DNA cloning in Bacillus subtilis.

Ehrlich, S. Dusko

doi:10.1073/pnas.75.3.1433

Cited by 175 publications

(53 citation statements)

References 15 publications

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“…Prior to this analysis, the inability of E. coli to express genes from C. crescentus was rather puzzling since the E. coli transcriptional machinery recognizes transcriptional signals from a wide range of microorganisms as divergent as Bacillus subtilis (6) and Saccharomyces cerevisiae (23). In another report (16), we show that when the C. crescentus rpoD gene is fused to the E. coli lacZ promoter, it is expressed in E. coli.…”

Section: Vol 177 1995 C Crescentus Biosynthetic Gene Promoter 4375mentioning

confidence: 99%

A consensus promoter sequence for Caulobacter crescentus genes involved in biosynthetic and housekeeping functions

1995

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Caulobacter crescentus differentiates prior to each cell division to form two different daughter cells: a monoflagellated swarmer cell and a nonmotile stalked cell. Thus, one might expect that developmentally expressed genes would be regulated by mechanisms different from those used to regulate the expression of the biosynthetic genes. To determine a consensus promoter sequence for genes involved in biosynthetic or housekeeping functions, DNA fragments containing the regulatory regions of the ilvD, ilvR, cysC, pleC, and fdxA genes were cloned. S1 nuclease protection mapping and primer extension techniques were used to identify the transcription initiation sites. Comparison of the regulatory regions of these genes with those of the published sequences of the ilvBN, rrnA, trpFBA, dnaA, dnaK, hemE, and rsaA genes has resulted in the identification of a putative promoter consensus sequence. The ؊35 region contains the sequence TTGACGS, which is similar to the Escherichia coli ؊35 region, while the ؊10 region, GCTANAWC, has a more balanced GC content than the corresponding region in E. coli. Oligonucleotide-directed site-specific mutagenesis of both the ilvBN and pleC promoters indicates that mutations that make a promoter more like the consensus result in increased promoter activity, while mutations decreasing similarity to the consensus result in decreased promoter activity.Caulobacter crescentus is a gram-negative bacterium that differentiates prior to each cell division to generate two dissimilar progeny cells. The new cells differ from one another morphologically and developmentally. Much of what is known about gene regulation in C. crescentus has resulted from studies of periodically expressed genes, especially the genes involved in flagellar biogenesis and function (21, 27). The 5Ј regulatory regions of some of the flagellar genes have been shown to contain a set of activated promoters that are transcribed by a 54 RNA polymerase holoenzyme (3,19). These promoters contain the consensus sequences recognized by the enteric 54 at positions Ϫ12 and Ϫ24 (4,19,22). Other fla genes may be regulated by a second alternative sigma factor (5,30,35).Very little information is available on the expression of genes that are expected to show no cell cycle-dependent regulation. For instance, biosynthetic genes are likely to be expressed throughout the cell cycle to fulfill the nutritional requirements of the bacterium. To understand the regulatory mechanisms involved in the expression of the biosynthetic and housekeeping genes, we investigated the cis-acting regulatory elements involved in the expression of a number of such genes. Analysis of the sequences within the 5Ј regulatory regions led us to propose a consensus promoter sequence for biosynthetic and housekeeping genes. Site-specific mutagenesis of the ilvBN and the pleC promoter regions was used to evaluate the role of the nucleotides composing the proposed consensus. MATERIALS AND METHODSBacterial growth. C. crescentus strains were grown in PYE medium (13), and Escheric...

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Section: Vol 177 1995 C Crescentus Biosynthetic Gene Promoter 4375mentioning

confidence: 99%

A consensus promoter sequence for Caulobacter crescentus genes involved in biosynthetic and housekeeping functions

1995

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“…A sequence was identified which is required for the efficient conversion of the single plus strand to the double-stranded form during plasmid replication. Deletion of this sequence resulted in a low level of segregational plasmid instability.The development of cloning vectors for use in Bacillus subtilis has relied on plasmids isolated from Staphylococcus aureus (6,7,14,15). Both structural (10,15,19,20,27) and segregational (1, 2, 10, 19, 28) instabilities were frequently observed when recombinant vectors were transformed into B. subtilis.…”

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confidence: 99%

“…The development of cloning vectors for use in Bacillus subtilis has relied on plasmids isolated from Staphylococcus aureus (6,7,14,15). Both structural (10,15,19,20,27) and segregational (1,2,10,19,28) instabilities were frequently observed when recombinant vectors were transformed into B. subtilis.…”

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confidence: 99%

Replication and segregational stability of Bacillus plasmid pBAA1

et al. 1989

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A cryptic plasmid, pBAA1, was identified in an industrial Bacillus strain. The plasmid is 6.8 kilobases in size and is present in cells at a copy number of approximately 5 per chromosome equivalent. The p!asmid has been maintained under industrial fermentation conditions without apparent selective pressure and so is assumed to be partition proficient. The minimal replicon was localized to a 1.4-kilobase fragment which also contains the functions required for copy number control. The very low level of segregational instability of the minimal replicon suggests that it also contains functions involved in plasmid maintenance. Comparison with other plasmids indicates that pBAA1 belongs to the group of small gram-positive plasmids which replicate by a rolling cycle-type mechanism. A sequence was identified which is required for the efficient conversion of the single plus strand to the double-stranded form during plasmid replication. Deletion of this sequence resulted in a low level of segregational plasmid instability.The development of cloning vectors for use in Bacillus subtilis has relied on plasmids isolated from Staphylococcus aureus (6,7,14,15). Both structural (10,15,19,20,27) and segregational (1, 2, 10, 19, 28) instabilities were frequently observed when recombinant vectors were transformed into B. subtilis. These instabilities led to intense investigation of the mode of replication and of the stability functions of these plasmids. It has emerged that many of these plasmids replicate by a rolling cycle-type mechanism (11,12,31). The essential features of this mode of replication are (i) an origin of plus-strand synthesis, (ii) a replication protein which interacts with the plus origin to generate a nick which allows displacement synthesis of the plus strand to occur, and (iii) a signal for efficient conversion of the single strand to the double-stranded form. These features have been identified for a variety of plasmids, including pT181 (12, 16, 25) and pC194 (11, 12).Our aim is to analyze the segregational instability of plasmids in B. subtilis. It was hypothesized that the segregational instability of many S. aureus plasmids in B. subtilis is due, at least in part, to a suboptimal host-plasmid relationship. Thus, we chose to analyze the stability functions of a plasmid, pBAA1, which was resident in an industrial strain of B. subtilis. pBAA1 has been maintained under industrial fermentation conditions without apparent selection pressure. Preliminary experiments demonstrated that the copy number was low (approximately 5 per chromosome equivalent), so it was assumed that pBAA1 must encode an active partitioning function. In this paper, we report that all of the functions required for replication, copy number control, and partitioning are located on a 2.2-kilobase (kb) fragment. The DNA sequence and other evidence show that pBAA1 replicates by a rolling-circle mechanism and that some features of its replication functions are related both to 4X174 and to the gram-positive plasmids pC194, pUB110, and pFTB14.MAT...

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“…Efforts have been made to apply the techniques of molecular cloning, which are highly developed for the Gram-negative enterobacterium Escherichia coli, to B. subtilis (Ehrlich 1978;Goebel et al 1979;Lovett and Keggins 1979;Dubnau et al 1980) and several plasmid vector systems have been reported. These involve plasmids capable of replication in B. subtilis alone or in B. subtilis and other Gram-positive microorganisms (Ehrlich 1977;Wilson and Baldwin 1978;Bernhard et al 1978;Ehrlich et al 1982) or altematively bifunctional plasmids able to replicate in both E. coli and B. subtilis (Ehrlich 1978;Kreft et al 1978;Kreft and Hughes 1982). In this paper we describe the construction of bifunctional plasmids which allow studies on the expression in B. subtilis of three antibiotic resistance genes from E. coli.…”

Section: Introducnonmentioning

confidence: 99%

Expression of antibiotic resistance genes from Escherichia coli in Bacillus subtilis

1983

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Summary. Bifunctional recombinant plasmids were constructed, comprised of the E. coli vectors pBR322, pBR325 and pACYC184 and different plasmids from Gram-positive bacteria, e.g. pBSU161-1 of B. subtilis and pUB110 and pC221 of S. aureus. The beta-lactamase (bla) gene and the chloramphenicol acetyltransferase (cat) gene from the. E. coli plasmids were not transcribed and therefore not expressed in B. subtilis. However, tetracycline resistance from the E. coli plasmids was expressed in B. subtilis. Transcription ofthe tetracycline resistance gene(s) started in B. subtilis at or near the original E. coli p~omoter, the sequence of which is almost identical with the sequence recognized by a 55 of B. subtilis RNA polymerase. IntroducnonThe Gram-positive soil bacterium Bacillus subtilis is considered harmless for man and animals and is widely used in basic and applied microbiology. Efforts have been made to apply the techniques of molecular cloning, which are highly developed for the Gram-negative enterobacterium Escherichia coli, to B. subtilis (Ehrlich 1978;Goebel et al. 1979;Lovett and Keggins 1979;Dubnau et al. 1980) and several plasmid vector systems have been reported. These involve plasmids capable of replication in B. subtilis alone or in B. subtilis and other Gram-positive microorganisms (Ehrlich 1977;Wilson and Baldwin 1978;Bernhard et al. 1978;Ehrlich et al. 1982) or altematively bifunctional plasmids able to replicate in both E. coli and B. subtilis (Ehrlich 1978;Kreft et al. 1978;Kreft and Hughes 1982). In this paper we describe the construction of bifunctional plasmids which allow studies on the expression in B. subtilis of three antibiotic resistance genes from E. coli. Materials and MethodsBacterial Strains. E. coli 5K, r-, m-was obtained from S. Glover, and JC1569 recA, thy-, arg-, his-, mer, Ieu-, Smr harbouring pACYC184 from S. Cohen. B. subtilis BR151CM1 trpC2, metBJO, lys3, spoCMJ was kindly provided by P. Lovett.

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DNA cloning in Bacillus subtilis.

Cited by 175 publications

References 15 publications

A consensus promoter sequence for Caulobacter crescentus genes involved in biosynthetic and housekeeping functions

A consensus promoter sequence for Caulobacter crescentus genes involved in biosynthetic and housekeeping functions

Replication and segregational stability of Bacillus plasmid pBAA1

Expression of antibiotic resistance genes from Escherichia coli in Bacillus subtilis

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