Search

Bacteria survive under various conditions by sensing stimuli triggering specific adaptive physiological responses, which are often based on membrane-integrated sensors connected to a cytoplasmic regulator. Recent studies reveal that mucus glycans may act as signal molecules for two-component systems involved in intestinal colonization. Bacillus cereus,a human and insect opportunistic pathogen was used to identify bacterial factors expressed in an insect gut infection model. The screen revealed a promoter involved in the expression of a gene with so far unknown functions. A search for gut-related compounds, inducing its transcription, identified glucose-6-phosphate as an activation signal. The gene is part of a five-gene cluster, including a two-component system. Interestingly such five gene loci are conserved in the pathogenic Bacillusgroup as well as in various Clostridiabacteria and are with analogy to other multi-component sensor systems in enteropathogenic bacteria, such as E. coli. Thus our results provide insights into the function of two-component and auxiliary sensor systems in host-microbe interactions and opens up possible investigations of such systems in other gut associated bacteria.

ABSTRACTThe Bacillus cereuspathogenic spectrum ranges from strains used as probiotics to human-lethal strains. However, prediction of the pathogenic potential of a strain remains difficult. Here, we show that food poisoning and clinical strains can be differentiated from harmless strains on the basis of host colonization phenotypes.

ABSTRACTBacillus cereusis found in food, soil, and plants, and the ability to cause food-borne diseases and opportunistic infection presumably varies among strains. Therefore, measuring harmful toxin production, in addition to the detection of the bacterium itself, may be key for food and hospital safety purposes. All previous studies have focused on the main known virulence factors, cereulide, Hbl, Nhe, and CytK. We examined whether other virulence factors may be specific to pathogenic strains. InhA1, NprA, and HlyII have been described as possibly contributing to B. cereuspathogenicity. We report the prevalence and expression profiles of these three new virulence factor genes among 57 B. cereusstrains isolated from various sources, including isolates associated with gastrointestinal and nongastrointestinal diseases. Using PCR, quantitative reverse transcriptase PCR, and virulence in vivoassays, we unraveled these factors as potential markers to differentiate pathogenic from nonpathogenic strains. We show that the hlyIIgene is carried only by strains with a pathogenic potential and that the expression levels of inhA1and nprAare higher in the pathogenic than in the nonpathogenic group of strains studied. These data deliver useful information about the pathogenicity of various B. cereusstrains.

Food packaging papers are not sterile, as the manufacturing is an open process, and the raw materials contain bacteria. We modeled the potential transfer of the Bacillus cereusspores from packaging paper to food by using a green fluorescent protein–expressing construct of Bacillus thuringiensisBt 407Cry−[pHT315ω (papha3-gfp)], abbreviated BT-1. Paper (260 g m−2) containing BT-1 was manufactured with equipment that allowed fiber formation similar to that of full-scale manufactured paper. BT-1 adhered to pulp during papermaking and survived similar to an authentic B. cereus. Rice and chocolate were exposed to the BT-1–containing paper for 10 or 30 days at 40 or 20°C at relative air humidity of 10 to 60%. The majority of the spores remained immobilized inside the fiber web; only 0.001 to 0.03% transferred to the foods. This amount is low compared with the process hygiene criteria and densities commonly found in food, and it does not endanger food safety. To measure this, we introduced BT- 1 spores into the paper in densities of 100 to 1,000 times higher than the amounts of the B. cereusgroup bacteria found in commercial paper. Of BT-1 spores, 0.03 to 0.1% transferred from the paper to fresh agar surface within 5 min of contact, which is more than to food during 10 to 30 days of exposure. The findings indicate that transfer from paper to dry food is restricted to those microbes that are exposed on the paper surface and readily detectable with a contact agar method.

The explosive, destructive course of Bacillus endophthalmitis has been attributed to the production of toxins during infection. In this study we analyzed the contribution of toxins controlled by the global regulator plcR to the pathogenesis of experimental Bacillus endophthalmitis. Isogenic plcR-deficient mutants of Bacillus cereus and Bacillus thuringiensis were constructed by insertional inactivation of plcR by the kanamycin resistance cassette, aphA3. Rabbit eyes were injected intravitreally with approximately 100 CFU of wild-type B. cereus or B. thuringiensis or a plcR-deficient mutant. The evolution of endophthalmitis resulting from each plcR-deficient mutant was considerably slower than that caused by each wild-type strain. Retinal function was not eliminated until 42 h postinfection in rabbits with endophthalmitis caused by the plcR-deficient mutants, whereas wild-type infections resulted in a complete loss of retinal function within 18 h. The intraocular inflammatory cell influx and retinal destruction in plcR-deficient endophthalmitis approached the severity observed in wild-ype infections, but not until 36 h postinfection. Gross and histological examinations of eyes infected with plcR mutants demonstrated that the anterior and posterior segment changes were muted compared to the changes observed in eyes infected with the wild types. The loss of plcR-regulated factors significantly attenuated the severity of Bacillus endophthalmitis. The results therefore suggest that plcR may represent a target for which adjunct therapies could be designed for the prevention of blindness during Bacillus endophthalmitis.

ABSTRACTThe explosive, destructive course of Bacillusendophthalmitis has been attributed to the production of toxins during infection. In this study we analyzed the contribution of toxins controlled by the global regulator plcRto the pathogenesis of experimental Bacillusendophthalmitis. Isogenic plcR-deficient mutants of Bacillus cereusand Bacillus thuringiensiswere constructed by insertional inactivation of plcRby the kanamycin resistance cassette, aphA3. Rabbit eyes were injected intravitreally with approximately 100 CFU of wild-type B. cereusor B. thuringiensisor a plcR-deficient mutant. The evolution of endophthalmitis resulting from each plcR-deficient mutant was considerably slower than that caused by each wild-type strain. Retinal function was not eliminated until 42 h postinfection in rabbits with endophthalmitis caused by the plcR-deficient mutants, whereas wild-type infections resulted in a complete loss of retinal function within 18 h. The intraocular inflammatory cell influx and retinal destruction in plcR-deficient endophthalmitis approached the severity observed in wild-ype infections, but not until 36 h postinfection. Gross and histological examinations of eyes infected with plcRmutants demonstrated that the anterior and posterior segment changes were muted compared to the changes observed in eyes infected with the wild types. The loss of plcR-regulated factors significantly attenuated the severity of Bacillusendophthalmitis. The results therefore suggest that plcRmay represent a target for which adjunct therapies could be designed for the prevention of blindness during Bacillusendophthalmitis.

Membrane-damaging toxins are thought to be responsible for the explosive clinical course of Bacillus endophthalmitis. This study analyzed the contribution of phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC) to the pathogenesis of experimental Bacillus endophthalmitis. Isogenic mutants were constructed by insertion of lacZ into Bacillus thuringiensis genes encoding PI-PLC (plcA) and PC-PLC (plcB). Rabbit eyes were injected intravitreally with 2 log(10) CFU of strain BT407 (wild type), the PI-PLC mutant (BTplcA::lacZ), or the PC-PLC mutant (BTplcB::lacZ). The rates of decrease in retinal responses of eyes infected with the isogenic mutants were similar to that of wild type, with all infections resulting in elimination of retinal function by 18 h. Strain BT407 caused a significant increase in the latency of retinal responses at 6 h, but strains BTplcA::lacZ and BTplcB::lacZ did not. All strains elicited significant inflammatory cell influx into the anterior chamber by 12 h. Histologically, eyes infected with each strain were indistinguishable throughout the infection course. In this model, neither PI-PLC nor PC-PLC had an effect on the course or severity of experimental Bacillus endophthalmitis. Alterations in retinal responses early in infection may mark the beginnings of specific photoreceptor or glial cell dysfunction.

ABSTRACTMembrane-damaging toxins are thought to be responsible for the explosive clinical course of Bacillusendophthalmitis. This study analyzed the contribution of phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC) to the pathogenesis of experimental Bacillusendophthalmitis. Isogenic mutants were constructed by insertion of lacZinto Bacillus thuringiensisgenes encoding PI-PLC (plcA) and PC-PLC (plcB). Rabbit eyes were injected intravitreally with 2 log10CFU of strain BT407 (wild type), the PI-PLC mutant (BTplcA::lacZ), or the PC-PLC mutant (BTplcB::lacZ). The rates of decrease in retinal responses of eyes infected with the isogenic mutants were similar to that of wild type, with all infections resulting in elimination of retinal function by 18 h. Strain BT407 caused a significant increase in the latency of retinal responses at 6 h, but strains BTplcA::lacZand BTplcB::lacZdid not. All strains elicited significant inflammatory cell influx into the anterior chamber by 12 h. Histologically, eyes infected with each strain were indistinguishable throughout the infection course. In this model, neither PI-PLC nor PC-PLC had an effect on the course or severity of experimental Bacillusendophthalmitis. Alterations in retinal responses early in infection may mark the beginnings of specific photoreceptor or glial cell dysfunction.

The survival and conjugation ability of sporogenic and asporogenic Bacillus thuringiensisstrains were investigated in broth, in non‐amended sterile clay soil monoculture and in mixed soil culture. The 75 kb pHT73 plasmid carrying an erythromycin resistance determinant and a cry1Ac gene was transferred in mating broth and soil microcosm. Survival of strains was assessed in soil monoculture and in mixed soil culture for up to 20 days. Sporogenic strains rapidly formed viable spores which were maintained until the end of the experiment. The asporogenic strains were no longer recovered after 8 days of incubation. This study shows that the environmental impact of asporogenic B. thuringiensisstrains is lower than that of sporogenic B. thuringiensisstrains. Thus, the use of asporogenic strains may significantly reduce any potential risk (gene transfer, soil and plant contamination) due to the dissemination of B. thuringiensis‐based biopesticides in the environment.

We describe a novel approach for the insertion of an insecticidal toxin gene into a resident plasmid in Bacillus thuringiensis (Bt). A gene encoding a coleopteran–specific toxin was cloned within a fragment of IS232 and inserted into a plasmid thermosensitive for replication in Bt. The plasmid was used to transform a Bt strain toxic to lepidoptera, and the transformants were then selected at non–permissive temperature for clones in which the vector had integrated into a copy of IS232 present on a resident plasmid. A second recombination event was selected such that the vector was eliminated and the newly introduced toxin gene was conserved. The resulting strain contained only DNA of Bt origin, and displayed insecticidal activity against both lepidoptera and coleoptera.

ThesigE andsigK genes, encoding the sporulation-specific sigma factorsσ35 andσ28 ofBacillus thuringiensis, were each disrupted by inserting a gene conferring resistance to kanamycin into their coding sequence. TheB. thuringiensis SigE− and SigK− mutant strains were blocked at different sporulation stages and were unable to sporulate. The SigE− strain was blocked at stage II of sporulation, whereas the SigK− strain was blocked at stage IV. The expression of acryIAa′-′lacZ transcriptional fusion was analysed in these genetic backgrounds and it was found that both sigma factors are involved in the in vivo transcription of this gene. However, the SigK− strain harbouring thecryIAa gene produced amounts of toxin similar to those produced by theB. thuringiensis Spo+ strain. The toxins accumulated in the mother cell compartment to form a crystal inclusion which remained encapsulated within the cell wall. Thus, transcription from theσE-dependent promoter alone (Bt I promoter) is sufficient to support high levels of toxin production inB. thuringiensis.

A Bacillus thuringiensisplasmid carrying an insecticidal crystal protein gene (cry1Ac) was genetically marked with a gene conferring resistance to erythromycin. The conjugative transfer of this plasmid between B. thuringiensisstrains was analyzed in broth culture, soil microcosms and infected larvae of the lepidopteran insect Anticarsia gemmatalis. Transconjugants appeared after 2 h in broth culture and after 4 h in soil systems. The lowest conjugation frequency was in soil (8.0×10−8–2.7×10−5) and the highest in infected larvae (1.0–8.4×10−1). Plasmid transfer was observed in non‐supplemented soils originating from sites with and without different plant covers. Thus non‐amended soil and insect larvae appear to be favorable environments for plasmid transfer by conjugation.

The transmissible plasmid pAMß1, which codes for resistance to erythromycin and lincomycin, was transferred from Streptococcus faecalis to several strains of Bacillus thuringiensis by a filter-mating process. Introduction of pAMß1 into the Emr transconjugant strains of B. thuringiensis was confirmed by Southern hybridisation using the 32P-labelled pAMß1 as a probe. In the B. thuringiensis transconjugant strains, used as donors, the ß plasmid conserved its ability to be transferred during intraspecific mating, with a frequency of 10-4 per recipient cell. In addition, the transconjugant clones acted as donors of the erythromycin resistance marker and permitted the transfer of cryptic plasmids present in the B. thuringiensis (ß) strains used as donors. From a transconjugant clone of B. thuringiensis a hybrid plasmid resulting from an in vivo insertion into pAMß1 of a 3 Md DNA sequence was isolated. This 3 Md DNA molecule originated from a 54 Md plasmid of a kurstaki strain and is related to several plasmids found in different serotypes of B. thuringiensis.

The spoOA gene of Bacillus subtilis encodes the key factor involved in the initiation of sporulation. It was previously shown that the B. thuringiensis (Bt) cryIIIA gene, encoding a toxin active against coleopteran larvae, is overexpressed in an spoOA mutant of B. subtilis. In this paper we describethe construction of a Bt spo0A mutant strain and its use to produce insecticidal crystal proteins. The spoOA gene of Bt was cloned and identified by its ability to transform a B. subtilis spoOA mutant to prototrophy. Its nucleotidesequence is homologous to the B. subtilis gene. The spo0A gene was replaced in the Bt genome with a dis rupted copy to give an Spo- strain unable to initiate sporulation. When the cryIIIA gene was cloned in the Bt spoOA mutant, large amounts of toxins were produced and accumulated to form a large crystal inclusion which remained encapsulated within the ghost cell. These encapsulatedtoxins were highly active against coleopteran larvae. We anticipate that the cryIIIA expression system and the Bt spoOA mutant will provide a convenient process to generate novel formulations of stabilized and environmentally safe Bt-based biopesticides.

A Bacillus thuringiensisplasmid carrying an insecticidal crystal protein gene (cry1Ac) was genetically marked with a gene conferring resistance to erythromycin. The conjugative transfer of this plasmid between B. thuringiensisstrains was analyzed in broth culture, soil microcosms and infected larvae of the lepidopteran insect Anticarsia gemmatalis. Transconjugants appeared after 2 h in broth culture and after 4 h in soil systems. The lowest conjugation frequency was in soil (8.0×10−8–2.7×10−5) and the highest in infected larvae (1.0–8.4×10−1). Plasmid transfer was observed in non-supplemented soils originating from sites with and without different plant covers. Thus non-amended soil and insect larvae appear to be favorable environments for plasmid transfer by conjugation.

Screening for the plasmid content of 11 strains belonging to nine different serotypes ofB. thuringiensis was carried out by electron microscope examination and electrophoresis in agarose gels. All the strains contained at least two covalently closed, circular (CCC) DNA species. In one strain (berliner 1715), 17 extrachromosomal elements could be distinguished with regard to their size, ranging from 3.9 to 180 Mdal. Southern hybridisation experiments showed that most of these plasmids fell into two categories (inferior to 15 Mdal and superior to 15 Mdal) which have no homology between them. Within these two size groups there is partial conservation of DNA sequences through various serotypes. Further relationships among the plasmids were investigated by a two dimensional version of the Southern's blotting technique.

The mobile genetic element Tn4430, originating from the gram-positive bacterium, Bacillus thuringiensis, and previously described as the Th-sequence, is the first transposon isolated from the genus Bacillus. In the present work a gene (APH-III) conferring resistance to kanamycin was inserted into this 4.2 kb transposon. Transposition experiments showed that Tn4430OAPH-III could transpose in the gram-negative host Escherichia coli when its insertion functions were supplied by an intact copy of Tn4430. By transposing Tn4430OAPH-III directly onto pBR322, it was possible to determine the nucleotide sequence of the terminal inverted repeats of Tn4430 and of the target DNA site. Identical 38 bp in inverted orientation are situated at each end of the transposon and there is a direct duplication of 5 bp at the insertion site. Thus, it is clear that Tn4430 is closely related to the transposons belonging to the Tn3 family (class II elements).

Bacilluspopilliae is an obligate pathogen for larvae of the insect family Scarabaeidae (Coleoptera). It forms parasporal crystals upon sporulation. The gene cry18Aa coding for the parasporal crystal protein and an upstream open reading frame, orf1, were previously isolated from B.popilliae. Here we report an analysis of cry18Aa transcription in Bacillusthuringiensis. The only transcriptional start site of cry18Aa was found 29 bp upstream of the open reading frame orf1, suggesting that orf1 and cry18Aa are transcribed as an operon. lacZ fusion to the cry18Aa promoter was used to follow the time‐course of cry18Aa transcription in wild type B.thuringiensis and in various B.thuringiensis sporulation‐deficient mutants (spo0A, sigE or sigK). In wild type B.thuringiensis, the cry18Aa promoter was activated 2 h after the end of exponential growth and the expression lasted to the late sporulation phase. The results of promoter activity in Spo+ or Spo– backgrounds together with the results of primer extension experiments suggest that the transcription from this promoter can be driven by both σE and σK types of RNA polymerase at a single start site. The promoter region of cry18Aa operon fits the consensus sequences of both σE and σK dependent promoters of Bacillus.

Endophthalmitis is a devastating infection that can cause blindness. Over half of Bacillusendophthalmitis cases result in significant loss of useful vision. Bacillusproduces many virulence factors that may contribute to retinal damage and robust inflammation. We analyzed Bacillusimmune inhibitor A (InhA) metalloproteases in the context of this disease, hypothesizing that InhAs contribute to Bacillusintraocular virulence and inflammation. We analyzed phenotypes and infectivity of wild type (WT), InhA1-deficient (ΔinhA1), InhA2-deficient (ΔinhA2), or InhA1, A2, and A3-deficient (ΔinhA1-3) Bacillus thuringiensis. In vitroanalysis of growth, proteolysis, and cytotoxicity were compared. WT and InhA mutants were similarly cytotoxic to retinal cells. Mutants ΔinhA1and ΔinhA2entered log phase growth earlier than WT. Proteolysis by the ΔinhA1-3mutant was decreased, but this strain grew similar to WT in vitro. Experimental endophthalmitis was initiated by intravitreally infecting C57BL/6J mice with 200 CFU of B. thuringiensisWT or InhA mutants. Eyes were analyzed for intraocular Bacillusand myeloperoxidase concentrations, retinal function loss, and gross histological changes. Eyes infected with ΔinhA1or ΔinhA2strains contained greater numbers of bacteria than eyes infected with WT throughout the infection course. Eyes infected with single mutants had inflammation and retinal function loss similar to eyes infected with WT strain. Eyes infected with ΔinhA1-3cleared the infection. RT-PCR results suggested that there may be compensatory expression of the other InhAs in the single InhA mutant. These results indicate that together, the InhA metalloproteases contribute to the severity of infection and inflammation in Bacillusendophthalmitis.

Quorum sensing describes the ability of microbes to alter gene regulation according to their local population size. Some successful theory suggests that this is a form of cooperation, namely, investment in shared products is only worthwhile if there are sufficient bacteria making the same product. This theory can explain the genetic diversity in these signaling systems in Gram-positive bacteria, such as Bacillusand Staphylococcussp. The possible advantages gained by rare genotypes (which can exploit the products of their more common neighbors) could explain why different genotypes can coexist. We show that while these social interactions can occur in simple laboratory experiments, they do not occur in naturalistic infections using an invertebrate pathogen, Bacillus thuringiensis. Instead, our results suggest that different genotypes are adapted to differently sized hosts. Overall, social models are not easily applied to this system, implying that a different explanation for this form of quorum sensing is required.

ABSTRACTBacillus thuringiensis(Bt) is armed to complete a full cycle in its insect host. During infection, virulence factors are expressed under the control of the quorum sensor PlcR to kill the host. After the host's death, the quorum sensor NprR controls a necrotrophic lifestyle, allowing the vegetative cells to use the insect cadaver as a bioincubator and to survive. Only a part of the Bt population sporulates in the insect cadaver, and the precise composition of the whole population and its evolution over time are unknown. Using fluorescent reporters to record gene expression at the single-cell level, we have determined the differentiation course of a Bt population and explored the lineage existing among virulent, necrotrophic, and sporulating cells. The dynamics of cell differentiation were monitored during growth in homogenized medium, biofilm formation, and colonization of insect larvae. We demonstrated that in the insect host and in planktonic culture in rich medium, the virulence, necrotrophism, and sporulation regulators are successively activated in the same cell. In contrast, in biofilms, activation of PlcR is dispensable for NprR activation and we observed a greater heterogeneity than under the other two growth conditions. We also showed that sporulating cells arise almost exclusively from necrotrophic cells. In biofilm and in the insect cadaver, we identified an as-yet-uncharacterized category of cells that do not express any of the reporters used. Overall, we showed that PlcR, NprR, and Spo0A act as interconnected integrators to allow finely tuned adaptation of the pathogen to its environment.IMPORTANCEBt is an entomopathogen found ubiquitously in the environment and is a widely used biopesticide. Studies performed at the population level suggest that the infection process of Bt includes three successive steps (virulence, necrotrophism, and sporulation) controlled by different regulators. This study aimed to determine how these phenotypes are activated at the cellular level and if they are switched on in all cells. We used an insect model of infection and biofilms to decipher the cellular differentiation of this bacterium under naturalistic conditions. Our study reveals the connection and lineage existing among virulent, necrotrophic, and sporulating cells. It also shows that the complex conditions encountered in biofilms and during infection generate great heterogeneity inside the population, which might reflect a bet-hedging strategy to ameliorate survival. These data generate new insights into the role of regulatory networks in the adaptation of a pathogen to its host.

Laboratory selection of a strain of Spodoptera littoralis Boisduval with spore-crystal preparations of the Bacillus thuringiensis CryIC toxin for 14 generations resulted in resistance ratios from 10- to >500-fold resistance by using 2 different methods of selection. Partial cross-resistance was exhibited to CryID, CryIE, CryIAb toxins and to the parental strain B. thuringiensis aizawai 7.29. No cross-resistance occurred to CryIF. Resistance declined from >500 times the resistance ratio to >74 times after 1 generation without selection pressure, remained ≈100 times after 3–6 generations and fell to 11 times after 8 generations. Sex ratio remained the same over selection period. Pupal weights of the selected strain varied, but we observed no trend in these differences. Developmental time increased from 5 to 6 d for treated, resistant insects.