Biomedical subjects
P Stragier
Publications and source records attributed to P Stragier.
Binding of the Bacillus subtilis spoIVCA product to the recombination sites of the element interrupting the sigma K-encoding gene.
The gene encoding sigma K, a transcription factor controlling mother-cell-specific gene expression at a late stage of sporulation, is interrupted by the skin element in Bacillus subtilis. The skin element is excised from the mother cell chromosome by a DNA rearrangement that depends on the spoIVCA gene product. This protein has no other role in sporulation than promoting skin excision and exhibits sequence similarity to a family of bacterial site-specific recombinases. An expression library of B. subtilis DNA in lambda gt11 was screened for the presence of a gene encoding a protein able to bind in vitro to an oligonucleotide matching the inverted repeat sequences present at the ends of the skin element. Several bacteriophages were found to contain the spoIVCA gene. A cell extract containing the SpoIVCA protein protected the inverted repeats and their neighboring sequences from DNase I digestion and methylation. SpoIVCA decreased the electrophoretic mobility of a DNA fragment containing its binding sequence and simultaneously bent the DNA. A single molecule of SpoIVCA bound initially to the repeat sequence followed by binding of a second molecule to create a complex straddling the recombination site.
Developmental regulation of transcription of the Bacillus subtilis ftsAZ operon.
The products of the ftsA and ftsZ genes play a major role in septum formation in Escherichia coli. Their homologues have been found in various bacterial species, such as Bacillus subtilis where they are involved in septation during vegetative growth as well as during sporulation, a developmental process that is initiated by the formation of an asymmetrically positioned septum. Transcription of the B. subtilis ftsAZ operon was studied during exponential growth and sporulation by monitoring beta-galactosidase synthesis in strains harboring fusions of the E. coli lacZ gene with various fragments of the ftsAZ regulatory region. Transcription of the ftsAZ operon was found to be controlled by three promoters which were mapped by primer extension and characterized by their temporal pattern of expression. Two of these promoters, P1 and P3, are dependent on sigma A, the major vegetative sigma factor, and are expressed mainly during growth. The third one, P2, is recognized by sigma H associated RNA polymerase and its activity increases three- to four-fold around the onset of sporulation. The post-exponential enhancement of P2-driven transcription is abolished in a spo0A mutant but partially restored in an abrB spo0A double mutant. After inactivation by oligonucleotide-directed mutagenesis mutated copies of P1 and P2 were introduced into the chromosome upstream from the ftsAZ operon. Transformants could be obtained only when ftsAZ transcription was controlled by a combination of two intact promoters, neither P1, P2 nor P3 being essential for viability. The sporulation efficiency was found to be dependent on the level of transcription of ftsAZ, the absence of P2 still allowing 30% of the normal sporulation rate. Therefore the post-exponential burst of synthesis of the FtsA and FtsZ proteins is not an absolute requirement for the successful completion of the asymmetric septum.
Crisscross regulation of cell-type-specific gene expression during development in B. subtilis.
Sporulation in Bacillus subtilis is a model for how cells of one type generate other differentiated cell types. During sporulation two cellular compartments arise that differ from each other and from the progenitor cell. Differential gene expression between the two is governed by the successive appearance of four transcription factors whose activities are coordinated in crisscross fashion between the two cells.
Cloning, characterization, and expression of the dapE gene of Escherichia coli.
The dapE gene of Escherichia coli encodes N-succinyl-L-diaminopimelic acid desuccinylase, an enzyme that catalyzes the synthesis of LL-diaminopimelic acid, one of the last steps in the diaminopimelic acid-lysine pathway. The dapE gene region was previously purified from a lambda bacteriophage transducing the neighboring purC gene (J. Parker, J. Bacteriol. 157:712-717, 1984). Various subcloning steps led to the identification of a 2.3-kb fragment that complemented several dapE mutants and allowed more than 400-fold overexpression of N-succinyl-L-diaminopimelic acid desuccinylase. Sequencing of this fragment revealed the presence of two closely linked open reading frames. The second one encodes a 375-residue, 41,129-M(r) polypeptide that was identified as N-succinyl-L-diaminopimelic acid desuccinylase. The first one encodes a 118-residue polypeptide that is not required for diaminopimelic acid biosynthesis, as judged by the wild-type phenotype of a strain in which this gene was disrupted. Expression of the dapE gene was studied by monitoring amylomaltase activity in strains in which the malPQ operon was under the control of various fragments located upstream of the dapE gene. The major promoter governing dapE transcription was found to be located in the adjacent orf118 gene, while a minor promoter allowed the transcription of both orf118 and dapE. Neither of these two promoters is regulated by the lysine concentration in the growth medium.
Nucleotide sequence of the lsp-dapB interval in Escherichia coli.
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A gene for a new lipoprotein in the dapA-purC interval of the Escherichia coli chromosome.
Cloning and sequence analysis of the region located downstream of the dapA gene of Escherichia coli has revealed the presence of an open reading frame that is cotranscribed with dapA. This gene codes for a 344-amino-acid polypeptide with a potential signal sequence characteristic of a lipoprotein. When this gene, called nlpB, is expressed from a multicopy plasmid in bacteria grown in the presence of [3H]palmitate, a labeled 37-kDa protein is produced. A slightly larger precursor molecule is detected when minicells expressing nlpB are treated with globomycin, a specific inhibitor of lipoprotein signal peptidase. Therefore, the nlpB gene encodes a new lipoprotein, designated NlpB. This lipoprotein is detected in outer membrane vesicles prepared from osmotically lysed spheroplasts and appears to be nonessential, since a strain in which the nlpB gene is disrupted by insertion of a chloramphenicol resistance gene is still able to grow and shows no discernible NlpB phenotype. The putative transcription termination signals of the dapA-nlpB operon overlap the promoter of the adjacent purC gene.
Cloning, characterization, and expression of the spoVB gene of Bacillus subtilis.
Mutation of the spoVB gene in Bacillus subtilis causes the production of spores containing a defective cortex and unable to acquire heat resistance. The spoVB locus is highly linked to another spo locus, spoIIIF, characterized by a single mutation (I. L. Lamont and J. Mandelstam, J. Gen. Microbiol. 130:1253-1261, 1984). A 18-kb DNA region overlapping the spoIIIF-spoVB region was cloned in successive steps starting from a Tn917 insertion in the nic locus. The exact location of the spoIIIF and spoVB loci was defined with various integrative plasmids carrying subfragments of that region. DNA sequencing established that spoIIIF and spoVB are a single monocistronic locus encoding a 518-amino-acid polypeptide with features of an integral membrane protein. The precise location of the spoIIIF590 and spoVB91 mutations in that unique open reading frame was determined, and both mutations were sequenced. A null mutation was engineered in the spoIIIF-spoVB locus and led to a typical spoVB phenotype, identical to the phenotype created by either spoIIIF590 or spoVB91, suggesting that the original spoIIIF mutant contained a secondary mutation arresting sporulation at an earlier stage. A transcriptional spoVB-lacZ fusion was constructed, and its expression was found to be directly dependent on RNA polymerase containing sigma E. A null mutation of spoVB had no effect on expression of sspB and cotA, members of the sigma G- and sigma K-controlled regulons respectively, while expression of cotC, a member of the latest known mother cell regulon, was delayed and strongly reduced. These results are consistent with SpoVB being involved in cortex biosynthesis and affecting only indirectly expression of late sporulation genes.
The Bacillus subtilis gene for the development transcription factor sigma K is generated by excision of a dispensable DNA element containing a sporulation recombinase gene.
The structural gene (sigK) for the mother-cell RNA polymerase sigma-factor sigma K in Bacillus subtilis is a composite of two truncated genes, named spoIVCB and spoIIIC, which are brought together by site-specific recombination during sporulation. We now show that the recombination event is compartmentalized in that the mother cell, but not the forespore chromosome, undergoes rearrangement. We also show that spoIIIC (encoding the carboxy-terminal portion of sigma K) lies approximately 42 kb downstream of spoIVCB (encoding the amino-terminal portion) and that the joining of the truncated coding sequences is a reciprocal recombination event in which intervening DNA is deleted from the chromosome as a circle. The rearrangement is governed by the product of a gene named spoIVCA located in the excised DNA, as demonstrated by the observations (1) that the product of spoIVCA, but not the product of any other stage-IV sporulation gene tested, is required for the rearrangement, and (2) that the presence of a cloned copy of the rearranged sigK gene in the chromosome bypasses the requirement for the spoIVCA gene product in sporulation. Because cells engineered to contain an intact copy of sigK sporulate normally, we conclude that the sigK rearrangement is not essential for the control of gene expression during sporulation, and we infer the existence of an additional mechanism for restricting sigma K-directed transcription to the mother-cell chamber of the sporangium. Finally, the construction of a strain deleted for the entire sigK intervening sequence shows that the 42-kb element contains no genes essential for viability.
Cascades of sigma factors revisited.
Programmed gene expression during the process of endospore formation in Bacillus subtilis is governed by the successive appearance of five developmental sigma factors. These sigma factors are encoded by genes in which mutations arrest sporulation at a defined stage. These genes are turned on sequentially and depend for their own transcription on the activity of a previously synthesized sigma factor. Superimposed on the regulation of synthesis of the sigma factors are post-transcriptional control mechanisms that couple the activation of the developmental sigma factors to the course of sporulation. Here we review evidence indicating that these developmental transcription factors comprise a regulatory cascade in the order sigma H----sigma F----sigma E----sigma G----sigma K in which the activity of each sigma factor depends on the action of the preceding sigma factor in the cascade.
The spoIIJ gene, which regulates early developmental steps in Bacillus subtilis, belongs to a class of environmentally responsive genes.
The Bacillus subtilis spoIIJ locus is defined by a Tn917 insertion which leads to an oligosporogenous phenotype. Here we show that this mutation severely decreases transcription of spoIIA, spoIIE, and spoIIG, three operons involved in asymmetric septation, the earliest morphological event of sporulation. A 14.3-kilobase region overlapping the site of the spoIIJ::Tn917 insertion was cloned and the exact location of the spoIIJ gene was defined with various integrative plasmids carrying subfragments of that region. DNA sequencing established that spoIIJ is a monocistronic locus encoding a 606-amino-acid polypeptide which contains a canonical "transmitter" domain, indicating that spoIIJ is a new member of the "sensor" class of signal-transducing systems in bacteria. Thus, spoIIj, which is transcribed during vegetative growth, presumably under the control of sigma H, encodes a protein that could interact with major regulators of early sporulation stages, such as SpoOA and/or SpoOF.
Chromosomal rearrangement generating a composite gene for a developmental transcription factor.
Differential gene expression in the mother cell chamber of sporulating cells of Bacillus subtilis is determined in part by an RNA polymerase sigma factor called sigma K (or sigma 27). The sigma K factor was assigned as the product of the sporulation gene spoIVCB on the basis of the partial aminoterminal amino acid sequence of the purified protein. The spoIVCB gene is now shown to be a truncated gene capable of specifying only the amino terminal half of sigma K. The carboxyl terminal half is specified by another sporulation gene, spoIIIC, to which spoIVCB becomes joined inframe at an intermediate stage of sporulation by site-specific recombination within a 5-base pair repeated sequence. Juxtaposition of spoIVCB and spoIIIC need not be reversible in that the mother cell and its chromosome are discarded at the end of the developmental cycle. The rearrangement of chromosomal DNA could account for the presence of sigma K selectively in the mother cell and may be a precedent for the generation of cell type-specific regulatory proteins in other developmental systems where cells undergo terminal differentiation.
Identification of a new sigma-factor involved in compartmentalized gene expression during sporulation of Bacillus subtilis.
During sporulation of Bacillus subtilis, two identical genomes segregate in two compartments, the forespore and mother cell. These genomes are expressed differentially, with some genes such as sspE turned on only in the forespore. In vitro transcription of sspE was obtained only with RNA polymerase extracted from sporulating cells. Fractionation of factors associated with this enzyme and reconstitution with core RNA polymerase from vegetative cells generated an enzyme accurately transcribing sspE in vitro and led to purification of a polypeptide with the amino-terminal sequence of the spoIIIG product. Inactivation of spoIIIG abolished expression of sspE and five other forespore-specific genes, whereas synthesis of the spoIIIG product in vegetative cells rapidly turned these genes on. Therefore, spoIIIG encodes a sigma-factor, sigma G, which controls the expression of multiple genes in the forespore compartment.
Tandem genes encoding sigma-factors for consecutive steps of development in Bacillus subtilis.
During sporulation, Bacillus subtilis undergoes successive morphological changes that can be arrested at various stages by mutations in many genes. One of these, spoIIGB, encodes a transcriptional factor, sigma E, which is necessary to proceed beyond stage II and to differentiate the cell in two compartments, the forespore and the mother cell. Mutations were introduced in an open reading frame located immediately downstream of spoIIGB. They block sporulation at stage III and define a new gene, spoIIIG, encoding a 260-amino-acid polypeptide highly similar to bacterial sigma-factors. A promoter was identified in the spoIIGB-spoIIIG interval by transcriptional fusion to lacZ. It is turned on 1 hr after the start of sigma E synthesis and is specifically activated in the forespore. The tandemly arranged spoIIGB and spoIIIG genes appear to encode homologous proteins that modulate transcription in a sequential fashion during sporulation.
Processing of a sporulation sigma factor in Bacillus subtilis: how morphological structure could control gene expression.
Sporulation of Bacillus subtilis is a primitive example of coupling between morphological changes and timing of gene expression during development. A major early control of transcriptional activity is dependent on a new sigma factor, sigma E, which is encoded by the sigE gene and synthesized as an inactive precursor, pro-sigma E. We show that mutations in the spoIIGA gene block the processing of pro-sigma E. Moreover, synthesis of both spoIIGA and sigE products in vegetative cells leads to expression of a sigma E-controlled promoter during growth, suggesting that SpoIIGA has pro-sigma E processing activity. The SpoIIGA polypeptide, which contains five potential transmembrane domains, is synthesized during sporulation 1 hr before processing activity can be detected. We propose that SpoIIGA processing activity is triggered by the presence of the sporulation septum, which is itself dependent on the spoIIAA and spoIIE products. These proteins are normally needed for pro-sigma E processing during sporulation but can be bypassed in vegetative cells. According to this model, a morphological structure would directly control the synthesis of a developmental sigma factor and would modify gene expression.
Pseudomonas aeruginosa diaminopimelate decarboxylase: evolutionary relationship with other amino acid decarboxylases.
The lysA gene encodes meso-diaminopimelate (DAP) decarboxylase (E.C.4.1.1.20), the last enzyme of the lysine biosynthetic pathway in bacteria. We have determined the nucleotide sequence of the lysA gene from Pseudomonas aeruginosa. Comparison of the deduced amino acid sequence of the lysA gene product revealed extensive similarity with the sequences of the functionally equivalent enzymes from Escherichia coli and Corynebacterium glutamicum. Even though both P. aeruginosa and E. coli are Gram-negative bacteria, sequence comparisons indicate a greater similarity between enzymes of P. aeruginosa and the Gram-positive bacterium C. glutamicum than between those of P. aeruginosa and E. coli enzymes. Comparison of DAP decarboxylase with protein sequences present in data bases revealed that bacterial DAP decarboxylases are homologous to mouse (Mus musculus) ornithine decarboxylase (E.C.4.1.1.17), the key enzyme in polyamine biosynthesis in mammals. On the other hand, no similarity was detected between DAP decarboxylases and other bacterial amino acid decarboxylases.
Identification of the genes in the Escherichia coli ileS-lsp operon. Analysis of multiple polycistronic mRNAs made in vivo.
The genes encoding isoleucyl-tRNA synthetase (ileS) and prolipoprotein signal peptidase (lsp) of Escherichia coli were previously shown to be co-transcribed (Miller, K. W., and Wu, H. C. (1987) J. Biol. Chem. 262, 389-393). However, the boundaries of this transcriptional unit have not been established. In this regard, DNA sequence determination has shown that ileS and lsp are closely flanked by four open reading frames, i.e. x-ileS-lsp-orf149-orf316-orf304. To define the boundaries of the operon, we applied Northern blotting hybridization and mRNA 5'-end mapping to analyze mRNA from a wild-type strain (SM31) and a mutant strain (SM31-2B4) that exhibits an increased expression of prolipoprotein signal peptidase. Four ileS-lsp co-transcripts were detected in RNA from the strain SM31. In addition to these four mRNAs, two new, highly abundant co-transcripts were also detected in RNA from the mutant. Based upon the determination of the 5'-ends of the mRNAs and analysis of their coding sequences, we conclude that the six mRNAs actually are comprised of three pairs of related mRNAs. The two mRNAs in a given pair have the same 5'-termini (all located upstream of or within gene x), but vary with respect to the identity of their 3'-terminal coding sequence (lsp or orf316). In conclusion, the ileS-lsp operon contains five genes, x-ileS-lsp-orf149-orf316, whose transcription probably is dependent upon promoter(s) located upstream of or within gene x. The next gene downstream, orf304, apparently does not reside in the operon.