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.

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.

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.

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.

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.

A novel anti-dipteran Bacillus thuringiensis strain: Unusual Cry toxin genes in a highly dynamic plasmid environment.

Bacillus thuringiensis emerged as a major bioinsecticide on the global market. It offers a valuable alternative to chemical products classically utilized to control pest insects. Despite the efficiency of several strains and products available on the market, the scientific community is always on the lookout for novel toxins that can replace or supplement the existing products. In this study, H3, a novel B. thuringiensis strain showing mosquitocidal activity, was isolated from Lebanese soil and characterized at an in vivo, genomic and proteomic levels. H3 parasporal crystal is toxic on its own but displays an unusual killing profile with a higher LC50 than the reference B. thuringiensis serovar israelensis crystal proteins. In addition, H3 has a different toxicity order: it is more toxic to Aedes albopictus and Anopheles gambiae than to Culex pipiens Whole genome sequencing and crystal analysis revealed that H3 can produce eleven novel Cry proteins, eight of which are assembled in genes with an orf1-gap-orf2 organization, where orf2 is a potential Cry4-type crystallization domain. Moreover, pH3-180, the toxin-carrying plasmid, holds a wide repertoire of mobile genetic elements that amount to ca 22% of its size., including novel insertion sequences and class II transposable elements Two other large plasmids present in H3 carry genetic determinants for the production of many interesting molecules - such as chitinase, cellulase and bacitracin - that may add up to H3 bioactive properties. This study therefore reports a novel mosquitocidal Bacillus thuringiensis strain with unusual Cry toxin genes in a rich mobile DNA environment.IMPORTANCE Bacillus thuringiensis, a soil entomopathogenic bacteria, is at the base of many sustainable eco-friendly bio-insecticides. Hence stems the need to continually characterize insecticidal toxins. H3 is an anti-dipteran B. thuringiensis strain, isolated from Lebanese soil, whose parasporal crystal contains eleven novel Cry toxins and no Cyt toxins. In addition to its individual activity, H3 showed potential as a co-formulant with classic commercialized B. thuringiensis products, to delay the emergence of resistance and to shorten the time required for killing. On a genomic level, H3 holds three large plasmids, one of which carries the toxin-coding genes, with four occurrences of the distinct orf1-gap-orf2 organization. Moreover, this plasmid is extremely rich in mobile genetic elements, unlike its two co-residents. This highlights the important underlying evolutionary traits between toxin-carrying plasmids and the adaptation of a B. thuringiensis strain to its environment and insect host spectrum.

Diversity of Bacillus cereus sensu lato mobilome.

BACKGROUND: Bacillus cereus sensu lato s.l.) is a group of bacteria displaying close phylogenetic relationships but a high ecological diversity. The three most studied species are Bacillus anthracis, Bacillus cereus sensu stricto and Bacillus thuringiensis. While some species are pathogenic to mammals or associated with food poisoning, Bacillus thuringiensis is a well-known entomopathogenic bacterium used as biopesticide worldwide. B. cereus s.l. also contains a large variety of mobile genetic elements (MGEs). RESULTS: In this study, we detail the occurrence and plasmid vs. chromosome distribution of several MGEs in 102 complete and annotated genomes of B. cereus s.l. These MGEs include 16 Insertion Sequence (IS) families, the Tn3 family, 18 different Bacillus cereus repeats (BCRs) and 30 known group II introns. CONCLUSIONS: Our analysis not only shows the diversity of these MGEs among strains of the same species and between different species within the B. cereus s.l. group, but also highlights the potential impact of these elements on the plasticity of the plasmid pool, and the TEs (Transposable Elements) - species relationship within B. cereus s.l.

pXO16, the large conjugative plasmid from Bacillus thuringiensis serovar israelensis displays an extended host spectrum.

pXO16, the large conjugative plasmid from Bacillus thuringiensis serovar israelensis is able to efficient self-transfer, to mobilize and retro-mobilize non-conjugative plasmids, including "non-mobilizable" plasmids, and to transfer chromosomal loci. It also displays a remarkable aggregation phenotype associated with conjugation under liquid conditions. However, it was recently shown that aggregation boosts pXO16 transfer but is not mandatory. In this paper, we have further explored pXO16 transfers under various mating conditions and with different members of the Bacillus cereus group. The results indicated that colony or filter mating largely compensate the transfer deficit observed when using a pXO16 aggregation-minus mutant. Using filter mating, pXO16 transfer efficiency and host range were both improved. For instance, pXO16 was shown to transfer itself, and to mobilize the small pUB110 plasmid, from B. thuringiensis serovar israelensis to the thermotolerant Bacillus cytotoxicus at frequencies of 3.3 × 10-3 and 5.2 × 10-4 transconjugants per donor (T/D), respectively. All together, these results indicate that pXO16 can potentially "circulate" among members of the Bacillus cereus group. Yet, this is contrasting with pXO16's known natural distribution, which is apparently limited to the israelensis serovar of B. thuringiensis.

Characterization and Whole Genome Sequencing of AR23, a Highly Toxic Bacillus thuringiensis Strain Isolated from Lebanese Soil.

The demand for sustainable and eco-friendly control methods of pests and insects is increasing worldwide. From this came the interest in Bacillus thuringiensis, an entomopathogenic bacterium capable of replacing chemical pesticides. However, the possibility of pests developing resistance to a particular strain may impair its use, and there is a need to identify novel strains of this species as potential commercial biopesticides. B. thuringiensis sv. israelensis is one of the most successful serovars, widely commercialized for its activity against black fly and mosquito larvae. In this study, we isolated, characterized, and sequenced a new Lebanese B. thuringiensis sv. israelensis isolate, strain AR23. Compared to the commercialized reference strain AM65-52 (Vectobac®, Sumitomo), AR23 showed an increased activity against several mosquito species. The genomic analysis revealed that this strain, compared to AM65-52, possesses a simplified plasmid content and an additional functional cry4Ba coding gene that most likely accounts for the increased effectiveness of this strain in mosquito larvae killing.

Bacterial Sexuality at the Nanoscale.

Understanding the basic mechanisms of bacterial sexuality is an important topic in current microbiology and biotechnology. While classical methods used to study gene transfer provide information on whole cell populations, nanotechnologies offer new opportunities for analyzing the behavior of individual mating partners. We introduce an innovative atomic force microscopy (AFM) platform to study and mechanically control DNA transfer between single bacteria, focusing on the large conjugative pXO16 plasmid of the Gram-positive bacterium Bacillus thuringiensis. We demonstrate that the adhesion forces between single donor and recipient cells are very strong (∼2 nN). Using a mutant plasmid, we find that these high forces are mediated by a pXO16 aggregation locus that contains two large surface protein genes. Notably, we also show that AFM can be used to mechanically induce plasmid transfer between single partners, revealing that transfer is very fast (<15 min) and triggers major cell surface changes in transconjugant cells. We anticipate that the single-cell technology developed here will enable researchers to mechanically control gene transfer among a wide range of Gram-positive and Gram-negative bacterial species and to understand the molecular forces involved. Also, the method could be useful in nanomedicine for the design of antiadhesion compounds capable of preventing intimate cell-cell contacts, therefore providing a means to control the resistance and virulence of bacterial pathogens.

A novel T4SS-mediated DNA transfer used by pXO16, a conjugative plasmid from Bacillus thuringiensis serovar israelensis.

The entomopathogenic Bacillus thuringiensis serovar israelensis displays peculiar conjugative transfer capabilities, accounted for by the large conjugative plasmid pXO16 (350 kb). The efficient and fast conjugative transfers are accompanied by a macroscopic aggregation of bacterial partners. Moreover, pXO16 has proven capable of effective mobilization and the retro-transfer of both mobilizable and 'non-mobilizable' plasmids. In this work, the aggregation phenomenon is shown to promote pXO16 transfer while not being mandatory for transfer. Transfer of pXO16 to B. thuringiensis recipient strains that do not display aggregation is observed as well, hence enlarging the previously defined host range. The use of variant calling analysis of transconjugants allowed for observation of up to 791 kb chromosomal regions mobilization. Previous analysis of pXO16 did not reveal any Type IV Secretion System (T4SS) homologs, which suggested the presence of an unusual conjugative system. A FtsK/SpOIIIE ATPase gene proved here to be necessary for conjugative transfer. Additionally, the analysis of natural restriction-modification systems in both conjugative partners gave credit to a ssDNA transfer mechanism. A 'transfer israelensis plasmid' (tip) region containing this ATPase gene was shown to code for other potential T4SS proteins, illustrating a conjugative system distantly related to the other known Gram-positive T4SSs.

Complete genome sequence of bacteriophage Deep-Purple, a novel member of the family Siphoviridae infecting Bacillus cereus.

Bacteriophage Deep-Purple, isolated from an agricultural soil in Belgium, lyses the emetic Bacillus weihenstephanensis strain LH002 and exhibits a lytic activity against 55% of emetic Bacillus cereus and B. weihenstephanensis strains. Deep-Purple is able to complete its lytic cycle within 45 min and is stable to a large range of pHs and temperatures below 60 °C. It possesses an icosahedral head of about 63 nm in diameter and a non-contractile tail of approximately 165 nm in length. The genome of this newly classifiable Siphoviridae family member is 36,278 bp long, with a G+C content of 38.36% and 40 putative CDSs. Most CDSs do not display similarity with other B. cereus group phages supporting the idea that Deep-Purple belongs to a new and currently uncharacterised Siphoviridae subfamily.

Complete genome sequence of two tomato-infecting begomoviruses in Venezuela: evidence of a putative novel species and a novel recombinant strain.

At least six begomovirus species have been reported infecting tomato in Venezuela. In this study the complete genomes of two tomato-infecting begomovirus isolates (referred to as Trujillo-427 and Zulia-1084) were cloned and sequenced. Both isolates showed the typical genome organization of New World bipartite begomoviruses, with DNA-A genomic components displaying 88.8% and 90.3% similarity with established begomoviruses, for isolates Trujillo-427 and Zulia-1084, respectively. In accordance to the guidelines for begomovirus species demarcation, the Trujillo-427 isolate represents a putative new species and the name "Tomato wrinkled mosaic virus" is proposed. Meanwhile, Zulia-1084 represents a putative new strain classifiable within species Tomato chlorotic leaf distortion virus, for which a recombinant origin is suggested.

A liquid bead array for the identification and characterization of fljB-positive and fljB-negative monophasic variants of Salmonella Typhimurium.

Salmonella1,4,[5],12:i:- accounts currently for one of the most common serotypes observed worldwide. These isolates do not express the FljB flagellin and mostly derive from Salmonella Typhimurium. They are therefore termed Salmonella Typhimurium monophasic variants (STMV) and are considered of comparable public health risk. Since serological identification of the somatic and flagellar antigens of STMV is not sufficient to demonstrate relatedness with Salmonella Typhimurium, additional assays detecting genetic markers unique to Salmonella Typhimurium are required. In addition, identification of the mutations affecting expression of the flagellar gene fljB can be useful to support the monophasic character observed phenotypically. Finally, genetic subtyping of the various mono- and biphasic Salmonella Typhimurium clonal groups can facilitate their epidemiological follow-up. Here, we present a home-made liquid bead array able to fulfill these requirements. This array confirmed the monophasic character of 240 STMV isolates collected in Belgium during 2014-2015 and identified 10 genetic subtypes. Microevolution in and around the fljB locus linked to IS26 insertions is probably one of the driven force accounting for STMV population diversity. Thanks to its open design, other genetic signatures could later be merged to the assay to subtype additional STMV clonal groups and to detect rare mutations.

Diversity and enzymatic potentialities of Bacillus sp. strains isolated from a polluted freshwater ecosystem in Cuba.

Genotypic and phenotypic characterization of Bacillus spp. from polluted freshwater has been poorly addressed. The objective of this research was to determine the diversity and enzymatic potentialities of Bacillus spp. strains isolated from the Almendares River. Bacilli strains from a polluted river were characterized by considering the production of extracellular enzymes using API ZYM. 14 strains were selected and identified using 16S rRNA, gyrB and aroE genes. Genotypic diversity of the Bacillus spp. strains was evaluated using pulsed field gel electrophoresis. Furthermore, the presence of genetic determinants of potential virulence toxins of the Bacillus cereus group and proteinaceous crystal inclusions of Bacillus thuringiensis was determined. 10 strains were identified as B. thuringiensis, two as Bacillus megaterium, one as Bacillus pumilus and one as Bacillus subtilis. Most strains produced proteases, amylases, phosphatases, esterases, aminopeptidases and glucanases, which reflect the abundance of biopolymeric matter in Almendares River. Comparison of the typing results revealed a spatio-temporal distribution among B. thuringiensis strains along the river. The results of the present study highlight the genotypic and phenotypic diversity of Bacillus spp. strains from a polluted river, which contributes to the knowledge of genetic diversity of Bacilli from tropical polluted freshwater ecosystems.

Horizontal transfer of chromosomal markers mediated by the large conjugative plasmid pXO16 from Bacillus thuringiensis serovar israelensis.

pXO16, a large plasmid originating from Bacillus thuringiensis serovar israelensis, displays unique conjugation capacities: besides efficient self-transfer, it is able to mobilize and retro-mobilize non-conjugative plasmids, including those missing an oriT and/or a mob gene, also known as "non-mobilizable" plasmids. In this paper, another peculiar transfer property of pXO16 is described. This element is indeed able to transfer chromosomal loci at frequencies of ca. 10-5-10-6 transconjugants/donor cell. Whereas most other chromosomal transfer systems occur via the integration of the conjugative elements into the chromosome prior to its transfer, pXO16 appears to transfer the chromosomal markers in the absence of physical integration, but rather through a "donation-type" mobilization.

Molecular and biological characterization of a new Tomato mild yellow leaf curl Aragua virus strain producing severe symptoms in tomato.

Tomato mild yellow leaf curl Aragua virus (ToMYLCV) is a begomovirus first reported infecting tomato (Solanum lycopersicum) and milkweed (Euphorbia heterophylla) in Venezuela. In this study, a ToMYLCV isolate (Zulia-219) was completely sequenced and its host range was evaluated. The DNA-A and DNA-B components of isolate Zulia-219 showed 93 and 85% nucleotide sequence identity with the respective counterparts of the ToMYLCV type strain. According to current demarcation criteria for begomovirus species, Zulia-219 is a new strain of ToMYLCV. Interestingly, tomato plants inoculated with ToMYLCV Zulia-219 displayed severe symptoms, including severe chlorotic leaf curling, in contrast to mild symptoms associated with the type strain of this begomovirus. These results indicate potential risks associated with this new ToMYLCV strain for tomato production in Venezuela.

Cereulide production by Bacillus weihenstephanensis strains during growth at different pH values and temperatures.

Besides Bacillus cereus, some strains of the psychrotolerant, potentially foodborne pathogen Bacillus weihenstephanensis can produce the emetic toxine (cereulide). This toxin is a heat- and acid-stable cyclic dodecadepsipeptide that causes food intoxication with vomiting. However, some severe clinical cases with lethal outcomes have been described. If cereulide can be produced during refrigerated storage, it will not be inactivated by reheating food, representing an important risk of food intoxication for consumers. In this paper, we determined the capacity of the B. weihenstephanensis strains BtB2-4 and MC67 to grow and produce cereulide on agar media at temperatures from 8 °C to 25 °C and at a pH from 5.4 to 7.0. At 8 °C, strain BtB2-4 produced quantifiable amounts of cereulide, whereas the limit of detection was reached for strain MC67. For BtB2-4, cereulide production increased 5-fold between 8 °C and 10-15 °C and by more than 100-fold between 15 °C and 25 °C. At temperatures of 10 °C and higher, cereulide concentrations were within the range of those reported by previous works in foods implicated in emetic poisoning. At 25 °C, decreasing the pH to 5.4 reduced cereulide production by strain BtB2-4 by at least 20-fold.

Role of Ionic Strength in Staphylococcal Cell Aggregation.

Cell aggregation plays a key role in biofilm formation and pathogenesis of Staphylococcus species. Although the molecular basis of aggregation in Staphylococci has already been extensively investigated, the influence of environmental factors, such as ionic strength, remains poorly understood. In this paper, we report a new type of cellular aggregation of Staphylococci that depends solely on ionic strength. Seven strains out of 14, all belonging to staphylococcal species, formed large cell clusters within minutes in buffers of ionic strength ranging from 1.5 to 50 mM, whereas isolates belonging to other Gram-positive species did not display this phenotype. Atomic force microscopy (AFM) with chemically functionalized tips provided direct evidence that ionic strength modulates cell surface adhesive properties through changes in cell surface charge. The optimal ionic strength for aggregation was found to be strain dependent, but in all cases, bacterial aggregates formed at an ionic strength of 1.5-50 mM were rapidly dispersed in a solution of higher ionic strength, indicating a reversibility of the cell aggregation process. These findings suggest that some staphylococcal isolates can respond to ionic strength as an external stimulus to trigger rapid cell aggregation in a way that has not yet been reported.

Extensive genetic variability linked to IS26 insertions in the fljB promoter region of atypical monophasic variants of Salmonella enterica serovar Typhimurium.

Fifty-nine monophasic Salmonella enterica serovar Typhimurium isolates, collected in Belgium during the period from 2008 to 2011, have been serotyped as 4,[5]:i:- and shown to harbor an fljB coding sequence. The genetic differences between these strains and phenotypically biphasic Salmonella Typhimurium were analyzed through PCR and DNA sequencing. Genetic alterations in the fljB promoter region affecting expression of the phase 2 flagellin were observed in 53 isolates. Other genetic events in the invertible region carrying the fljB promoter were observed in 2 isolates. For the remaining 4 isolates, no molecular differences with a reference biphasic Salmonella Typhimurium strain could be observed. Next-generation sequencing of one representative isolate affected in the fljB promoter region revealed a 26-kb IS26 composite transposon insertion along with a local genomic rearrangement. Several other IS26 element-mediated alterations of this genomic region were observed. This group of monophasic Salmonella Typhimurium isolates was genetically heterogeneous, as revealed by multilocus variable-number tandem-repeat analysis (MLVA), PCR, and sequencing. Pigs and pork represented a major source of such monophasic isolates in Belgium, as reported in other countries. Three out of 5 isolates of human origin presented genetic profiles identical to those of food isolates, demonstrating the pathogenic potential of the newly characterized variants and potential dissemination along the food chain. This study highlighted the key role played by IS26 insertions in the loss of phase 2 flagellin expression and the subsequent generation of multiple monophasic variant lineages from biphasic Salmonella Typhimurium ancestors.