Identification of Quorum-Quenching N-Acyl Homoserine Lactonases from Bacillus Species

A range of gram-negative bacterial species use N-acyl homoserine lactone (AHL) molecules as quorum-sensing signals to regulate different biological functions, including production of virulence factors. AHL is also known as an autoinducer. An autoinducer inactivation gene, aiiA, coding for an AHL lactonase, was cloned from a bacterial isolate, Bacillus sp. strain 240B1. Here we report identification of more than 20 bacterial isolates capable of enzymatic inactivation of AHLs from different sources. Eight isolates showing strong AHL-inactivating enzyme activity were selected for a preliminary taxonomic analysis. Morphological phenotypes and 16S ribosomal DNA sequence analysis indicated that these isolates probably belong to the species Bacillus thuringiensis. Enzymatic analysis with known Bacillus strains confirmed that all of the strains of B. thuringiensis and the closely related species B. cereus and B. mycoides tested produced AHL-inactivating enzymes but B. fusiformis and B. sphaericus strains did not. Nine genes coding for AHL inactivation were cloned either by functional cloning or by a PCR procedure from selected bacterial isolates and strains. Sequence comparison of the gene products and motif analysis showed that the gene products belong to the same family of AHL lactonases.     

带cry3Aa启动子的aiiA基因在苏云金芽胞杆菌中的表达

N 乙酰高丝氨酸内酯 (N acyl homoserinelactones,AHLs) ,是一类数量感知 (Quorum sensing)系统中的信号分子 ,它参与诱导调控许多植物病原菌致病基因的表达。苏云金芽胞杆菌的AiiA蛋白能降解这类AHLs分子 ,进而可减弱病原菌致病基因表达产生的病害。苏云金芽胞杆菌杀虫晶体蛋白基因cry3Aa的启动子是一种不依赖芽胞形成的启动子 ,它相对于其它cry类基因的启动子有启动基因转录时间早 ,转录时间长的优点。通过重叠延伸PCR ,用杀虫晶体蛋白基因cry3Aa启动子替换编码AiiA蛋白的基因aiiA自身的启动子 ,构建了融合基因pro3A aiiA。将融合基因装入穿梭载体pHT3 0 4的BamHI SphI位点 ,得到重组质粒pBMB686并转化苏云金芽胞杆菌无晶体突变株BMB171,重组菌株BMB686的AiiA蛋白表达量在各个生长时期均高于对照菌株 ,对AHLs分子的降解活性和对胡萝卜软腐欧文氏菌感染马铃薯产生病害的抑制能力也明显优于对照菌株     

Fluorescent amplified fragment length polymorphism analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis isolates

DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. Department of Agriculture collection. Twenty-four diverse B. anthracis isolates were also included. Phylogenetic analysis of AFLP data revealed extensive diversity within B. thuringiensis and B. cereus compared to the monomorphic nature of B. anthracis. All of the B. anthracis strains were more closely related to each other than to any other Bacillus isolate, while B. cereus and B. thuringiensis strains populated the entire tree. Ten distinct branches were defined, with many branches containing both B. cereus and B. thuringiensis isolates. A single branch contained all the B. anthracis isolates plus an unusual B. thuringiensis isolate that is pathogenic in mice. In contrast, B. thuringiensis subsp. kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B. anthracis isolates. The interspersion of B. cereus and B. thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes. B. thuringiensis isolates of a particular subspecies tended to cluster together.     

Insecticidal Bacillus thuringiensis silences Erwinia carotovora virulence by a new form of microbial antagonism, signal interference

It is commonly known that bacteria may produce antibiotics to interfere with the normal biological functions of their competitors in order to gain competitive advantages. Here we report that Bacillus thuringiensis suppressed the quorum-sensing-dependent virulence of plant pathogen Erwinia carotovora through a new form of microbial antagonism, signal interference. E. carotovora produces and responds to acyl-homoserine lactone (AHL) quorum-sensing signals to regulate antibiotic production and expression of virulence genes, whereas B. thuringiensis strains possess AHL-lactonase, which is a potent AHL-degrading enzyme. B. thuringiensis did not seem to interfere with the normal growth of E. carotovora; rather, it abolished the accumulation of AHL signal when they were cocultured. In planta, B. thuringiensis significantly decreased the incidence of E. carotovora infection and symptom development of potato soft rot caused by the pathogen. The biocontrol efficiency is correlated with the ability of bacterial strains to produce AHL-lactonase. While all the seven AHL-lactonase-producing B. thuringiensis strains provided significant protection against E. carotovora infection, Bacillus fusiformis and Escherichia coli strains that do not process AHL-degradation enzyme showed little effect in biocontrol. Mutation of aiiA, the gene encoding AHL-lactonase in B. thuringiensis, resulted in a substantial decrease in biocontrol efficacy. These results suggest that signal interference mechanisms existing in natural ecosystems could be explored as a new version of antagonism for prevention of bacterial infections.     

Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis

Gram-negative bacteria can communicate with each other by N-acyl homoserine lactones (AHLs), which are quorum-sensing autoinducers. Recently, the aiiA gene (encoding an enzyme catalyzing the degradation of AHL) has been cloned from Bacillus sp. strain 240B1. During investigations in the course of the ongoing Bacillus thuringiensis subsp. morrisoni genome project, an aiiA homologue gene in the genome sequence was found. These results led to consideration of the possibility of the widespread existence of the gene in B. thuringiensis. aiiA homologue genes were found in 16 subspecies of B. thuringiensis, and their sequences were determined. Comparison of the Bacillus sp. strain 240B1 aiiA gene with the B. thuringiensis aiiA homologue genes showed high homologies of 89 to 95% and 90 to 96% in the nucleotide sequence and deduced amino acid sequence, respectively. Among the subspecies of B. thuringiensis having an aiiA gene, the subspecies aizawai, galleriae, kurstaki, kyushuensis, ostriniae, and subtoxicus were shown to degrade AHL. It was observed that recombinant Escherichia coli producing AiiA proteins also had AHL-degrading activity and could also attenuate the plant pathogenicity of Erwinia carotovora. These results indicate that insecticidal B. thuringiensis strains might have potential to compete with gram-negative bacteria in natural ecosystems by autoinducer-degrading activity.     

Genetic differentiation between sympatric populations of Bacillus cereus and Bacillus thuringiensis

Little is known about genetic exchanges in natural populations of bacteria of the spore-forming Bacillus cereus group, because no population genetics studies have been performed with local sympatric populations. We isolated strains of Bacillus thuringiensis and B. cereus from small samples of soil collected at the same time from two separate geographical sites, one within the forest and the other at the edge of the forest. A total of 100 B. cereus and 98 B. thuringiensis strains were isolated and characterized by electrophoresis to determine allelic composition at nine enzymatic loci. We observed genetic differentiation between populations of B. cereus and B. thuringiensis. Populations of a given Bacillus species--B. thuringiensis or B. cereus--were genetically more similar to each other than to populations of the other Bacillus species. Hemolytic activity provided further evidence of this genetic divergence, which remained evident even if putative clones were removed from the data set. Our results suggest that the rate of gene flow was higher between strains of the same species, but that exchanges between B. cereus and B. thuringiensis were nonetheless possible. Linkage disequilibrium analysis revealed sufficient recombination for B. cereus populations to be considered panmictic units. In B. thuringiensis, the balance between clonal proliferation and recombination seemed to depend on location. Overall, our data indicate that it is not important for risk assessment purposes to determine whether B. cereus and B. thuringiensis belong to a single or two species. Assessment of the biosafety of pest control based on B. thuringiensis requires evaluation of the extent of genetic exchange between strains in realistic natural conditions.     

Unusually High Frequency of Genes Encoding Vegetative Insecticidal Proteins in an Australian <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Collection

Of 188 Australian Bacillus thuringiensis strains screened for genes encoding soluble insecticidal proteins by polymerase chain reaction/restriction-length fragment polymorphism (RFLP) analysis, 87% showed the presence of such genes. Although 135 isolates (72%) produced an RFLP pattern identical to that expected for vip3A genes, 29 isolates possessed a novel vip-like gene. The novel vip-like gene was cloned from B. thuringiensis isolate C81, and sequence analysis demonstrated that it was 94% identical to the vip3Ba1 gene. The new gene was designated vip3Bb2. Cell-free supernatants from both the B. thuringiensis strain C81 and from Escherichia coli expressing the Vip3Bb2 protein were toxic for the cotton bollworm, Helicoverpa armigera.     

Diversity of cry genes and genetic characterization of Bacillus thuringiensis isolated from Brazil

Two hundred and eighteen Bacillus thuringiensis isolates from Brazil were characterized by the presence of crystal protein genes by PCR with primers specific to different cry and cyt genes. Among these isolates, 95 were selected according to their geographic origin for genetic characterization with the 16S rRNA gene, RAPD, and plasmid profile. Isolates containing cry1 genes were the most abundant (48%) followed by the cry11 and cyt (7%) and cry8 genes (2%). Finally, 40.3% of the isolates did not produce any PCR product. The plasmid profile and RAPD analysis showed a remarkable diversity among the isolates of B. thuringiensis not observed in the 16S rRNA gene. These results suggest that the genetic diversity of B. thuringiensis species results from the influence of different ecological factors and spatial separation between strains generated by the conquest of different habitats.     

Identification and characterization of a previously undescribed cyt gene in Bacillus thuringiensis subsp. israelensis.

Mosquitocidal Bacillus thuringiensis strains show as a common feature the presence of toxic proteins with cytolytic and hemolytic activities, Cyt1Aa1 being the characteristic cytolytic toxin of Bacillus thuringiensis subsp. israelensis. We have detected the presence of another cyt gene in this subspecies, highly homologous to cyt2An1, coding for the 29-kDa cytolytic toxin from B. thuringiensis subsp. kyushuensis. This gene, designated cyt2Ba1, maps upstream of cry4B coding for the 130-kDa crystal toxin, on the 72-MDa plasmid of strain 4Q2-72. Sequence analysis revealed, as a remarkable feature, a 5' mRNA stabilizing region similar to those described for some cry genes. PCR amplification and Southern analysis confirmed the presence of this gene in other mosquitocidal subspecies. Interestingly, anticoleopteran B. thuringiensis subsp. tenebrionis belonging to the morrisoni serovar also showed this gene. On the other hand, negative results were obtained with the anti-lepidopteran strains B. thuringiensis subsp. kurstaki HD-1 and subsp. aizawai HD-137. Western analysis failed to reveal Cyt2A-related polypeptides in B. thuringiensis subsp. israelensis 4Q2-72. However, B. thuringiensis subsp. israelensis 1884 and B. thuringiensis subsp. tenebrionis did show cross-reactive products, although in very small amounts.     

Arbitrary primer polymerase chain reaction, a powerful method to identify Bacillus thuringiensis serovars and strains.

Arbitrary primer polymerase chain reaction technology has been applied to the identification of commercial strains of Bacillus thuringiensis by using total DNAs extracted from single bacterial colonies as templates. Characteristic DNA banding patterns can be readily and reproducibly obtained by agarose gel electrophoresis. This method has been used to distinguish commercial products containing B. thuringiensis serovar kurstaki (3a3b). When a single primer was used this method was capable of producing discriminating DNA fingerprints for 33 known serovars. Differentiation from the closely related species Bacillus cereus is also readily achieved. This technique should prove to be a powerful tool for identification and discrimination of individual B. thuringiensis strains.     

Occurrence and significance of Bacillus cereus and Bacillus thuringiensis in ready-to-eat food

Among 48,901 samples of ready-to-eat food products at the Danish retail market, 0.5% had counts of Bacillus cereus-like bacteria above 10(4) cfu g(-1). The high counts were most frequently found in starchy, cooked products, but also in fresh cucumbers and tomatoes. Forty randomly selected strains had at least one gene or component involved in human diarrhoeal disease, while emetic toxin was related to only one B. cereus strain. A new observation was that 31 out of the 40 randomly selected B. cereus-like strains could be classified as Bacillus thuringiensis due to crystal production and/or content of cry genes. Thus, a large proportion of the B. cereus-like organisms present in food may belong to B. thuringiensis.     

Population structure and evolution of the Bacillus cereus group

Representative strains of the Bacillus cereus group of bacteria, including Bacillus anthracis (11 isolates), B. cereus (38 isolates), Bacillus mycoides (1 isolate), Bacillus thuringiensis (53 isolates from 17 serovars), and Bacillus weihenstephanensis (2 isolates) were assigned to 59 sequence types (STs) derived from the nucleotide sequences of seven alleles, glpF, gmk, ilvD, pta, pur, pycA, and tpi. Comparisons of the maximum likelihood (ML) tree of the concatenated sequences with individual gene trees showed more congruence than expected by chance, indicating a generally clonal structure to the population. The STs followed two major lines of descent. Clade 1 comprised B. anthracis strains, numerous B. cereus strains, and rare B. thuringiensis strains, while clade 2 included the majority of the B. thuringiensis strains together with some B. cereus strains. Other species were allocated to a third, heterogeneous clade. The ML trees and split decomposition analysis were used to assign STs to eight lineages within clades 1 and 2. These lineages were defined by bootstrap analysis and by a preponderance of fixed differences over shared polymorphisms among the STs. Lineages were named with reference to existing designations: Anthracis, Cereus I, Cereus II, Cereus III, Kurstaki, Sotto, Thuringiensis, and Tolworthi. Strains from some B. thuringiensis serovars were wholly or largely assigned to a single ST, for example, serovar aizawai isolates were assigned to ST-15, serovar kenyae isolates were assigned to ST-13, and serovar tolworthi isolates were assigned to ST-23, while other serovars, such as serovar canadensis, were genetically heterogeneous. We suggest a revision of the nomenclature in which the lineage and clone are recognized through name and ST designations in accordance with the clonal structure of the population.     

The clonal structure of Bacillus thuringiensis isolates from north-east Poland does not correlate with their cry gene diversity

The genetic relationship among 103 natural Bacillus thuringiensis isolates was investigated on the basis of polymerase chain reaction amplification of their specific crystal (cry) protein type genes and chromosomal DNA profiling by pulsed-field gel electrophoresis (PFGE). The strains were recovered from the intestines of small wild rodents and insectivores from the Biebrza National Park and the Lomza Landscape Park of the Narew River Valley in north-east Poland. The percentage of B. thuringiensis strains harbouring genes coding for toxins active against Lepidoptera (cry1, cry2, cry9) was very high (64%) compared with that of Diptera-specific strains (cry4, 14%). No strain with cry genes coding for proteins directed against coleopteran larvae and nematodes was found. After digestion with NotI and AscI, only nine PFGE pulsotypes were observed among all isolates, indicating a clonal structure for the B. thuringiensis population from NE Poland. Interestingly, no correlation was observed between the DNA pulsotype strains and their crystal gene content and diversity. These results therefore emphasize the importance of cry gene horizontal transfer occurring among natural isolates of B. thuringiensis.     

Natural isolates of Bacillus thuringiensis display genetic and psychrotrophic properties characteristic of Bacillus weihenstephanensis

Aim:  To determine the potential of Bacillus thuringiensis , known primarily for its entomopathogenicity, to be a psychrotolerant contaminant of stored products.
Methods and Results:  We determined the genetic properties and diversity of cold-adapted isolates of B. thuringiensis based on (i) the presence of cspA , a genetic determinant that confers psychrotolerance in Bacillus weihenstephanensis , (ii) 16S rRNA genes, and (iii) pulse-field gel electrophoretic (PFGE) genome profiles. We assessed the pathogenic potential of these isolates based on whether they harboured various combinations of known toxigenic-associated determinants ( nheA , hblA , cytK ). Of 36 nonclonal B. thuringiensis cultured from soil and milk, 21 harboured cspA , and of these, 16 (76%) were psychrotolerant and possessed genetic signatures typical of psychrotrophic Bacillus species. The majority of psychrotolerant isolates contained various combinations of nheA , hblA , and cytK .
Conclusion:  Our results show that natural isolates of psychrotolerant B. thuringiensis occur in soil and milk, and suggest that psychrotolerance is determined by cspA .
Significance and Impact of the study:  The presence of cspA in combination with nheA , hblA , and cytK could be of concern if commercial products are contaminated with strains that harbour these determinants.     

Virulent spores of Bacillus anthracis and other Bacillus species deposited on solid surfaces have similar sensitivity to chemical decontaminants

AIMS: To compare the relative sensitivity of Bacillus anthracis and spores of other Bacillus spp. deposited on different solid surfaces to inactivation by liquid chemical disinfecting agents. METHODS AND RESULTS: We prepared under similar conditions spores from five different virulent and three attenuated strains of B. anthracis, as well as spores of Bacillus subtilis, Bacillus atrophaeus (previously known as Bacillus globigii), Bacillus cereus, Bacillus thuringiensis and Bacillus megaterium. As spore-surface interactions may bias inactivation experiments, we evaluated the relative binding of different spores to carrier materials. The survival of spores deposited on glass, metallic or polymeric surfaces were quantitatively measured by ASTM standard method E-2414-05 which recovers spores from surfaces by increasing stringency. The number of spores inactivated by each decontaminant was similar and generally within 1 log among the 12 different Bacillus strains tested. This similarity among Bacillus strains and species was observed through a range of sporicidal efficacy on spores deposited on painted metal, polymeric rubber or glass. CONCLUSIONS: The data obtained indicate that the sensitivity of common simulants (B. atrophaeus and B. subtilis), as well as spores of B. cereus, B. thuringiensis, and B. megaterium, to inactivation by products that contain either: peroxide, chlorine or oxidants is similar to that shown by spores from all eight B. anthracis strains studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The comparative results of the present study suggest that decontamination and sterilization data obtained with simulants can be safely extrapolated to virulent spores of B. anthracis. Thus, valid conclusions on sporicidal efficacy could be drawn from safer and less costly experiments employing non-pathogenic spore simulants.     

Screening of Bacillus thuringiensis serotypes by polymerase chain reaction (PCR) for insecticidal crystal genes toxic against coffee berry borer

Using PCR,257 isolates of Bacillus thuringiensis(Bt) were screened for cry-type genes. Of 257 isolates/strains, 60 isolates were identified as cry7/8, 10 isolates as cry3 and 36 isolates as cry 1I. One specific strain of B. thuringiensis (sumiyoshiensis; T03B 001) was investigated for the presence of cry7 and cry8 genes. Genes Cry7 and cry8 were first detected in this strain using family primers prior to analysis by exclusion polymerase chain reaction (E-PCR) using specific type primers. E-PCR conducted with the above said primers led to the identification by agarose gel electrophoresis of a remaining 1.5 Kb family band indicating a potentially novel gene. This PCR product, (1.5 Kb), was purified from the gel and cloned in pGEM-T Easy vector. Twenty recombinant colonies bearing 1.5 Kb insert were identified and three randomly selected representatives of the group, clones 7, 8 and 10, were sequenced and compared to all cry7 and cry8 sequences available from Gene Bank. Alignments with available DNA and protein sequences showed that all these clones contained a gene related to cry8Aa1. Analysis using protein sequence alignment showed that the sequence from clone 7 differed from the closest relative, known under the new nomenclature as cry 8Aa1, by 44%. The crystal proteins from B. thuringiensis sumiyoshiensis (T03B 001) was toxic to coffee berry borer larvae.     

Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene

A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis delta-endotoxin nomenclature committee.     

Screening of cry gene contents of Bacillus thuringiensis strains isolated from avocado orchards in Mexico, and their insecticidal activity towards Argyrotaenia sp. (Lepidoptera: Tortricidae) larvae

Aims:  To screen for Bacillus thuringiensis strains from avocado orchards in two Mexican states with lepidopteran-specific cry gene content and evaluate their insecticidal activity against Argyrotaenia sp., an undescribed species present in avocado orchards.
Methods and Results:  Lepidopteran-active cry 1, cry 2 and cry 9 genes were detected by PCR analysis in 37 isolates. cry 1 genes were more frequent in Michoacán, but were undetected in Nayarit isolates. cry 9 and cry 2 genes were detected in isolates from both states, although cry 2 genes were less frequent. A variety of crystal shapes were observed among the isolates. According to gene profile, eight isolates were selected and tested against 2-day old Argyrotaenia sp. larvae. Standard strain HD-125 caused the highest mortality followed by strain MR-26 from Michoacán at a concentration of 500 μg ml⁻¹, respectively.
Conclusions:  Bacillus thuringiensis strains isolated from avocado orchards exhibit a low toxic activity towards Argyrotaenia sp. larvae, in spite of their specific cry gene content.
Significance and Impact of the Study:  Toxic activity of B. thuringiensis is not necessarily related to insect pest habitat and neither to specific cry gene content associated to other lepidopterans.     

Diversity and polymorphism in AHL-lactonase gene (aiiA) of Bacillus

To explore bacterial diversity for elucidating genetic variability in acylhomoserine lactone (AHL) lactonase structure, we screened 800 bacterial strains. It revealed the presence of a quorum quenching (QQ) AHL-lactonase gene (aiiA) in 42 strains. These 42 strains were identified using rrs (16S rDNA) sequencing as Bacillus strains, predominantly B. cereus. An in silico restriction endonuclease (RE) digestion of 22 AHL lactonase gene (aiiA) sequences (from NCBI database) belonging to 9 different genera, along with 42 aiiA gene sequences from different Bacillus spp. (isolated here) with 14 type II REs, revealed distinct patterns of fragments (nucleotide length and order) with four REs; AluI, DpnII, RsaI, and Tru9I. Our study reflects on the biodiversity of aiiA among Bacillus species. Bacillus sp. strain MBG11 with polymorphism (115Alanine > Valine) may confer increased stability to AHL lactonase, and can be a potential candidate for heterologous expression and mass production. Microbes with ability to produce AHL-lactonases degrade quorum sensing signals such as AHL by opening of the lactone ring. The naturally occurring diversity of QQ molecules provides opportunities to use them for preventing bacterial infections, spoilage of food, and bioremediation.