ANNUAL REVIEWS
Annu. Rev. Genet. 1992.26:113-130. Downloaded from www.annualreviews.org by University of California - San Francisco UCSF on 01/28/15. For personal use only.
Annu.Rev.Genet. 1992.26:113-30 Copyright © by Annual Reviews Inc.All rights reserved
Further
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DEVELOPMENTALLY REGULATED GENE REARRANGEMENTS IN PROKARYOTES Robert Haselkorn Department of Molecular Genetics and Cell Biology, University of Chicago,
Chicago, Illinois 60637
KEY WORDS: gene rearrangement, heterocys t, s porulation, Anabaena, Bacillus
CONTENTS INTRODUCTION. ..... . . . . . ..... . . . . . . .. . . . . . . . . . . . . . . . . . . STOCHASTIC INVERSIONS
. . . .. . ... ... . ...... ... . . . .... . ....
1 13 1 15
NITROGEN FIXATION AND HETEROCYST DIFFERENTIATION . . . . . . . . . .
1 16
NifGENE REARRANGEMENT IN ANABAENA . . . . . . . . . . . . . . . . . . . . . . .
1 19
Spo GENE REARRANGEMENT IN B. SUBT/US . . . . . . . . . . . . . . . . . . . . . .
125
WHAT PRESERVES THE INTERRUPTING ELEMENTS? ....... . . . ......
128
INTRODUCTION Gene rearrangements are endemic in bacteria. Transposing elements and inverting elements have been described in virtually every bacterial species for which transposition or inversion provides a scorable phenotype. Rearrange ment in these cases provides a small fraction of the cell population with a phenotype, usually a surface property, that allows these few cells to evade surveillance or to respond immediately to a new environment. In contrast to the inducible systems that require signal transduction, gene activation, transcription, translation and possibly dilution or turnover of a prior manifes tation of gene expression, the cells containing rearranged genes are, like boy scouts, always prepared. If the eventuality for which they are prepared does not occur, no problem. Even if they are disadvantaged in the climate of the 1 13
0066-4197/92/1215-0113$02.00
Annu. Rev. Genet. 1992.26:113-130. Downloaded from www.annualreviews.org by University of California - San Francisco UCSF on 01/28/15. For personal use only.
1 14
HASELKORN
time and would normally be diluted by the faster growth of cells with unrearranged genomes, they are recreated each�genei"ation by the low-fre quency, spontaneous rearrangement event. These phenomena can be contrasted with the developmentally regulated rearrangements that are the subject of this review. Two of these occur during the differentiation of heterocysts, cells specialized for nitrogen fixation in filamentous cyanobacteria (6, 10-12). A third is observed in the chromosome of the mother cell during sporulation of Bacillus subtilis (20, 29, 33). All three of these events result in the excision of DNA elements that interrupt coding regions of genes required for further development, in the case of B. subtilis, or for nitrogen fixation in the cyanobacteria. The three elements have sizes of II, 55,' and 42 kb (6, 8, 16). Each encodes a site-specific recombinase that recognizes short, directly repeated sequences at its own ends and catalyzes,.the. excision of a nonreplicating circular DNA molecule and the fusion oHhct.previously distant parts of a gene. Each of the rearrangements occurs,after�a series of signal transductions, gene activations, and even morphological differentiations that have occurred in response to environmental cues. For cyanobacteria, these developmental rearrangements are seen in every chromosome in developing heterocysts (between 10 and 20 per cell in Anabaena), but never in vegetative cells. Similarly, in B. subtilis the rearrangements are seen in every mother cell but never in the forespore. Both the heterocyst and the mother cell are terminal cells, providing no DNA for the next generation. The vegetative cells of cyanobacteria and the forespores that give rise to the next generation of bacilli retain the interrupting elements and must therefore excise them every time they differentiate. One well-studied example in Escherichia coli can be considered a devel opmentally regulated gene rearrangement of this type, but it does not inure to the benefit of the cell. A lambda lysogen subjected to environmental insult that results in DNA damage will destroy the lambda repressor. As a consequence, the integrated lambda chromosome is excised by recombination between directly repeated sequences at the ends of the lambda chromosome. The essential difference here is that the excised element replicates and encodes proteins that package the viral chromosome. The host is killed but the virus can survive and eventually find another home. We might therefore view the cyanobacterial and B. subtilis elements as the descendants of temperate viruses that have lost the ability to replicate independently of the host chromosome and perhaps to encode and/or transcribe viral structural proteins. Of course, they would have to retain the ability to excise or they would have disappeared long ago. The matter of selective advantage they may confer on their hosts is considered later.
PROKARYOTIC GENE REARRANGEMENTS
115
STOCHASTIC INVERSIONS
Annu. Rev. Genet. 1992.26:113-130. Downloaded from www.annualreviews.org by University of California - San Francisco UCSF on 01/28/15. For personal use only.
Having emphasized the differences between developmentally regulated gene rearrangements and the stochastic inversions and transpositions that provide a few cells with new flagella or pili or polysaccharide, it is useful nevertheless to review the molecular details of some of the latter phenomena. One of the best studied is the inversion of the DNA element carrying the promoter for one of the flagellar protein operons in
Salmonella typhimurium (13, 36). In
one orientation, the element promotes transcription of a gene encoding one flagellar protein and a repressor of a distant gene encoding an alternative flagellar protein. Inversion of the element blocks transcription of both the first antigen and the repressor genes, permitting expression of the second antigen gene instead. Inversion requires three cis-acting elements in the DNA and two proteins, one encoded by the element (Hin) and one encoded by the host (Fis). Hin protein dimers bind to sites at the ends of the element. Fis dimers bind to two closely spaced sites within the element, called the recombinational enhancer. Fis-Hin interaction results in the formation of an "invertasome" at the enhancer site containing two Fis dimers, two Hin dimers, and both of the directly repeated target sequences. Special in vitro conditions permit the identification of intermediates in the formation of the invertasome and several partial reaction products, one of which contains Hin protein covalently attached to DNA cleaved at the recombination site. The DNA must be negatively supercoiled for invertasome formation. Fis binding at the enhancer results in bending the DNA, determined experimentally by gel electrophoresis and methylation protection (14). The Fis protein structure has been determined by high resolution X-ray crystallography. Efficient docking of a model of the Fis dimer with DNA requires that the DNA be bent at discrete sites (14). Bending may be necessary for enhancer function. This may be a widespread pathway for this class of recombinational event. In Mu transposition, it is possible that the recombinase recognizes both the enhancer and the recombination sites, but a Fis function supplied by the host is still necessary (5). In lambda excision, the Int and Xis proteins require both Fis and IHF (Integration Host Factor) for efficient excision, but in this case the only function of IHF is to bend the DNA and Fis has not been shown to bind to lnt or Xis, although a complex structure, the "intasome" is indeed formed. Both Mu transposition and lambda integration and excision require negatively supercoiled DNA. With this background, we proceed to a description of the rearrangements
B. subtiUs. The plural cyanobacteria is used because, (Anabaena sp. strain PCC 7120), elements similar to the ll-kb interruption of the nifD coding region in Anabaena 71 20 have been detected at the same location in a large in cyanobacteria and
although most information is available for a single strain
116
HASELKORN
number of Anabaena and Nostoc strains collected in many parts of the world
Annu. Rev. Genet. 1992.26:113-130. Downloaded from www.annualreviews.org by University of California - San Francisco UCSF on 01/28/15. For personal use only.
(23). NITROGEN FIXATION AND HETEROCYST DIFFERENTIATION Nitrogen fixation, the reduction of atmospheric nitrogen to ammonia, occurs only in prokaryotes. Molecular analysis of the genes required for nitrogen
Klebsiella pneumoniae, but nif gene organization, sequence, regulation,and function is also available for two species of Azotobacter, Rhodobacter capsulatus, and fixation is most extensive for the soil bacterium
detailed analysis of
several species each of root-nodulating Rhizobium and Bradyrhizobium
(32).
Substantial amounts of nitrogen are fixed also by cyanobacteria which inhabit soil surfaces,lakes,streams,and the oceans, as well as the roots of cycads, the leaves of small ferns and homworts,and the mucus glands of the giant angiosperm
Gunnera (12).
Some nitrogen-fixing cyanobacteria are unicellu
lar, but most are filamentous. Many species of filamentous nitrogen-fixing cyanobacteria differentiate specialized cells called heterocysts, where nitrogen fixation occurs under conditions of nitrogen limitation. Heterocysts are usually spaced at regular intervals along the filament, with up to
10% of the cells
Anabaena or Nostoc. In the symbiotic associa tions,such as Nostoc-Gunnera, for example, the heterocyst frequency can be
differentiating in free-living much higher,up to
60% (31). The symbiotic association can support such
high frequencies because the host plant provides the carbohydrate needed to fuel nitrogen reduction; in the free-living case,the vegetative cells must do this. Certain aspects of the biochemistry of nitrogen fixation are common to every system studied. The nitrogenase complex has two separable compo nents: nitrogenase itself, an
(12132
tetramer of molecular weight
-240,000,
which includes several Fe
Annu. Rev. Genet. 1992.26:113-130. Downloaded from www.annualreviews.org by University of California - San Francisco UCSF on 01/28/15. For personal use only.
Annu.Rev.Genet. 1992.26:113-30 Copyright © by Annual Reviews Inc.All rights reserved
Further
Quick links to online content
DEVELOPMENTALLY REGULATED GENE REARRANGEMENTS IN PROKARYOTES Robert Haselkorn Department of Molecular Genetics and Cell Biology, University of Chicago,
Chicago, Illinois 60637
KEY WORDS: gene rearrangement, heterocys t, s porulation, Anabaena, Bacillus
CONTENTS INTRODUCTION. ..... . . . . . ..... . . . . . . .. . . . . . . . . . . . . . . . . . . STOCHASTIC INVERSIONS
. . . .. . ... ... . ...... ... . . . .... . ....
1 13 1 15
NITROGEN FIXATION AND HETEROCYST DIFFERENTIATION . . . . . . . . . .
1 16
NifGENE REARRANGEMENT IN ANABAENA . . . . . . . . . . . . . . . . . . . . . . .
1 19
Spo GENE REARRANGEMENT IN B. SUBT/US . . . . . . . . . . . . . . . . . . . . . .
125
WHAT PRESERVES THE INTERRUPTING ELEMENTS? ....... . . . ......
128
INTRODUCTION Gene rearrangements are endemic in bacteria. Transposing elements and inverting elements have been described in virtually every bacterial species for which transposition or inversion provides a scorable phenotype. Rearrange ment in these cases provides a small fraction of the cell population with a phenotype, usually a surface property, that allows these few cells to evade surveillance or to respond immediately to a new environment. In contrast to the inducible systems that require signal transduction, gene activation, transcription, translation and possibly dilution or turnover of a prior manifes tation of gene expression, the cells containing rearranged genes are, like boy scouts, always prepared. If the eventuality for which they are prepared does not occur, no problem. Even if they are disadvantaged in the climate of the 1 13
0066-4197/92/1215-0113$02.00
Annu. Rev. Genet. 1992.26:113-130. Downloaded from www.annualreviews.org by University of California - San Francisco UCSF on 01/28/15. For personal use only.
1 14
HASELKORN
time and would normally be diluted by the faster growth of cells with unrearranged genomes, they are recreated each�genei"ation by the low-fre quency, spontaneous rearrangement event. These phenomena can be contrasted with the developmentally regulated rearrangements that are the subject of this review. Two of these occur during the differentiation of heterocysts, cells specialized for nitrogen fixation in filamentous cyanobacteria (6, 10-12). A third is observed in the chromosome of the mother cell during sporulation of Bacillus subtilis (20, 29, 33). All three of these events result in the excision of DNA elements that interrupt coding regions of genes required for further development, in the case of B. subtilis, or for nitrogen fixation in the cyanobacteria. The three elements have sizes of II, 55,' and 42 kb (6, 8, 16). Each encodes a site-specific recombinase that recognizes short, directly repeated sequences at its own ends and catalyzes,.the. excision of a nonreplicating circular DNA molecule and the fusion oHhct.previously distant parts of a gene. Each of the rearrangements occurs,after�a series of signal transductions, gene activations, and even morphological differentiations that have occurred in response to environmental cues. For cyanobacteria, these developmental rearrangements are seen in every chromosome in developing heterocysts (between 10 and 20 per cell in Anabaena), but never in vegetative cells. Similarly, in B. subtilis the rearrangements are seen in every mother cell but never in the forespore. Both the heterocyst and the mother cell are terminal cells, providing no DNA for the next generation. The vegetative cells of cyanobacteria and the forespores that give rise to the next generation of bacilli retain the interrupting elements and must therefore excise them every time they differentiate. One well-studied example in Escherichia coli can be considered a devel opmentally regulated gene rearrangement of this type, but it does not inure to the benefit of the cell. A lambda lysogen subjected to environmental insult that results in DNA damage will destroy the lambda repressor. As a consequence, the integrated lambda chromosome is excised by recombination between directly repeated sequences at the ends of the lambda chromosome. The essential difference here is that the excised element replicates and encodes proteins that package the viral chromosome. The host is killed but the virus can survive and eventually find another home. We might therefore view the cyanobacterial and B. subtilis elements as the descendants of temperate viruses that have lost the ability to replicate independently of the host chromosome and perhaps to encode and/or transcribe viral structural proteins. Of course, they would have to retain the ability to excise or they would have disappeared long ago. The matter of selective advantage they may confer on their hosts is considered later.
PROKARYOTIC GENE REARRANGEMENTS
115
STOCHASTIC INVERSIONS
Annu. Rev. Genet. 1992.26:113-130. Downloaded from www.annualreviews.org by University of California - San Francisco UCSF on 01/28/15. For personal use only.
Having emphasized the differences between developmentally regulated gene rearrangements and the stochastic inversions and transpositions that provide a few cells with new flagella or pili or polysaccharide, it is useful nevertheless to review the molecular details of some of the latter phenomena. One of the best studied is the inversion of the DNA element carrying the promoter for one of the flagellar protein operons in
Salmonella typhimurium (13, 36). In
one orientation, the element promotes transcription of a gene encoding one flagellar protein and a repressor of a distant gene encoding an alternative flagellar protein. Inversion of the element blocks transcription of both the first antigen and the repressor genes, permitting expression of the second antigen gene instead. Inversion requires three cis-acting elements in the DNA and two proteins, one encoded by the element (Hin) and one encoded by the host (Fis). Hin protein dimers bind to sites at the ends of the element. Fis dimers bind to two closely spaced sites within the element, called the recombinational enhancer. Fis-Hin interaction results in the formation of an "invertasome" at the enhancer site containing two Fis dimers, two Hin dimers, and both of the directly repeated target sequences. Special in vitro conditions permit the identification of intermediates in the formation of the invertasome and several partial reaction products, one of which contains Hin protein covalently attached to DNA cleaved at the recombination site. The DNA must be negatively supercoiled for invertasome formation. Fis binding at the enhancer results in bending the DNA, determined experimentally by gel electrophoresis and methylation protection (14). The Fis protein structure has been determined by high resolution X-ray crystallography. Efficient docking of a model of the Fis dimer with DNA requires that the DNA be bent at discrete sites (14). Bending may be necessary for enhancer function. This may be a widespread pathway for this class of recombinational event. In Mu transposition, it is possible that the recombinase recognizes both the enhancer and the recombination sites, but a Fis function supplied by the host is still necessary (5). In lambda excision, the Int and Xis proteins require both Fis and IHF (Integration Host Factor) for efficient excision, but in this case the only function of IHF is to bend the DNA and Fis has not been shown to bind to lnt or Xis, although a complex structure, the "intasome" is indeed formed. Both Mu transposition and lambda integration and excision require negatively supercoiled DNA. With this background, we proceed to a description of the rearrangements
B. subtiUs. The plural cyanobacteria is used because, (Anabaena sp. strain PCC 7120), elements similar to the ll-kb interruption of the nifD coding region in Anabaena 71 20 have been detected at the same location in a large in cyanobacteria and
although most information is available for a single strain
116
HASELKORN
number of Anabaena and Nostoc strains collected in many parts of the world
Annu. Rev. Genet. 1992.26:113-130. Downloaded from www.annualreviews.org by University of California - San Francisco UCSF on 01/28/15. For personal use only.
(23). NITROGEN FIXATION AND HETEROCYST DIFFERENTIATION Nitrogen fixation, the reduction of atmospheric nitrogen to ammonia, occurs only in prokaryotes. Molecular analysis of the genes required for nitrogen
Klebsiella pneumoniae, but nif gene organization, sequence, regulation,and function is also available for two species of Azotobacter, Rhodobacter capsulatus, and fixation is most extensive for the soil bacterium
detailed analysis of
several species each of root-nodulating Rhizobium and Bradyrhizobium
(32).
Substantial amounts of nitrogen are fixed also by cyanobacteria which inhabit soil surfaces,lakes,streams,and the oceans, as well as the roots of cycads, the leaves of small ferns and homworts,and the mucus glands of the giant angiosperm
Gunnera (12).
Some nitrogen-fixing cyanobacteria are unicellu
lar, but most are filamentous. Many species of filamentous nitrogen-fixing cyanobacteria differentiate specialized cells called heterocysts, where nitrogen fixation occurs under conditions of nitrogen limitation. Heterocysts are usually spaced at regular intervals along the filament, with up to
10% of the cells
Anabaena or Nostoc. In the symbiotic associa tions,such as Nostoc-Gunnera, for example, the heterocyst frequency can be
differentiating in free-living much higher,up to
60% (31). The symbiotic association can support such
high frequencies because the host plant provides the carbohydrate needed to fuel nitrogen reduction; in the free-living case,the vegetative cells must do this. Certain aspects of the biochemistry of nitrogen fixation are common to every system studied. The nitrogenase complex has two separable compo nents: nitrogenase itself, an
(12132
tetramer of molecular weight
-240,000,
which includes several Fe
