromosomal element carries mosquitocidal toxin-, conjugation-, and recombinase-encoding genes, together with a putative arbitrium system, a genetic module recently discovered in temperate phages controlling lysogeny-lysis transition and in mobile genetic elements (MGEs) where its function remains clarified. Using conjugation experiments, pTAND672-2 is shown to be a novel integrative and conjugative element (ICE), which can horizontally transfer from B. thuringiensis serovar israelensis to Lysinibacillus sphaericus, another mosquitocidal bacterium, where it integrates into the chromosome. Its integration and circularization are reversible and involve a single-cross recombination between 33-bp specific sites, attB in the chromosome of L. sphaericus and attP in pTAND672-2. CDS143, coding for the putative tyrosine integrase Int143 distantly related to site-specific tyrosine Xer recombinases and phage integrases, can mediate the integration of pTAND672-2 to attB. The B. thuringiensis mosquito-killing genes carried by pTAND672-2 are efficiently transcribed and expressed in L. sphaericus, displaying a slight increased toxicity in this bacterium against Aedes albopictus larvae. The occurrence of pTAND672-2-like plasmids within the Bacillus cereus group was also explored and indicated that they all share a similar genetic backbone with diverse plasmid sizes, ranging from 58 to 225 kb. Interestingly, among them, the pEFR-4-4 plasmid of Bacillus paranthracis EFR-4 and p5 of B. thuringiensis BT-59 also display conjugative capability; moreover, like pTAND672-2 displays a chimeric structure between the pCH_133-e- and pBtoxis-like plasmids, pBTHD789-3 also appears to be mosaic of two plasmids. IMPORTANCE Horizontal transfer of mobile genetic elements carrying mosquitocidal toxin genes may play a driving role in the diversity of mosquitocidal bacteria. Here, the 144-kb mosquitocidal toxin-encoding plasmid pTAND672-2 is the first verified integrative and conjugative element (ICE) identified in Bacillus thuringiensis serovar israelensis. The key tyrosine integrase Int143, involved in the specific integration, is distantly related to other tyrosine recombinases. The study also reports the occurrence and potential interspecies transmission of pTAND672-2-like plasmids with varied sizes in B. thuringiensis, Bacillus paranthracis, and Bacillus wiedmannii isolates belonging to the Bacillus cereus group. This study is important for further understanding the evolution and ecology of mosquitocidal bacteria, as well as for providing new direction for the genetic engineering of biopesticides in the control of disease-transmitting mosquitoes.

Phage Adsorption to Gram-Positive Bacteria.

The phage life cycle is a multi-stage process initiated by the recognition and attachment of the virus to its bacterial host. This adsorption step depends on the specific interaction between bacterial structures acting as receptors and viral proteins called Receptor Binding Proteins (RBP). The adsorption process is essential as it is the first determinant of phage host range and a sine qua non condition for the subsequent conduct of the life cycle. In phages belonging to the Caudoviricetes class, the capsid is attached to a tail, which is the central player in the adsorption as it comprises the RBP and accessory proteins facilitating phage binding and cell wall penetration prior to genome injection. The nature of the viral proteins involved in host adhesion not only depends on the phage morphology (i.e., myovirus, siphovirus, or podovirus) but also the targeted host. Here, we give an overview of the adsorption process and compile the available information on the type of receptors that can be recognized and the viral proteins taking part in the process, with the primary focus on phages infecting Gram-positive bacteria.

Conjugation across Bacillus cereus and kin: A review.

Horizontal gene transfer (HGT) is a major driving force in shaping bacterial communities. Key elements responsible for HGT are conjugation-like events and transmissible plasmids. Conjugative plasmids can promote their own transfer as well as that of co-resident plasmids. Bacillus cereus and relatives harbor a plethora of plasmids, including conjugative plasmids, which are at the heart of the group species differentiation and specification. Since the first report of a conjugation-like event between strains of B. cereus sensu lato (s.l.) 40 years ago, many have studied the potential of plasmid transfer across the group, especially for plasmids encoding major toxins. Over the years, more than 20 plasmids from B. cereus isolates have been reported as conjugative. However, with the increasing number of genomic data available, in silico analyses indicate that more plasmids from B. cereus s.l. genomes present self-transfer potential. B. cereus s.l. bacteria occupy diverse environmental niches, which were mimicked in laboratory conditions to study conjugation-related mechanisms. Laboratory mating conditions remain nonetheless simplistic compared to the complex interactions occurring in natural environments. Given the health, economic and ecological importance of strains of B. cereus s.l., it is of prime importance to consider the impact of conjugation within this bacterial group.

Conjugation-mediated transfer of pXO16, a large plasmid from Bacillus thuringiensis sv. israelensis, across the Bacillus cereus group and its impact on host phenotype.

pXO16, the 350 kb-conjugative plasmid from Bacillus thuringiensis sv. israelensis promotes its own transfer at high efficiency, triggers the transfer of mobilizable and non-mobilizable plasmids, as well as the transfer of host chromosomal loci. Naturally found in B. thuringiensis sv. israelensis, pXO16 transfers to various strains of Bacillus cereus sensu lato (s.l.) at a wide range of frequencies. Despite this host diversity, a paradox remains between the relatively large host spectrum and the natural occurrence of pXO16, so far restricted to B. thuringiensis sv. israelensis. Proposing first insights exploring this paradox, we investigated the behaviour of pXO16 amongst different members of the B. cereus group. We first looked at the transfer of pXO16 to two new host clusters of B. cereus s.l., Bacillus mycoides and Bacillus anthracis clusters. This examination brought to light the impairment of the characteristic rhizoidal phenotype of B. mycoides in presence of pXO16. We also explored the stability of pXO16 at different temperatures as some B. cereus group members are well-known for their psychro- or thermo-tolerance. This shed light on the thermo-sensitivity of the plasmid. The influence of pXO16 on its host cell growth and on swimming capacity also revealed no or limited impact on its natural host B. thuringiensis sv. israelensis. On the contrary, pXO16 affected more strongly both the growth and swimming capacity of other B. cereus s.l. hosts. This reinforced the running hypothesis of a co-evolution between pXO16 and B. thuringiensis sv. israelensis, enabling the plasmid maintenance without impairing the host strain development.

Getting Outside the Cell: Versatile Holin Strategies Used by Distinct Phages to Leave Their Bacillus thuringiensis Host.

Holins are small transmembrane proteins involved in the final stage of the lytic cycle of double-stranded DNA (dsDNA) phages. They cooperate with endolysins to achieve bacterial lysis, thereby releasing the phage progeny into the extracellular environment. Besides their role as membrane permeabilizers, allowing endolysin transfer and/or activation, holins also regulate the lysis timing. In this work, we provide functional characterization of the holins encoded by three phages targeting the Bacillus cereus group. The siphovirus Deep-Purple has a lysis cassette in which holP30 and holP33 encode two proteins displaying holin properties, including a transmembrane domain. The holin genes were expressed in Escherichia coli and induced bacterial lysis, with HolP30 being more toxic than HolP33. In Bacillus thuringiensis, the simultaneous expression of both holins was necessary to observe lysis, suggesting that they may interact to form functional pores. The myoviruses Deep-Blue and Vp4 both encode a single candidate holin (HolB and HolV, respectively) with two transmembrane domains, whose genes are not located near the endolysin genes. Their function as holin proteins was confirmed as their expression in E. coli impaired cell growth and viability. The HolV expression in B. thuringiensis also led to bacterial lysis, which was enhanced by coexpressing the holin with its cognate endolysin. Despite similar organizations and predicted topologies, truncated mutants of the HolB and HolV proteins showed different toxicity levels, suggesting that differences in amino acid composition influence their lysis properties. IMPORTANCE The phage life cycle ends with the host cell lysis, thereby releasing new virions into the environment for the next round of bacterial infection. Nowadays, there is renewed interest in phages as biocontrol agents, primarily due to their ability to cause bacterial death through lysis. While endolysins, which mediate peptidoglycan degradation, have been fairly well described, the pore-forming proteins, referred to as holins, have been extensively characterized in only a few model phages, mainly infecting Gram-negative bacteria. In this work, we characterized the holins encoded by a siphovirus and two myoviruses targeting members of the Gram-positive Bacillus cereus group, which comprises closely related species, including the well-known Bacillus anthracis, B. cereus sensu stricto, and Bacillus thuringiensis. Overall, this paper provides the first experimental characterization of holins encoded by B. cereus phages and reveals versatile lysis mechanisms used by these phages.

Viral Proteins Involved in the Adsorption Process of Deep-Purple, a Siphovirus Infecting Members of the Bacillus cereus Group.

The infection of a bacterium by a tailed phage starts from the adsorption process, which consists of a specific and strong interaction between viral proteins called receptor binding proteins (RBPs) and receptors located on the bacterial surface. In addition to RBPs, other tail proteins, such as evolved distal tail (evoDit) proteins and tail lysins, harboring carbohydrate binding modules (CBMs) have been shown to facilitate the phage adsorption by interacting with host polysaccharides. In this work, the proteins involved in the adsorption of Deep-Purple, a siphovirus targeting bacteria of the Bacillus cereus group, were studied. Bioinformatic analysis of Deep-Purple tail protein region revealed that it contains two proteins presenting CBM domains: Gp28, an evoDit protein, and Gp29, the potential RBP. The implication of both proteins in the adsorption of Deep-Purple particles was confirmed through cell wall decoration assays. Interestingly, whereas RBP-Gp29 exhibited the same host spectrum as Deep-Purple, evoDit-Gp28 was able to bind to many B. cereus group strains, including some that are not sensitive to the phage infection. Using immunogold microscopy, both proteins were shown to be located in the phage baseplate. Additionally, an in silico analysis of the tail regions encoded by several Siphoviridae infecting the B. cereus group was performed. It revealed that although the tail organization displayed by Deep-Purple is the most prevalent, different tail arrangements are observed, suggesting that distinct baseplate organization and adsorption mechanisms are encountered in siphoviruses targeting the B. cereus group. IMPORTANCE The B. cereus group is a complex cluster of closely related species, among which certain strains can be pathogenic (i.e., Bacillus anthracis, Bacillus cereus sensu stricto, and Bacillus cytotoxicus). Nowadays, phages are receiving increasing attention for applications in controlling and detecting such pathogens. Thus, understanding the molecular mechanisms governing the phage adsorption to its bacterial host is paramount as this step is a key determinant of the phage host spectrum. Until now, the knowledge regarding the adsorption process of tailed phage targeting the B. cereus groups was mainly restricted to the phage gamma infecting B. anthracis. With this work, we provide novel insights into the adsorption of Deep-Purple, a siphovirus infecting the B. cereus group. We showed that this phage recognizes polysaccharides and relies on two different viral proteins for its successful adsorption.

The CovRS Environmental Sensor Directly Controls the ComRS Signaling System To Orchestrate Competence Bimodality in Salivarius Streptococci.

In bacteria, phenotypic heterogeneity in an isogenic population compensates for the lack of genetic diversity and allows concomitant multiple survival strategies when choosing only one is too risky. This powerful tactic is exploited for competence development in streptococci where only a subset of the community triggers the pheromone signaling system ComR-ComS, resulting in a bimodal activation. However, the regulatory cascade and the underlying mechanisms of this puzzling behavior remained partially understood. Here, we show that CovRS, a well-described virulence regulatory system in pathogenic streptococci, directly controls the ComRS system to generate bimodality in the gut commensal Streptococcus salivarius and the closely related species Streptococcus thermophilus. Using single-cell analysis of fluorescent reporter strains together with regulatory mutants, we revealed that the intracellular concentration of ComR determines the proportion of competent cells in the population. We also showed that this bimodal activation requires a functional positive-feedback loop acting on ComS production, as well as its exportation and reinternalization via dedicated permeases. As the intracellular ComR concentration is critical in this process, we hypothesized that an environmental sensor could control its abundance. We systematically inactivated all two-component systems and identified CovRS as a direct repression system of comR expression. Notably, we showed that the system transduces its negative regulation through CovR binding to multiple sites in the comR promoter region. Since CovRS integrates environmental stimuli, we suggest that it is the missing piece of the puzzle that connects environmental conditions to (bimodal) competence activation in salivarius streptococci. IMPORTANCE Combining production of antibacterial compounds and uptake of DNA material released by dead cells, competence is one of the most efficient survival strategies in streptococci. Yet, this powerful tactic is energy consuming and reprograms the metabolism to such an extent that cell proliferation is transiently impaired. To circumvent this drawback, competence activation is restricted to a subpopulation, a process known as bimodality. In this work, we explored this phenomenon in salivarius streptococci and elucidated the molecular mechanisms governing cell fate. We also show that an environmental sensor controlling virulence in pathogenic streptococci is diverted to control competence in commensal streptococci. Together, those results showcase how bacteria can sense and transmit external stimuli to complex communication devices for fine-tuning collective behaviors.

Performances of the barrel kiln used in cottage industry for fish processing and effects on physicochemical characteristics and safety of smoked fish products.

BACKGROUND: In the traditional food sector, the smoking process and smoking-drying process are widely used to increase the shelf-life of seafood products. The smoking process and smoking-drying process are mainly performed using barrel kiln and wood as fuel in many West African countries. The present study evaluated the performances of the barrel kiln and its effects on physicochemical characteristics and safety of smoked fish (SF) and smoked-dried fish (SDF). Twelve follow-ups were conducted with three experimental processors and 24 samples of fish collected at different steps of processing were analyzed in a laboratory using standard methods. RESULTS: The extreme values of combustion temperature recorded during the smoking process (456.4 °C) and smoking-drying process (482.8 °C) were higher than 450 °C, the temperature at which wood pyrolysis generates polycyclic aromatic hydrocarbons (PAHs). Smoked fish were highly contaminated with PAHs, and showed maximal levels of benzo[a]pyrene (52.7 µg kg-1 ) and PAH4 (i.e. sum of benzo[a]pyrene, chrysene, benzo[b]fluoranthene and benz[a]anthracene) (290.9 µg kg-1 ) exceeding the European Union limits by about 25-fold. After smoking of Scomber scombrus and smoking-drying of Cypselurus cyanopterus, no significant differences were recorded for lipid, protein and biogenic amine contents between fresh and processed fish, even if the histamine content of both fish exceeded the limit fixed by the European Union regulation. CONCLUSION: The results obtained in the present study showed that smoked fish and smoked-dried fish produced using barrel kiln and wood fuel are highly contaminated by PAHs. Therefore, there is a need to improve the preservation practices of raw fish and smoking conditions to limit the contamination of end-products by PAHs known to be carcinogenic components for humans and to ensure consumer safety. © 2021 Society of Chemical Industry.

Chemical hazards in smoked meat and fish.

This review aims to give an insight into the main hazards currently found in smoked meat and fish products. Literature research was carried out on international databases such as Access to Global Online Research in Agriculture (AGORA) database, Science direct, and Google scholar to collect and select 92 relevant publications included in this review. The smoking process was described and five hazards mostly found in smoked fish and meat were presented. The heat-induced compounds such as polycyclic aromatic hydrocarbons, heterocyclic amines, and nitrosamines were found in smoked fish and meat. Other hazards such as biogenic amines and heavy metals were also present in smoked fish and meat. The levels of these hazards reported from the literature exceeded the maximal limits of European Union. A brief description of risk assessment methodology applicable to such toxic compounds and risk assessment examples was also presented in this review. As most of the hazards reported in this review are toxic and even carcinogenic to humans, actions should be addressed to reduce their presence in food to protect consumer health and to prevent public health issue.

Cyclical Patterns Affect Microbial Dynamics in the Water Basin of a Nuclear Research Reactor.

The BR2 nuclear research reactor in Mol, Belgium, runs in successive phases of operation (cycles) and shutdown, whereby a water basin surrounding the reactor vessel undergoes periodic changes in physico-chemical parameters such as flow rate, temperature, and radiation. The aim of this study was to explore the microbial community in this unique environment and to investigate its long-term dynamics using a 16S rRNA amplicon sequencing approach. Results from two sampling campaigns spanning several months showed a clear shift in community profiles: cycles were mostly dominated by two Operational Taxonomic Units (OTUs) assigned to unclassified Gammaproteobacterium and Pelomonas, whereas shutdowns were dominated by an OTU assigned to Methylobacterium. Although 1 year apart, both campaigns showed similar results, indicating that the system remained stable over this 2-year period. The community shifts were linked with changes in physico-chemical parameters by Non-metric Multidimensional Scaling (NMDS) and correlation analyses. In addition, radiation was hypothesized to cause a decrease in cell number, whereas temperature had the opposite effect. Chemoautotrophic use of H2 and dead cell recycling are proposed to be used as a strategies for nutrient retrieval in this extremely oligotrophic environment.

New Insights into the Potential Cytotoxic Role of Bacillus cytotoxicus Cytotoxin K-1.

The thermotolerant representative of the Bacillus cereus group, Bacillus cytotoxicus, reliably harbors the coding gene of cytotoxin K-1 (CytK-1). This protein is a highly cytotoxic variant of CytK toxin, initially recovered from a diarrheal foodborne outbreak that caused the death of three people. In recent years, the cytotoxicity of B. cytotoxicus has become controversial, with some strains displaying a high cytotoxicity while others show no cytotoxicity towards cell lines. In order to better circumscribe the potential pathogenic role of CytK-1, knockout (KO) mutants were constructed in two B. cytotoxicus strains, E8.1 and E28.3. The complementation of the cytK-1 KO mutation was implemented in a mutant strain lacking in the cytK-1 gene. Using the tetrazolium salt (MTT) method, cytotoxicity tests of the cytK-1 KO and complemented mutants, as well as those of their wild-type strains, were carried out on Caco-2 cells. The results showed that cytK-1 KO mutants were significantly less cytotoxic than the parental wild-type strains. However, the complemented mutant was as cytotoxic as the wild-type, suggesting that CytK-1 is the major cytotoxicity factor in B. cytotoxicus.

Comparative Genomics of Prophages Sato and Sole Expands the Genetic Diversity Found in the Genus Betatectivirus.

Tectiviruses infecting the Bacillus cereus group represent part of the bacterial "plasmid repertoire" as they behave as linear plasmids during their lysogenic cycle. Several novel tectiviruses have been recently found infecting diverse strains belonging the B. cereus lineage. Here, we report and analyze the complete genome sequences of phages Sato and Sole. The linear dsDNA genome of Sato spans 14,852 bp with 32 coding DNA sequences (CDSs), whereas the one of Sole has 14,444 bp comprising 30 CDSs. Both phage genomes contain inverted terminal repeats and no tRNAs. Genomic comparisons and phylogenetic analyses placed these two phages within the genus Betatectivirus in the family Tectiviridae. Additional comparative genomic analyses indicated that the "gene regulation-genome replication" module of phages Sato and Sole is more diverse than previously observed among other fully sequenced betatectiviruses, displaying very low sequence similarities and containing some ORFans. Interestingly, the ssDNA binding protein encoded in this genomic module in phages Sato and Sole has very little amino acid similarity with those of reference betatectiviruses. Phylogenetic analyses showed that both Sato and Sole represent novel tectivirus species, thus we propose to include them as two novel species in the genus Betatectivirus.

At the Gate of Mutualism: Identification of Genomic Traits Predisposing to Insect-Bacterial Symbiosis in Pathogenic Strains of the Aphid Symbiont Serratia symbiotica.

Mutualistic associations between insects and heritable bacterial symbionts are ubiquitous in nature. The aphid symbiont Serratia symbiotica is a valuable candidate for studying the evolution of bacterial symbiosis in insects because it includes a wide diversity of strains that reflect the diverse relationships in which bacteria can be engaged with insects, from pathogenic interactions to obligate intracellular mutualism. The recent discovery of culturable strains, which are hypothesized to resemble the ancestors of intracellular strains, provide an opportunity to study the mechanisms underlying bacterial symbiosis in its early stages. In this study, we analyzed the genomes of three of these culturable strains that are pathogenic to aphid hosts, and performed comparative genomic analyses including mutualistic host-dependent strains. All three genomes are larger than those of the host-restricted S. symbiotica strains described so far, and show significant enrichment in pseudogenes and mobile elements, suggesting that these three pathogenic strains are in the early stages of the adaptation to their host. Compared to their intracellular mutualistic relatives, the three strains harbor a greater diversity of genes coding for virulence factors and metabolic pathways, suggesting that they are likely adapted to infect new hosts and are a potential source of metabolic innovation for insects. The presence in their genomes of secondary metabolism gene clusters associated with the production of antimicrobial compounds and phytotoxins supports the hypothesis that S. symbiotia symbionts evolved from plant-associated strains and that plants may serve as intermediate hosts. Mutualistic associations between insects and bacteria are the result of independent transitions to endosymbiosis initiated by the acquisition of environmental progenitors. In this context, the genomes of free-living S. symbiotica strains provide a rare opportunity to study the inventory of genes held by bacterial associates of insects that are at the gateway to a host-dependent lifestyle.

Pan-Genome Portrait of Bacillus mycoides Provides Insights into the Species Ecology and Evolution.

Bacillus mycoides is poorly known despite its frequent occurrence in a wide variety of environments. To provide direct insight into its ecology and evolutionary history, a comparative investigation of the species pan-genome and the functional gene categorization of 35 isolates obtained from soil samples from northeastern Poland was performed. The pan-genome of these isolates is composed of 20,175 genes and is characterized by a strong predominance of adaptive genes (∼83%), a significant amount of plasmid genes (∼37%), and a great contribution of prophages and insertion sequences. The pan-genome structure and phylodynamic studies had suggested a wide genomic diversity among the isolates, but no correlation between lineages and the bacillus origin was found. Nevertheless, the two B. mycoides populations, one from Bialowieza National Park, the last European natural primeval forest with soil classified as organic, and the second from mineral soil samples taken in a farm in Jasienówka, a place with strong anthropogenic pressure, differ significantly in the frequency of genes encoding proteins enabling bacillus adaptation to specific stress conditions and production of a set of compounds, thus facilitating their colonization of various ecological niches. Furthermore, differences in the prevalence of essential stress sigma factors might be an important trail of this process. Due to these numerous adaptive genes, B. mycoides is able to quickly adapt to changing environmental conditions. IMPORTANCE This research allows deeper understanding of the genetic organization of natural bacterial populations, specifically, Bacillus mycoides, a psychrotrophic member of the Bacillus cereus group that is widely distributed worldwide, especially in areas with continental cold climates. These thorough analyses made it possible to describe, for the first time, the B. mycoides pan-genome, phylogenetic relationship within this species, and the mechanisms behind the species ecology and evolutionary history. Our study indicates a set of functional properties and adaptive genes, in particular, those encoding sigma factors, associated with B. mycoides acclimatization to specific ecological niches and changing environmental conditions.