Minireplicon from pBtoxis of Bacillus thuringiensis subsp. israelensis

Abstract

A 2.2-kb fragment containing a replicon from pBtoxis, the large plasmid that encodes the insecticidal endotoxins of Bacillus thuringiensis subsp. israelensis, was identified, cloned, and sequenced. This fragment contains cis elements, including iterons, found in replication origins of other large plasmids and suggests that pBtoxis replicates by a type A theta mechanism. Two genes, pBt156 and pBt157, encoding proteins of 54.4 kDa and 11.8 kDa, respectively, were present in an operon within this minireplicon, and each was shown by deletion analysis to be essential for replication. The deduced amino acid sequences of the 54.4-kDa and 11.8-kDa proteins showed no substantial homology with known replication (Rep) proteins. However, the 54.4-kDa protein contained a conserved FtsZ domain, and the 11.8 kDa protein contained a helix-turn-helix motif. As FtsZ proteins have known functions in bacterial cell division and the helix-turn-helix motif is present in Rep proteins, it is likely that these proteins function in plasmid replication and partitioning. The minireplicon had a copy number of two or three per chromosome equivalent in B. thuringiensis subsp. israelensis but did not replicate in B. cereus, B. megaterium, or B. subtilis. A plasmid constructed to synthesize large quantities of the Cry11A and Cyt1A endotoxins demonstrated that this minireplicon can be used to engineer vectors for cry and cyt gene expression.The various subspecies of Bacillus thuringiensis are characterized by the synthesis of parasporal crystals during sporulation. These crystals are typically composed of one or more highly specific insecticidal or nematocidal endotoxin proteins (28). Most isolates of B. thuringiensis harbor an array of plasmids with sizes ranging from 2 kb to 600 kb, and the genes coding for endotoxins are typically located on large plasmids (10, 16). The cis elements and genes required for replication of small plasmids, such as pTX14-3 (2), have been well characterized. However, little is known about the cis elements or mechanisms involved in replicating and partitioning the large endotoxin-encoding plasmids.One of the most important subspecies of B. thuringiensis is B. thuringiensis subsp. israelensis. This subspecies is highly insecticidal for the larvae of mosquitoes and blackflies and is presently used in many countries to control pest and vector species of these flies. The insecticidal activity of B. thuringiensis subsp. israelensis results principally from synergistic interactions of major endotoxin proteins, Cry11A (previously CryIVD), Cry4A, Cry4B, and Cyt1A (14, 27, 28). Recently, the nucleotide sequence of the large 128-kb plasmid, pBtoxis, that encodes these proteins was reported (7). Interestingly, comparative analyses of the 158 known and putative proteins encoded by pBtoxis revealed no homology with known plasmid replication proteins. However, comparative nucleotide analysis of pBtoxis and pXO1 of B. anthracis (25) suggested that a putative replication origin is present near the assigned first nucleotide position...

Evolution of the cytoskeleton

“…(59,60) These appear to function in plasmid partitioning by forming a cytoplasmic filament bundle that binds a plasmid at each end and pushes them to opposite poles. Additional distant relatives of FtsZ in archaea are discussed by Vaughan et al(30) …”

Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition

“…Specifically, it forms a cytokinetic ring called the Z ring at midcell, which mediates septation (5,6). Recently, however, prokaryotic proteins encoded on large plasmids harbored in bacilli showing 15-20% sequence similarity to both FtsZ and tubulin have been identified and dubbed TubZ (7)(8)(9)(10)(11)(12). Studies showed that the Bacillus thuringiensis TubZ protein from the pBtoxis plasmid is essential for plasmid DNA segregation.…”

“…The best studied of these systems is that found on the pBtoxis plasmid in B. thuringiensis. This plasmid stability system is represented by an operon encoding two proteins: ORF156 (TubZ) and ORF157 (TubR) (7)(8)(9)11). TubR is a 11.6 kDa DNAbinding protein that shows no sequence homology to any known protein.…”

Dynamics of bacterial cytoskeletal elements

“…Emphasizing the spectrum of different functions performed by related cytoskeletal proteins, a tubulin/ FtsZ-like protein, TubZ, has recently been identified in the Gram positive bacteria Bacillus anthracis and B. thuringienis, where it is plasmid-encoded and is essential for the distribution of the plasmid copies onto the daughter cells [Tinsley and Khan, 2006;Tang et al, 2006]. In B. thuringienis, TubZ forms filaments of several lm (2-5) length [Larsen et al, 2007].…”