Plasmid Patterns of Bacillus thuringiensis Type Strains

Abstract

Practically all Bacillus thuringiensis strains contain a set of self-replicating, extrachromosomal DNA molecules or plasmids, which vary in number and size in the different strains. The plasmid patterns obtained from gel electrophoresis have previously been used as a tool to characterize strains, but comparison of the plasmid patterns has been limited in the number and diversity of strains analyzed. In this report, we were able to compare the plasmid patterns of 83 type strains (out of 84) and 47 additional strains from six serotypes. The information obtained from this comparison showed the importance of this tool as a strain characterization procedure and indicates the complexity and uniqueness of this feature. For example, with one exception, all type strains showed a unique plasmid pattern. All were unique in such a way that none showed even a single comigrating plasmid in the agarose gels, and therefore, cluster analysis was impossible, indicating that plasmid patterns are qualitative rather than quantitative features. Furthermore, comparison between strains belonging to the same serotype showed a great difference in variability. Some serotypes (e.g., israelensis) showed the same basic pattern among all its strains, while other serotypes (e.g., morrisoni) showed a great diversity of patterns. These results indicate that plasmid patterns are valuable tools to discriminate strains below the serotype level.Bacillus thuringiensis is a ubiquitous bacterium (25) isolated from a great diversity of habitats, which include soil, insects, stored crop products (31), phylloplane (32), and aquatic habitats (18). Its great biotechnological success resides in the production of highly specific insecticidal proteins (Cry proteins) simultaneously with the sporulation phase. These Cry proteins are coded by genes (cry genes) harbored in megaplasmids (10,14,21,34), although it has also been suggested that they are present in the chromosome (21). Plasmids have also been associated with the production of a different toxin called ␤-exotoxin (23). The relevance of plasmids in B. thuringiensis strains is assumed by the regular presence of a set of plasmids, which can vary in number from 1 to 17 and in size from 2 to 80 MDa (2, 9). The set of plasmids harbored in a strain are normally visualized by agarose gel electrophoresis, where they form an electrophoretic pattern of bands, according to their differential migration in the gel. Although B. thuringiensis plasmids have been studied either to locate cry genes (9, 21) or to transfer them between different strains and species (3,5,10,15,30), plasmid patterns have frequently been used to characterize strains (2, 29, 33), especially compared to those of standard strains (16,17,27). A plasmid pattern seems to be related to each strain, serotype, or any other subspecific group.In a plasmid pattern, two different groups of plasmids can be recognized: those that are Յ30 MDa and those that are Ն30 MDa, called megaplasmids. For practical purposes, each group is divided by the so-called chromos...

Detection of New cry Genes of Bacillus thuringiensis by Use of a Novel PCR Primer System

“…thuringiensis shows great variability, as has been demonstrated by the huge number of strains isolated around the world (19), by the number of serotypes known to date (a total of 84) (20), and by the great number of different cry gene sequences accumulated so far (a total of 492) (8), as well as by the number of molecular characterization tools that have been developed, such as sequencing of the flagellin gene and of the gyrB and aroE genes, the band patterns from repetitive extragenic palindromic-PCR analyses, and the plasmid patterns, among others (17,18,25,27), all indicating the great variability within this species.…”

Characterization of a native Bacillus thuringiensis isolates from Malaysia that produces exosporium-enclosed parasporal inclusion

“…Plasmid patterns of B. thuringiensis can be divided into two groups; the small plasmids and the megaplasmids. According to Reyes-Ramirez and Ibarra (2008) and Fagundes et al (2011), these two groups were separated in the agarose gel electrophoresis by a chromosomal band. The small plasmids were less than 30 MDa and located below the chromosomal band whereas the megaplasmids were larger than 30 MDa and located above the chromosomal band.…”

“…In the characterization of B. thuringiensis plasmid profi les, strains are usually fi rstly distinguished by observing the migration patterns of the small plasmids. Megaplasmids are always the second option for characterization due to their limited migration in the agarose gel during electrophoresis, thus causing diffi culties in discriminating the comigrating bands (Reyes-Ramirez and Ibarra, 2008;Fagundes et al, 2011). In addition, megaplasmids tend to degrade during storage that leads to information loss on the plasmid patterns (Reyes-Ramirez and Ibarra, 2008).…”

“…Plasmids were extracted using a modifi ed protocol of Reyes-Ramirez and Ibarra (2008). Bt-S84-13a was precultured on Nutrient agar for 14-16 h at 28°C.…”

“…Plasmid patterns of B. thuringiensis can be divided into two different groups: small plasmids which are ≤30 MDa, and megaplasmids which are ≥30 MDa. In agarose gel electrophoresis, the small plasmids (usually present in high copy numbers) and the megaplasmids (usually present in low copy numbers) are separated by a chromosomal band (Reyes-Ramirez and Ibarra, 2008;Fagundes et al, 2011). The δ-endotoxin genes of B. thuringiensis are located on megaplasmids and/or chromosomes (Kronstad et al, 1983;Lereclus et al, 1993;Glare and O'Callaghan, 2000;Zujiao et al, 2008), whilst the smaller plasmids are referred as cryptic plasmids because no specifi c function has been found in them yet.…”