A Rare 920-Kilobase Chromosomal Inversion Mediated by IS1 Transposition Causes Constitutive Expression of the yiaK-S Operon for Carbohydrate Utilization in Escherichia coli

Abstract

The regulator of the yiaK-S operon, currently assigned a carbohydrate utilization function in Escherichia coli, is inactivated by a genome rearrangement that leads to the constitutive expression of the operon. the yiaK-S constitutive cells acquire the ability to utilize the rare pentose l-lyxose. Restriction analysis and sequencing of the regulator gene indicate that it is disrupted by foreign DNA. The insert consists of a large inverted fragment of DNA of 920 kilobases flanked by two IS1 elements with opposite polarity. One corresponds to that found naturally at min 0.4 of the bacterial chromosome and the other to a new copy transposed into the regulator gene located at min 80.6. This insertion-inversion could be the result of the intramolecular transposition mechanism itself, a gene rearrangement rarely originated by IS1. Alternatively, it could be attributed to the homologous recombination between the IS1 at min 0.4 and the IS1 transposed intermolecularly into the yiaK-S regulator gene. The participation of a rare IS1-mediated inversion in the evolution of a stable phenotype is thus identified. The regulator of the yiaK-S operon, currently assigned a carbohydrate utilization function in Escherichia coli, is inactivated by a genome rearrangement that leads to the constitutive expression of the operon. the yiaK-S constitutive cells acquire the ability to utilize the rare pentose l-lyxose. Restriction analysis and sequencing of the regulator gene indicate that it is disrupted by foreign DNA. The insert consists of a large inverted fragment of DNA of 920 kilobases flanked by two IS1 elements with opposite polarity. One corresponds to that found naturally at min 0.4 of the bacterial chromosome and the other to a new copy transposed into the regulator gene located at min 80.6. This insertion-inversion could be the result of the intramolecular transposition mechanism itself, a gene rearrangement rarely originated by IS1. Alternatively, it could be attributed to the homologous recombination between the IS1 at min 0.4 and the IS1 transposed intermolecularly into the yiaK-S regulator gene. The participation of a rare IS1-mediated inversion in the evolution of a stable phenotype is thus identified. Most known mobile IS 1The abbreviations used are: IS, insertion sequence; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s). can be inserted at a large number of chromosomal sites. They can subsequently be recombined and excised precisely or imprecisely, and they can engender inversions or deletions in neighboring chromosomal regions (1Galas D.J. Chandler M. Berg D.E. Howe M.M. Mobile DNA. American Society for Microbiology, Washington, DC1989: 109-162Google Scholar, 2Kleckner N. Annu. Rev. Cell Biol. 1990; 6: 297-327Crossref PubMed Scopus (75) Google Scholar). The presence of these insertion elements in the chromosome of Escherichia coli and other bacteria such asSalmonella typhimurium or Bacillus subtilisoriginally became apparent as a result of the transposition of these sequences from their natural positions into functional genes, which can lead to recognizable mutant phenotypes. As a corollary, their derived deletions, inversions, and other gene rearrangements could also result in phenotypic changes that could normally be stable and easily identifiable. Relevant models of cryptic gene activation by insertion of IS elements into the regulator or promoter region have been reported by several authors. The cryptic cel operon is activated in E. coli by insertion of IS1, IS2 or IS5 into a region 72–180 bp upstream from the transcription start site due to an unknown mechanism that does not involve provision of a promoter sequence (3Parker L.L. Hall B.G. Genetics. 1990; 124: 473-482PubMed Google Scholar). It has also been described that certain excisions of inserted elements function as activation mechanisms. For example, phnE is activated by excision of an 8-bp insertion, which causes a frameshift mutation present in the crypticphn operon (4Makino K. Kim S.K. Shinagawa H. Amemura M. Nakata A. J. Bacteriol. 1991; 173: 2665-2672Crossref PubMed Google Scholar). In another context, transpositions and subsequent deletions or inversions cause changes in the regular expression of several gene systems. Among the mutations mapped in the promoter region of the araBAD operon (5Eleuterio M. Griffin B. Sheppard D.E. J. Bacteriol. 1972; 111: 383-391Crossref PubMed Google Scholar) two insertions of IS1 were found, which resulted in reduced expression of this operon and hence the inability to utilize arabinose (6Miyada G.C. Sheppard D.E. Wilcox G. J. Bacteriol. 1983; 156: 765-772Crossref PubMed Google Scholar). Inversion, of uncertain origin in this case, of a 6-kb fragment containingslpA and slpB in Lactobacillus acidophilus, leads to the positioning of the silentslpB gene behind the slpA promoter, which is located outside the inverted segment, whereas the slpA gene is stored at the silent position of the slpB gene (7Boot H.J. Kolen C.P.A.M. Pouwels P.H. Mol. Microbiol. 1996; 21: 799-809Crossref PubMed Scopus (50) Google Scholar). Various chromosomal rearrangements subsequent to Tn10-mediated inversions that connect the udpgene and promoters of rrn operons were characterized in E. coli (8Fonstein M. Nikolskaya T. Zaporojets D. Nikolsky Y. Kulakauskas S. Mironov A. J. Bacteriol. 1994; 176: 2265-2271Crossref PubMed Google Scholar), some of them involving more complicated events such as partial internal reinversions. Overexpression of udpin these mutants complemented thymine auxotrophy. Another example of these events is a rearrangement subsequent to a REP-mediated gene duplication, which placed the his operon under the control of argR in Salmonella typhimurium (9Anderson R.P. Roth J.R. J. Mol. Biol. 1978; 119: 147-166Crossref PubMed Scopus (37) Google Scholar, 10Shyamala V. Schneider E. Ames G.F.-L. EMBO J. 1990; 9: 939-946Crossref PubMed Scopus (56) Google Scholar). Unexplained inversions and chromosomal rearrangements of up to one-third of the total length have been found in E. coli(11Xia X.M. Enomoto M. Mol. Gen. Genet. 1986; 205: 376-379Crossref PubMed Scopus (6) Google Scholar, 12Hill C.W. Harnish B.W. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7069-7072Crossref PubMed Scopus (175) Google Scholar) or Neisseria gonorrhoeae (13Gibbs C.P. Meyer T.F. FEMS Microbiol. Lett. 1996; 145: 173-179Crossref PubMed Google Scholar). Although precise phenotypic changes were not determined in these models, the effects of these major chromosomal rearrangements on cell fitness have been studied by Hill and Gray (14Hill C.W. Gray J.A. Genetics. 1988; 119: 771-778PubMed Google Scholar) following the growth of two E. coli strains with inversions between rRNA operons. According to this and other studies (15Mahan M.J. Roth J.R. Genetics. 1991; 129: 1021-1032Crossref PubMed Google Scholar) establishment of the inversions appears to depend on the type of genes present within the chromosomal interval. In thinking about evolutionary mechanisms, rearrangements have not been given the consideration that point mutations have received (16Roth J.R. Benson N. Galitski T. Haack K. Lawrence J.G. Miesel L. Neidhardt F.C. Ingraham J.L. Low K.B. Magasanik B. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. American Society for Microbiology, Washington, DC1996: 2256-2276Google Scholar). Nevertheless, rearrangements subsequent to insertion element-jumping events appear to play a role in evolution. Among the insertion sequences able to participate in genome rearrangements, IS1 is one of the smallest autonomous transposable elements known. It can transpose in both a replicative and a conservative manner, but it rarely gives rise to inversions (17Turlan C. Chandler M. EMBO J. 1995; 14: 5410-5421Crossref PubMed Scopus (75) Google Scholar). An inversion induced by a resident IS1 of the lactose transposon Tn951 (18Cornelis G. Saedler H. Mol. Gen. Genet. 1980; 178: 367-374Crossref PubMed Scopus (16) Google Scholar) may be mentioned as one of the few instances of IS1-mediated inversions documented so far. Here the yiaK-S operon (locus lyx at 80.6 min of the E. coli chromosome) (19Blattner F.R. Plunkett III, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1462Crossref PubMed Scopus (6056) Google Scholar), has been found to be constitutively expressed in the mutant strain JA134 selected for its ability to grow on the rare pentose l-lyxose (20Sánchez J.C. Giménez R. Schneider A. Fessner W.D. Baldomà L. Aguilar J. Badı́a J. J. Biol. Chem. 1994; 269: 29665-29669Abstract Full Text PDF PubMed Google Scholar). The constitutive expression of the operon has been shown to be the result of a chromosomal inversion secondary to an IS1 transposition into the repressor gene of the yiaK-S operon. The metabolic consequences of yiaK-S repressor disruption were found to be stable. The bacterial strains used in this work are E. coli K12 derivatives. ECL1 is HfrCphoA8 relA1 tonA22 T2r (λ) (21Lin E.C.C. Annu. Rev. Microbiol. 1976; 30: 535-578Crossref PubMed Scopus (374) Google Scholar). JA134 is Lyx+ rhaB but is otherwise isogenic to ECL1 (20Sánchez J.C. Giménez R. Schneider A. Fessner W.D. Baldomà L. Aguilar J. Badı́a J. J. Biol. Chem. 1994; 269: 29665-29669Abstract Full Text PDF PubMed Google Scholar). JA161 obtained from ECL1 in this work isyiaJ::cat. NM539 is F−, supF, hsd (rk−, mk+),lacY (P2) (22Frischauf A.M. Lehrach H. Poustka A. Murray N. J. Mol. Biol. 1983; 170: 827-842Crossref PubMed Scopus (1087) Google Scholar), and JC7623 is arg thi thr leu pro his strA recB21 recC22 sbcB15 (23Wackernagel W. Biochem. Biophys. Res. Commun. 1973; 51: 306-311Crossref PubMed Scopus (52) Google Scholar). Cells were grown aerobically on Luria broth or minimal medium (24Boronat A. Aguilar J. J. Bacteriol. 1979; 140: 320-326Crossref PubMed Google Scholar). For growth on minimal medium, carbon sources were added at concentrations of 12 mm for l-lyxose, 20 mm for glycerol, and 0.5% for casein acid hydrolysate. When necessary, the antibiotics were used at the following concentrations: ampicillin, 100 μg/ml; tetracycline, 12.5 μg/ml; chloramphenicol, 30 μg/ml. 5-Bromo-4-chloro-3-indolyl-β-d-galactoside and isopropyl-β-d-thiogalactoside were used at 30 and 10 μg/ml, respectively. Cell extracts were prepared as described previously (24Boronat A. Aguilar J. J. Bacteriol. 1979; 140: 320-326Crossref PubMed Google Scholar).l-Xylulose kinase activity was determined from the rate of NADH oxidation. The reaction mixture (1 ml) contained 100 mm Tris-HCl buffer (pH 8.0), 4 mml-xylulose, 1 mm ATP, 1.75 mmphosphoenolpyruvate, 3.5 mm MgCl2, 0.25 mm NADH, 0.35 mm reduced glutathione and 2 units/ml of pyruvate kinase and lactate dehydrogenase. Chromosomal DNA was obtained using the genomic DNA purification kit (Promega, Madison, WI). Plasmid DNA was routinely prepared by the boiling method (25Holmes D.S. Quigley M. Anal. Biochem. 1981; 114: 193-197Crossref PubMed Scopus (2019) Google Scholar). For a large scale preparation, a crude DNA sample was purified through a column (Qiagen GmbH, Düsseldorf, Germany). Other standard DNA manipulations were performed essentially as described by Sambrook et al. (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The DNA sequence was determined by the dideoxy chain termination procedure of Sanger et al. (27Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52769) Google Scholar) by using the T7 sequencing kit from Amersham Pharmacia Biotech. Double-stranded plasmid DNA was used as the template. Ordered deletions were obtained with the Erase-a-Base system (Promega). Universal or T3 primers were used. To avoid sequencing errors, both strands were sequenced by regular dGTP as well as dITP or 7-deaza-dGTP reactions. Primer A (TTTGGTGGTCAGGCGAT) and primer B (CACACTTTGCGCTACCA) were also used to confirm the sequence of the two insertional points in the regulator gene. The GenBankTM data base was searched with the TFASTA algorithm (28Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9393) Google Scholar). For Southern blot analysis, restriction fragments were separated by agarose gel electrophoresis, blotted onto nylon filters, and fixed by incubating at 80 for 2 A was used in the of Chromosomal DNA from strains ECL1 and JA134 were with and with the with as described by al. (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The of in was performed by using the system and on NM539 (P2) for of For strain JA134 a partial genomic was also prepared in a For of total cells of a grown to of were by at for 10 min and as described by et al. J.G. T. C.W. A. Full Text PDF PubMed Scopus Google Scholar). blot was performed with sample by following the procedure described previously by et al. E. Aguilar J. J. Bacteriol. PubMed Google Scholar). The chloramphenicol was used in the gene Google Scholar) by it into the restriction site This in the region of the gene and thus not cause inserted in the as the gene. The inactivated gene was by and used to strain JC7623 to chloramphenicol This strain DNA into its chromosome G.C. J. Bacteriol. PubMed Google Scholar). obtained from the selected chloramphenicol were used to the insertions into the strain ECL1 or the mutant strain were performed on purified chromosomal DNA with DNA polymerase to the and 100 of of the primers in 100 of reaction The reaction routinely of 30 The primers used to the region to the origin in the mutant strain and and used to the region were and For Southern analysis the of inverted of 1 at of were prepared by using of primers of bp which were following the E. coli genome sequence in were by agarose gel and purified by purification kit (Qiagen strain JA134 is an ECL1 selected for its ability to grow on the rare which does not growth of This mutant was characterized by constitutively kinase activity in (20Sánchez J.C. Giménez R. Schneider A. Fessner W.D. Baldomà L. Aguilar J. Badı́a J. J. Biol. Chem. 1994; 269: 29665-29669Abstract Full Text PDF PubMed Google Scholar). The gene kinase was in plasmid Here have the sequencing of the insert present in This sequence total with that of the located between positions and in the of kinase gene was thus as of a gene which to its at 80.6 min of the E. coli genome has been as Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). on the genome analysis by et al. H.J. Burland V. Plunkett G. R. Res. 1994; PubMed Scopus Google Scholar) this is by genes for carbohydrate to and one analysis of total of strain JA134 using of the genes and that transcription of the operon was in growth used. 2 A a with a internal The by the method (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) using a fragment as template. The of several due to of the In was found in strain in kinase in crude extracts of the used for of activity of to kinase as a activity of the expression of the yiaK-S operon. The role of gene as regulator of by and of this gene. analysis of the regulator gene and with other regulator sequences a of to DNA and a of unknown function were also found in regulator such as repressor or repressor in in to the known S. W. J. Mol. Microbiol. 1991; PubMed Scopus Google Scholar). The regulator gene upstream of the operon was inactivated by a insertion in To this a the operon was from a chromosomal of strain ECL1 prepared as under and The was selected by with a by the method (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) using as a internal fragment obtained from The fragment the regulator gene was in of the regulator gene was by insertion of in the restriction site present in plasmid plasmid and the mutant gene to the ECL1 strain The of the gene was by Southern blot analysis The effects of the of the regulator gene were in cell extracts of strain JA161 by kinase which was found to be present in growth that not this activity in analysis of total of strain JA161 using of the genes and were from obtained with total of strain When a internal to the regulator gene was analysis a constitutive in of strain ECL1 2 and in the strain JA161 The derived in the expression of the yiaK-S operon in strain JA161 that the and that this system is under the control of a repressor by this gene. The constitutive expression of yiaK-S operon in strain JA134 thus that the mutation could this operon To the a by the method using a fragment to was used to Southern of the type and mutant JA134 DNA with or The obtained in these for of the restriction was in the type and mutant strain are with the presence of an insertion or in the region the regulator and the yiaK-S operon the of the regulator gene using internal to the regulator of 2 with the type of mutation that could of transcription or an not with the used. The of of the regulator gene reported was the result of a mutation in this gene and is not with a mutation in the promoter region of the For this the of the mutation was studied in the regulator gene. A of the regulator was from a of strain using a prepared with the fragment insert was to Restriction analysis of the insert in plasmid that foreign DNA sequences were present in the To the DNA fragment not to the regulator of plasmid were prepared and their were analysis with sequences to and to the and genes, located at 0.4 The the fragment with the site for regulator and foreign DNA was obtained and the insert was of the sequences of with GenBankTM sequences to an between sequences to the regulator gene and to the 0.4 which were found to be inverted with to their natural genome To the other not in a from a of strain JA134 was using a prepared with the internal a fragment of the was to Restriction analysis of the insert in plasmid that DNA sequences not to the regulator region were of derived from sequences of the regulator and of another IS1 insertion the other point in the repressor gene. This an IS1 transposition into the This insertion in this between the sequences of the regulator and sequences to located between positions and in the min region The IS1 transposed opposite and a sequence with the IS1 naturally found in the 0.4 min of the E. coli chromosome T. H. H. T. A. N. K. K. Nakata A. Res. PubMed Scopus Google Scholar). The two IS1 elements the derived from the regulator sequences on one The of the inversion and changes in the gene the inverted fragment were by Southern To this fragments of about 1 were used to the inverted of the in the Southern using DNA from strain ECL1 and mutant strain JA134 with restriction It was thus that the inversion was up to 920 in length and that other internal rearrangement in the inverted To confirm the mutation two of the of the inversion and were used in of genomic DNA obtained from type and mutant strain A of was found mutant but not was the template. The of the yiaK-S operon expression subsequent to the IS1 transposition into its regulator phenotypic consequences by the of the ability to The of this mutation is of for its evolutionary To the of the constitutive expression of yiaK-S operon in strain the of the l-lyxose utilization as a phenotype was in its To this a of 1 of strain JA134 was grown on broth up to This was and for a growth to the cell The more 30 for l-lyxose the of the were on and on l-lyxose. the l-lyxose Among the changes that may phenotypic present a in which the regulator gene of an operon is by an inserted DNA This of an length of 920 kb, is flanked by two IS1 analysis of the sequences of and that the insertion into the regulator was the result of an intramolecular the with opposite polarity. an inverted gene of the foreign DNA is in plasmid whereas sequences of the repressor and 0.4 min region are found to of the of the IS1 in plasmid to these it is that a genome inversion from 0.4 to 80.6 the regulator gene has in mutant strain The region could be inverted in the of IS1 intramolecular transposition as a of in opposite of IS1 with the in the precisely located in 80.6 min as by and Chandler (17Turlan C. Chandler M. EMBO J. 1995; 14: 5410-5421Crossref PubMed Scopus (75) Google Scholar) for IS1 and by et al. Science. 1983; PubMed Scopus Google Scholar) for these insertion-inversion in intramolecular are present at The mutation the of this type of rearrangements, its participation in genome evolution. present have on the of this or the well known of IS1, its mechanism of transposition and the rearrangement are As has been (1Galas D.J. Chandler M. Berg D.E. Howe M.M. Mobile DNA. American Society for Microbiology, Washington, DC1989: 109-162Google Scholar, Science. 1983; PubMed Scopus Google Scholar) another mechanism be as an to the described in this case, and subsequent recombination between IS1 at min 0.4 and the IS1 inserted into the could also one such the of of the two are the of the of the at present between Nevertheless, the of two events in this mechanism the of its in the It has been by several that insertion-inversion events that the of certain genes with to the origin of may be or A. M.J. Roth J.R. Science. 1988; PubMed Scopus Google Scholar). This is as one of the functional to inversion To several functional consequences are They changes in copy number of some genes, or of the positioning of the one of the to copy more of the The fragment inverted in mutant strain JA134 from 80.6 to 0.4 min and thus the origin located at It is of that in not was the insertion-inversion mutant and able to be but also growth on carbon sources and were from that obtained with In this a genome rearrangement of phenotypic consequences is as well as the mechanism to This is in to of the or of unknown origin and phenotypic The of yiaK-S operon, as by the ability to utilize l-lyxose, is a stable that the mutation the repressor does not easily In this it is of to that of insertion mutations such as the one described are to be the IS1 or other of the transposed DNA in the gene rise to stable phenotypes. for of this and for