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.     

Changes in the phyllosphere community of the resurrection fern, Polypodium polypodioides, associated with rainfall and wetting

A combination of analyses were used to characterize the changes that occur in a bacterial community present in the phyllosphere of the epiphytic resurrection fern, Polypodium polypodioides, as the fern rehydrates from a desiccation-resistant, physiologically inactive state. Enrichment assays showed an increase in the viable count of bacteria using labile organic substrates following rainfall. Isolates obtained from enrichments were predominantly Gram-positive bacteria affiliated with various groups of the Actinobacteria and Firmicutes. In contrast, sequencing of 16S rRNA genes clones obtained from whole community DNA revealed that much of the community was dominated by other taxa, particularly the Alphaproteobacteria. Similar isolates were obtained from both dry and hydrated P. polypodioides fronds, whereas 16S rRNA gene sequencing of community DNA revealed different ribotypes on the dry and wet fern, and an overall reduction in richness following wetting. Wetting also produced changes in phyllosphere extracellular enzyme activity, with an initial burst of activity following rainfall and a subsequent burst approximately 48 h later. These findings suggest that the resurrection fern harbors a complex phyllosphere community, and that rehydration of the fern following rainfall may act as an enrichment culture stimulating certain bacterial populations and changing the overall community structure and activity.     

Taxonomic insights into the phylogeny of Bacillus badius and proposal for its reclassification to the genus Pseudobacillus as Pseudobacillus badius comb. nov. and reclassification of Bacillus wudalianchiensis Liu et al., 2017 as Pseudobacillus wudalianchiensis comb. nov.

The species Bacillus badius is one of the oldest members of the genus Bacillus isolated from faeces of children and was classified based on its ability to form endospores [8]. In 16S rRNA gene sequence and phylogenetic analysis, Bacillus badius DSM 23^T shared low similarity (93.0%) and distant relationship with B. subtilis, the type species of the genus Bacillus indicating that it does not belong to this genus. Additional strains of the species, B. badius DSM 5610, DSM 30822 and B. encimensis SGD-V-25 (which has been recently reclassified as a member of this species) were included in the study to consider intraspecies diversity. Detailed molecular phylogenetic and comparative genome analysis clearly showed that the strains of B. badius were consistently retrieved outside the cluster of Bacillus sensu stricto and also distantly related to the genera Domibacillus and Quasibacillus. Further, the data from biochemical reactions (inability to ferment most carbohydrates), polar lipids profile (presence of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an aminophosphoglycolipid) and fatty acids supported the molecular analysis. Thus the four B. badius strains; DSM 23^T, DSM 5610, DSM 30822 and SGD-V-25 displayed sufficient demarcating phenotypic characteristics that warrant their classification as members of a novel genus and single species, for which the name Pseudobacillus badius gen. nov. comb. nov. is proposed with Pseudobacillus badius DSM 23^T (= ATCC 14574^T) as the type strain. Additionally, based on our findings from phenotypic, chemotaxonomic and genotypic parameters, Bacillus wudalianchiensis DSM 100757^T was reclassified as Pseudobacillus wudalianchiensis comb. nov.     

ITS序列同源性在苏云金芽孢杆菌分型中的应用研究

ＰＣＲ扩增了苏云金芽孢杆菌９个亚种的１６Ｓ－２３Ｓ ｒＲＮＡ基因转录间隔（ＩＴＳ）片段,它们的长度均为１４４碱基;序列同源性分析结果批出这９个亚种及其它亚种的ＩＴＳ序列高度相似,说明１６Ｓ－２３Ｓ ｒＲＮＡ基因的ＩＴＳ序列不适于苏云金孢杆菌亚种的分型。     

Spatial variation in Bacillus thuringiensis/cereus populations within the phyllosphere of broad-leaved dock (Rumex obtusifolius) and surrounding habitats

The aim of this study was to determine the prevalence and toxin gene diversity of Bacillus thuringiensis/B. cereus in the phyllosphere of broad-leaved dock (Rumex obtusifolius) at a small spatial scale. B. thuringiensis/cereus populations were isolated from the phyllosphere of dock and neighbouring grass and in neighbouring soil using commercially available selective media which avoided the disadvantageous heat-shock selection procedure. The maximum density of B. thuringiensis/cereus in the dock phyllosphere was 1.9 x 10(4) CFU g(-1) but the between-leaf variation in numbers was found to follow a lognormal distribution. B. thuringiensis/cereus was also found at significant densities in soil and the phyllosphere of grass adjacent to the dock plants. PCR screening indicated that genes encoding cry1 toxin were present in the plasmids of 36.9% of B. thuringiensis/cereus isolates tested, 11.9% contained cry2, and none of the dock leaf isolates tested contained cry3, cry4, cry7 or cry8 genes. The diversity of cry genes is similar to that found from other studies focused on other parts of the world. This work is the first concerning the prevalence of B. thuringiensis/cereus on leaves in the UK, finding population sizes of previously unrecorded levels and a greater relative proportion of B. thuringiensis. We have also illustrated that before any ecological function can be investigated, suitable sampling scales need to be considered -- here we have shown that the minimum sampling unit should be individual leaves, to account for the log-normal distribution.     

Effect of Bacillus thuringiensis on Habrobracon hebetor during combined biological control of Plodia interpunctella

Abstract  The suitability of combining microbial pesticides and an insect parasitoid for pest management of stored cereal in China was evaluated using laboratory assays. For this purpose, interactions between Bacillus thuringiensis ( Bt ), Bt -intoxicated host larvae and the parasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae) were tested against Plodia interpunctella Hübner (Lepidoptera: Pyralidae) . Bt or H. hebetor alone caused 41.67% and 35.35% P. interpunctella larval mortality respectively. The Bt –parasitoid combined treatment significantly increased mortality of P. interpunctella (86%). Progeny development of H. hebetor was dependent upon its susceptibility to Bt . Fewer parasitoids emerged from Bt –parasitoid combined treatment than in non- Bt treatments. However, since Bt did not prevent parasitoid development, a combined treatment with Bt and parasitoid release could produce better protection against P. interpunctella than either treatments when used singly, because their lethal effects were additive to each other.     

Culture-dependent and culture-independent assessment of bacteria in the apple phyllosphere

Aims: Bacterial communities in the apple phyllosphere were examined quantitatively and qualitatively by applying culture‐dependent and culture‐independent methods. Methods and Results: Populations estimated by viewing cells stained with 4′,6‐diamidino‐2‐phenylindole generally were at least 100–1000 times greater than populations estimated by culturing on tryptic soy agar (TSA). Of the 44 operational taxonomic units (OTUs; cut‐off threshold of 97%) detected in total, five bacterial orders containing 23 OTUs were identified by culturing on TSA, whereas nine orders containing 33 OTUs were identified by 16S rRNA gene cloning of DNA extracted from apple leaf surfaces. Twelve of the 44 OTUs were shared between cultured isolates and 16S rRNA gene clones and included the orders Burkholderiales, Pseudomonadales, Rhizobiales and Sphingomonadales. Three OTUs within the genus Sphingomonas accounted for 40% of isolates and 68% of clones. The Actinomycetales were found only among isolates, whereas the Bacteroidales, Enterobacteriales, Myxococales and Sphingobacteriales were represented in the 16S rRNA gene clone libraries but were absent among isolates. Conclusions: Culture‐independent methods revealed greater numbers and greater richness of bacteria on apple leaves than found by culturing. Significance and Impact of the Study: This is the first study to directly compare culture‐dependent and independent approaches for assessing bacterial communities in the phyllosphere. The biases introduced by different methods will have a significant impact on studies related to phyllosphere ecology, biological control of plant diseases, reservoirs of antibiotic resistance genes and food safety.     

Physiological and molecular detection of crystalliferous Bacillus thuringiensis strains from habitats in the South Central United States

Gram-positive, endospore-forming Bacillus thuringiensis-like strains were isolated from 95 of 413 samples collected at the 0–5 cm depth of noncultivated soils and stagnant or dried-up ponds as well as from dust from stored grain products in South Central United States. Based on the production of parasporal crystals, 25 isolates were identified as B. thuringiensis after examining 227 B. thuringiensis-like colonies. The greatest proportion of samples yielding B. thuringiensis were from the dust from grain storage. The sodium acetate selective medium, heat processing, and crystal staining used in the initial screening revealed diverse populations of B. thuringiensis, which were categorized into distinct crystal morphological groups. Sugar fermentation, antibiotic sensitivity, growth characteristics and PCR studies showed diversity among the isolates that were distributed among 25 of the 58 known strains. The most frequently isolated strains were kurstaki, aizawai, morrisoni, thuringiensis, sotto and kenyae that together represented more than 90% of the characterized isolates. PCR analysis using 30 family primer pairs for cry and cyt genes showed that the frequency of the cry1 gene (62%) was predominant followed by the cry2 genes (30%), and the rest (8%) were other cry gene types, such as cry3, cry4, cry10, cry11, cry14, cry15, cry20, cry24 and cry26. Both cyt1 and -2 genes were also detected. Several isolates showed PCR products on the gel that were not consistent with the expected sizes of nucleotides targeted by the primers. These were suggestive of nonspecific amplifications and were not used in the characterization process. Journal of Industrial Microbiology & Biotechnology (2002) 28, 284–290 DOI: 10.1038/sj/jim/7000244 Received 30 May 2001/ Accepted in revised form 10 January 2002     

利用ERIC-PCR对苏云金芽胞杆菌与蜡状芽胞杆菌进行鉴定的研究

为了探索ERIC-PCR技术在苏云金芽胞杆菌和蜡状芽胞杆菌的鉴定及分型中的应用价值,本研究采用PCR方法初步检测苏云金芽胞杆菌杀虫晶体蛋白基因的组成,并对苏云金芽胞杆菌和蜡状芽胞杆菌的总DNA进行ERIC-PCR扩增,分析ERIC-PCR指纹图谱的特点并采用NTSYS2.10软件对其进行聚类。结果显示,各菌株的ERIC指纹图谱表现出不同程度的多态性,但图谱与菌株所含cry基因的类型存在一定的相关性。聚类分析结果显示,含有相同或相近cry基因类型的Bt菌株在进化树上趋向聚为一类,而不含cry基因的蜡状芽胞杆菌趋向于与不含cry基因的Bt菌株聚为一类或单独聚类。若在多种模式菌株的参考下,该方法可用于苏云金芽胞杆菌的初步鉴定和分型。     

Expression of chloramphenicol acetyltransferase in Bacillus subtilis under the control of a phytoplasma promoter

A cloned putative promoter region upstream of the 16S rRNA gene of the western X-disease phytoplasma was inserted behind the promoterless chloramphenicol acetyltransferase gene of plasmid pPL603. The DNA construct was used to transform Bacillus subtilis cells. The transformants were assayed for chloramphenicol acetyltransferase activity, showing that the phytoplasma promoter is efficiently expressed in a B. subtilis background.     

Purification of the Cry1Ac protein of Bacillus thuringiensis and assessment against the Plutella xylostella and soil microbial community

The insecticidal toxin gene of Bacillus thuringiensis (Bt) is the most commonly used to develop insect‐resistant living modified organisms (LMOs). Insecticidal proteins produced in transgenic plants are released into the soil from the roots. In this study, possible effects of crystal 1Ac (Cry1Ac) protein on the soil microbial community in Korea were studied. To purify the insoluble Cry1Ac protein expressing Escherichia coli cells, we performed repeated sonication and PBS washing of the insoluble part and Cry1Ac protein was isolated in soluble form from the insoluble form using 100 mM Na₂CO₃ buffer (pH 9.6) without affinity bead. Also, size‐exclusion chromatography (SEC) was performed to increase the purity of the isolated Cry1Ac protein. The final protein product was identified as Cry1Ac protein through MALDI‐TOF. Insecticidal activity of Cry1Ac protein was demonstrated through the death of Plutella xylostella treated with Cry1Ac protein. Purely isolated Cry1Ac protein showed the same insecticidal activity as Cry1Ac expressed in LM crops. To investigate the change of soil microbial distribution using maize field soils treated with Cry1Ac protein, we isolated high quality metagenomic DNAs from buffer‐ and Cry1Ac protein‐treated soil groups, and analyzed the distribution of soil microorganisms through next‐generation sequencing (NGS) analysis. NGS results showed a similar microbial distribution in both buffer‐ and Cry1Ac protein‐treated samples. These results suggest a useful risk assessment method for domestic targeted insect and soil microorganisms using the Cry1Ac protein.     

Bt杀虫蛋白处理后二化螟幼虫中肠细菌群落的变化

【目的】为探讨经Bt杀虫蛋白处理后二化螟Chilo suppressalis(Walker)幼虫中肠细菌群落的差异。【方法】本研究对采自北京(BJ)和福州(FZ)2个地区在室内分别经过未用Bt杀虫蛋白和使用Bt杀虫蛋白(Cry1Ab,Cry1Ac和Cry1Ca)多代汰选条件下的二化螟幼虫中肠进行解剖,采用变性梯度凝胶电泳(DGGE)和Illumina Mi Seq技术测序平台对3个处理组(BJCry1Ab,BJCry1Ac和FZ1Ca)和2个对照组(BJCK和FZCK)中肠细菌的16S r DNA V3可变区进行电泳检测和高通量测序。【结果】DGGE图谱显示,5个不同处理组的细菌不仅丰富度存在差异,同时同种细菌在不同处理组中的比例也存在差异。高通量测序结果表明,优势菌为厚壁菌门(Firmicutes)的肠球菌属Enterococcus,其次为乳杆菌属Lactobacillus和芽孢杆菌属Bacillus,以及变形菌门(Proteobacteria)、绿弯菌门(Chloroflexi)和拟杆菌门(Bacteroidetes)。同一地区的二化螟种群,Bt杀虫蛋白处理组(BJCry1Ab,BJCry1Ac和FZ1Ca)的二化螟幼虫与对照组(BJCK和FZCK)相比,优势菌肠球菌属Enterococcus比重均有所增加,而乳杆菌属Lactobacilluss所占比重均有所降低。北京和福州这2个地区未用Bt杀虫蛋白处理的对照组之间肠道菌落的结构也存在一定差异。【结论】经Bt杀虫蛋白处理后二化螟幼虫中肠细菌群落的丰富度出现变化,推测可能与二化螟取食不同Bt杀虫蛋白、地理位置差异以及饲养代数不同有关。     

5种中国苏云金芽孢杆菌的伴孢 晶体蛋白基因分析

利用聚合酶联反应(PCR)和聚丙烯酰胺凝胶电泳(SDS-PAGE)技术分析了5种中国苏云金杆菌制剂菌株的伴孢晶体蛋白及其基因组成。结果发现,5种菌株均含有cry1Aa和/或c和/或d和/或b基因,只有Bt+Virus菌株含有cry1Ab基因,cry1A基因编码的伴孢晶体蛋白分子量约为130 kD;仅有JS-Bt C菌株含有cry1B基因,其编码的伴孢晶体蛋白分子量约为138 kD;除HB Bt C菌株外,其余4个菌株均含有cry2Aa和/或b基因,这类基因编码分子量为70 kD的伴孢晶体蛋白;所有5个菌株都含有cry1I基因,其编码的伴孢晶体蛋白分子量应为81.2 kD,但实验中未曾检测到cry1I基因的表达;所有的菌株都不含有cry1C和cry1D基因。     

DIVERSITY IN GUT MICROFLORA OF Helicoverpa armigera POPULATIONS FROM DIFFERENT REGIONS IN RELATION TO BIOLOGICAL ACTIVITY OF Bacillus thuringiensis δ‐ENDOTOXIN Cry1Ac

Transgenic crops expressing toxin proteins from Bacillus thuringiensis (Bt) have been deployed on a large scale for management of Helicoverpa armigera. Resistance to Bt toxins has been documented in several papers, and therefore, we examined the role of midgut microflora of H. armigera in its susceptibility to Bt toxins. The susceptibility of H. armigera to Bt toxin Cry1Ac was assessed using Log‐dose‐Probit analysis, and the microbial communities were identified by 16S rRNA sequencing. The H. armigera populations from nine locations harbored diverse microbial communities, and had some unique bacteria, suggesting a wide geographical variation in microbial community in the midgut of the pod borer larvae. Phylotypes belonging to 32 genera were identified in the H. armigera midgut in field populations from nine locations. Bacteria belonging to Enterobacteriaceae (Order Bacillales) were present in all the populations, and these may be the common members of the H. armigera larval midgut microflora. Presence and/or absence of certain species were linked to H. armigera susceptibility to Bt toxins, but there were no clear trends across locations. Variation in susceptibility of F₁ neonates of H. armigera from different locations to the Bt toxin Cry1Ac was found to be 3.4‐fold. These findings support the idea that insect migut microflora may influence the biological activity of Bt toxins.     

苏云金芽孢杆菌cry基因研究进展

从cry基因的分类、cry基因与转座因子的关系、cry基因的表达调控以及cry基因的作用机理等 4个方面综述了cry基因的研究进展 ,并简要展望了其研究和应用前景。     

Comparative 16S rDNA sequence analysis of some alkaliphilic bacilli and the establishment of a sixth rRNA group within the genus Bacillus

Abstract Comparative sequence analysis of the 16S rDNA of 14 alkaliphilic or alkalitolerant, Gram-positive, aerobic, endo-spore forming bacterial strains was performed. Bacillus alcalophilus DSM 485^T and Bacillus cohnii DSM 6307^T were included to represent the two validly described alkaliphiles assigned to the genus Bacillus . The majority of isolates (8 strains) clustered with B. alcalophilus DSM 485^T forming a distinct phylogenetic group (rRNA group 6) within the radiation of the genus Bacillus and related taxa. Bacillus cohnii DSM 6307^T and two of the isolates, DSM 8719 and DSM 8723, grouped with B. fastidiosus and B. megaterium and are allocated to rRNA group 1. The remaining two strains DSM 8720 and DSM 8721 show an equidistant relationship to both groups.