Chitinolytic activity of phylogenetically diverse Bacillus cereus sensu lato from natural environments.

Bacillus cereus s.l., Gram-positive endospore-forming bacilli, persist ubiquitously in different natural habitats and play various ecological roles. Nevertheless, although chitin is one of the most abundant polymer on Earth, the study of the ability of B. cereus s.l. to hydrolyze this polymer were limited to individual B. cereus and B. thuringiensis strains only. Thus, to fill this gap in this research we focused on (i) the linkage between the capability to chitin degradation and the phylogenetic relatedness of B. cereus s.l. strains, and (ii) the genetic background of chitinolytic properties of these bacilli. Our results showed that chitin degradation is common among the B. cereus group members, yet strains clustered into particular phylogenetic groups differ in their chitinolytic capacity. Separate clustering of chitinolytic and non-chitinolytic strains in the phylogenetic tree indicates the ecotypic structure of these isolates. Two proteins belonging to subfamily A (ChiA) and subfamily B (ChiB) of the glycoside hydrolase GH18 family exhibited simultaneous chitobiosidase and endochitinase activities, and are responsible for chitin utilization by environmental B. cereus s.l. isolates.

Antimicrobial activity of broccoli (Brassica oleracea var. italica) cultivar Avenger against pathogenic bacteria, phytopathogenic filamentous fungi and yeast.

AIMS: The objective of this study was to show whether the edible part of broccoli has antibacterial and antifungal activity against micro-organism of importance in human health and vegetable spoilage, and to test if this effect was partially due to antimicrobial peptides (AMPs). METHODS AND RESULTS: Crude extracts were obtained from florets and stems of broccoli cultivar Avenger and the inhibitory effect was demonstrated against pathogenic bacteria (Bacillus cereus, Staphylococcus xylosus, Staphylococcus aureus, Shigella flexneri, Shigella sonnei, Proteus vulgaris), phytopathogenic fungi (Colletotrichum gloeosporioides, Asperigillus niger) and yeasts (Candida albicans and Rhodotorula sp.). It was shown that samples treated with proteolytic enzymes had a reduction of approximately 60% in antibacterial activity against Staph. xylosus, suggesting that proteinaceous compounds might play a role in the inhibitory effect. Antimicrobial components in crude extracts were thermoresistant and the highest activity was observed under acidic conditions. It was shown that antifungal activity of broccoli's crude extracts might not be attributed to chitinases. CONCLUSIONS: Organic broccoli cultivar Avenger has antimicrobial activity against pathogenic bacteria, yeast and phytophatogenic fungi. Data suggest that this effect is partially due to AMPs. SIGNIFICANCE AND IMPACT OF THE STUDY: Broccoli's crude extracts have activity not only against pathogenic bacteria but also against phytophatogenic fungi of importance in agriculture. We suggest for first time that the inhibitory effect is probably due to AMPs.

Potential use of Bacillus thuringiensis bacteriocins to control antibiotic-resistant bacteria associated with mastitis in dairy goats.

Mastitis caused by microbial infections in dairy goats reduces milk yield, modifies milk composition, and potentially contributes to morbidity in herds and consumers of dairy products. Microorganisms associated with mastitis in dairy goats are commonly controlled with antibiotics, but it is known that continued use of these chemical agents promotes antibiotic resistance among bacterial populations. Recently, it has been shown that bacteriocins of Bacillus thuringiensis inhibit growth of food-borne pathogens and also bacteria associated with bovine mastitis. However, there is no report on their ability to inhibit microorganisms linked to mastitis in dairy goats. In this study, using 16S rDNA and ITS regions of rDNA, we identified nine bacterial isolates and an encapsulated yeast associated with mastitis in dairy goats. Enterococcus durans, Brevibacillus sp., and Staphylococcus epidermidis 2 were resistant to, respectively, 75, ~67, ~42, and ~42 % of the antibiotics screened. In addition, 60 % of the bacterial isolates were resistant to penicillin, ampicillin, vancomycin, and dicloxacillin. Importantly, 60 % of the isolates were inhibited by the bacteriocins, but S. epidermidis 1, Enterobacter sp., Escherichia vulneris, and Cryptococcus neoformans were not susceptible to these antimicrobial peptides. Using Brevibacillus sp. and Staphylococcus chromogenes as indicator bacteria, we show that peptides of ~10 kDa that correspond to the molecular mass of bacteriocins used in this study are responsible for the inhibitory activity. Our results demonstrate that multiple antibiotic-resistant bacteria associated with subclinical mastitis in dairy goats from Guanajuato, Mexico, are susceptible to bacteriocins produced by B. thuringiensis.

Bacillus thuringiensis subsp. israelensis producing endochitinase ChiA74Δsp inclusions and its improved activity against Aedes aegypti.

AIMS: The objective of this study was to produce stable inclusions of chitinase ChiA74Δsp in Bacillus thuringiensis subsp. israelensis (Bti) and to assay its insecticidal activity against Aedes aegypti larvae. METHODS AND RESULTS: Bti was transformed with chiA74Δsp regulated by its own promoter or by the strong chimeric cytAp/STAB-SD promoter system to generate two recombinant Bti strains. These recombinants produced their native parasporal bodies composed of Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa and ChiA74Δsp inclusions, and showed a approx. threefold increase in both endochitinase activity and viable spore count when compared with the parental strain. Both recombinants were approximately twofold more toxic (LC50s 8·02, 9·6 ng ml(-1) ) than parental Bti (19·8 ng ml(-1) ) against 4(th) instars of A. aegypti larvae. CONCLUSIONS: ChiA74Δsp inclusions, together with the insecticidal crystals and spores of Bti increased the toxicity against A. aegypti larvae by at least twofold. SIGNIFICANCE AND IMPACT OF THE STUDY: We report for the first time the engineering of Bti to produce spore-parasporal body-ChiA74∆sp inclusions in the same sporangium, which are released together following autolysis. Our work lays a foundation for engineering Bti to produce more efficacious combinations of Cry4Aa, Cry4Ba, Cry11Aa, Cyt1Aa and chitinase inclusions.

Detection of ß-exotoxin synthesis in Bacillus thuringiensis using an easy bioassay with the nematode Caenorhabditis elegans.

UNLABELLED: The insecticidal activity of Bacillus thuringiensis is owing to the action of Cry and Cyt proteins. In addition to the synthesis of insecticidal proteins, some strains are able to synthesize ß-exotoxin, which is highly toxic to humans. In this regard, it is very important to have a simple method to detect ß-exotoxin to avoid the commercial production of this type of strains. In this work, we developed a simple and fast method, using the nematode Caenorhabditis elegans to detect indirectly the synthesis of ß-exotoxin by B. thuringiensis strain. Using this assay, we detected that ~60% of Mexican native strains (i.e. LBIT-471, 491, 492, 497, 507, 511, 515, 536 and 537) were toxic to the nematode (44-97% mortalities) and their ß-exotoxin (ßEx(+) ) production, including a positive control (NRD-12), was confirmed by HPLC. In addition, the negative controls (ßEx(-) ) LBIT-436 (HD-1) and LBIT-438 and also the native strains LBIT-499, 500, 521, 522, 533 and 542, did not show a detrimental effect against nematodes larvae, neither the synthesis of ß-exotoxin as determined by HPLC. Finally, we did not find a correlation between B. thuringiensis strains with similar plasmid patterns and the ß-exotoxin production. SIGNIFICANCE AND IMPACT OF THE STUDY: In this work, we implemented a qualitative and fast bioassay using the nematode Caenorhabditis elegans to detect the production of ß-exotoxin in different strains of Bacillus thuringiensis. We show that this assay is useful to detect ß-exotoxin in B. thuringiensis with high reliability, helping to discriminate strains that could not be used as bioinsecticides because of their putative risk to humans. Data show that qualitative bioassay with nematodes is a potential alternative to fly larvae bioassays, and correlated with the determination of ß-exotoxin by HPLC.

Correlation between Pr1 and Pr2 gene content and virulence in Metarhizium anisopliae strains.

Metarhizium anisopliae is a widely studied model to understand the virulence factors that participate in pathogenicity. Proteases such as subtilisin-like enzymes (Pr1) and trypsin-like enzymes (Pr2) are considered important factors for insect cuticle degradation. In four M. anisopliae strains (798, 6342, 6345, and 6347), the presence of pr1 and pr2 genes, as well as the enzymatic activity of these genes, was correlated with their virulence against two different insect pests. The 11 pr1 genes (A, B, C, D, E, F, G, H, I, J, and K) and pr2 gene were found in all strains. The activity of individual Pr1 and Pr2 proteases exhibited variation in time (24, 48, 72, and 96 h) and in the presence or absence of chitin as the inductor. The highest Pr1 enzymatic activity was shown by strain 798 at 48 h with chitin. The highest Pr2 enzymatic activity was exhibited by the 6342 and 6347 strains, both grown with chitin at 24 and 48 h, respectively. Highest mortality on S. exigua was caused by strain 6342 at 48 h, and strains 6342, 6345, and 6347 caused the highest mortality 7 days later. Mortality on Prosapia reached 30% without variation. The presence of subtilisin and trypsin genes and the activity of these proteases in M. anisopliae strains cannot be associated with the virulence against the two insect pests. Probably, subtilisin and trypsin enzyme production is not a vital factor for pathogenicity, but its contribution is important to the pathogenicity process.

Isolation of a new Mexican strain of Bacillus subtilis with antifungal and antibacterial activities.

Although several strains of B. subtilis with antifungal activity have been isolated worldwide, to date there are no published reports regarding the isolation of a native B. subtilis strain from strawberry plants in Mexico. A native bacterium (Bacillus subtilis 21) demonstrated in vitro antagonistic activity against different plant pathogenic fungi. Under greenhouse conditions, it was shown that plants infected with Rhizoctonia solani and Fusarium verticillioides and treated with B. subtilis 21 produced augment in the number of leaves per plant and an increment in the length of healthy leaves in comparison with untreated plants. In addition, B. subtilis 21 showed activity against pathogenic bacteria. Secreted proteins by B. subtilis 21 were studied, detecting the presence of proteases and bacteriocin-like inhibitor substances that could be implicated in its antagonistic activity. Chitinases and zwittermicin production could not be detected. Then, B. subtilis 21 could potentially be used to control phytopathogenic fungi that infect strawberry plants.

Generation of chitin-derived oligosaccharides toxic to pathogenic bacteria using ChiA74, an endochitinase native to Bacillus thuringiensis.

AIMS: To demonstrate that an endochitinase (ChiA74) native to Bacillus thuringiensis can be used to generate chitin-derived oligosaccharides (OGS) with antibacterial activity against a number of aetiological agents of disease, including bacteria that cause diarrhoeal and emetic syndromes in humans. METHODS AND RESULTS: The intact chiA74 with its cis elements was cloned into high and moderately high copy number Escherichia coli expression vectors. Functionally secreted ChiA74 was produced, and the endochitinase cleaved substrate colloidal chitin to produce OGS with 3, 5 and 6 degrees of polymerization. The enzyme was active for an extended period of incubation (24 h), but its activity showed a decrement of 73% and 87%, respectively, after 24 h of incubation at 37 and 55 degrees C. OGS showed inhibitory activity against Bacillus cereus, Listeria inoccua, E. coli, Staphylococcus xylosus, Salmonella species, Staphylococcus aureus, Pseudomona aeruginosa, Shigella flexneri, and Proteus vulgaris. CONCLUSIONS: Endochitinase ChiA74 is able to stably maintain hydrolytic activity during prolonged incubation in a mix reaction with chitin to produce bioactive OGS with inhibitory activity against important food-borne pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study showing that an endochitinase (ChiA74) native of the most important bioinsecticide used worldwide (B. thuringiensis), but here produced in E. coli, is able to generate chitin-derived OGS with antibacterial activity against clinically significant food-borne pathogenic bacteria.

Enhanced synthesis and antimicrobial activities of bacteriocins produced by Mexican strains of Bacillus thuringiensis.

Recently, we reported the synthesis of five bacteriocin-like inhibitor substances (Bt-BLIS: morricin 269, kurstacin 287, kenyacin 404, entomocin 420, and tolworthcin 524) by Mexican strains of Bacillus thuringiensis. Here we show that, collectively, these Bt-BLIS have a moderate to broad spectrum of antibacterial activity, being toxic to clinically significant against Gram-positive and Gram-negative bacteria, including common etiological agents of human diseases, such as strep throat and scarlet fever, septicemia, pneumonia, urinary tract infection, and emetic and gastrointestinal syndromes. Although synthesis of the five Bt-BLIS was independent of the presence of a target inducing bacterium, we demonstrated for the first time that a proteinaceous component(s) secreted by, or liberated by proteolytic cleavage of Bacillus cereus 183 following treatment with proteinase K, enhanced Bt-BLIS synthesis.

Generation of antibacterial oligosaccharides derived from chitin using heterologous endochitinase synthesized in Escherichia coli.

AIMS: To synthesize two heterologous endochitinases in Escherichia coli and demonstrate their potential for applied use in generating antibacterial chitin-derived oligosaccharides (OGS). METHODS AND RESULTS: Heterologous endochitinase genes, chiA Nima and chiA74, were expressed in E. coli. Endochitinases were secreted by the E. coli export machinery and by approximately 20 h maximal chitinolytic activity was observed. The highest chitinolytic activity was observed with ChiA Nima, which produced antibacterial OGS with activities against Enterobacter cloacae, Escherichia coli, Staphylococcus aureus and S. xylosus. CONCLUSIONS: It was shown that the export machinery of E. coli is well suited for the secretion of bioactive ChiA74 and ChiA Nima endochitinases, and that the latter can generate antibacterial OGS. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study suggests that it is feasible to synthesize endochitinases ChiA Nima and ChiA74 codified by E. coli and mass-produce these enzymes in culture supernatants. As signal peptides in native ChiA Nima and ChiA74 were recognized by the protein export molecular apparatus in E. coli, these short peptides could be included as signal sequences for transport in E. coli of other proteins with applied value. This is the first report suggesting that ChiA Nima can be used to produce OGS to control food-borne pathogenic bacteria.

Chitinases from Serratia marcescens Nima.

Chitinolytic activity of Serratia marcescens Nima (130 U ml(-1)) was up to 43 times higher than those produced by other S. marcescens strains. This strain synthesized an endochitinase (Chi-60), an exochitinase (Chi-50) and a novel N-acetylglucosaminidase. This latter showed two putative isoforms (Chi-180.5 and Chi-180.8) with isoelectric points of 5 and 8.1, respectively.