Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids

Ballester, Sara; López, Paloma; Alonso, Juan C.; Espinosa, Manuel; Lacks, Sanford A.

doi:10.1016/0378-1119(86)90094-6

Cited by 57 publications

(31 citation statements)

References 21 publications

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“…The location of the fixed border of the deleted derivatives of pJS37 within the plasmid replication region of the pC194 moiety suggested involvement of RepH activity in generation of these deletions, as has been indicated for a different class of deletions (24). This idea was supported by the finding that pJS140, an AccI-EcoRI-deleted derivative of pJS37 that lacks RepH, seg, and OrfC but conserves the RepH target sequence, generated only one deleted plasmid (pJS5), in which the deletion endpoint in the pC194 moiety was far from positions 5872 to 5880 in pJS37 (3).…”

Section: Resultsmentioning

confidence: 54%

“…Two kinds of intramolecular recombination events have been reported. In the first kind, the endpoints of deletions occur within short direct repeats (1,3,11,20); in the second, deletions occur between nonidentical sequences. The latter kind has been observed in Bacillus subtilis (20) In addition to these mechanisms, the plasmid replication machinery may be responsible for generating deletions (11,24).…”

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confidence: 99%

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Plasmid structural instability associated with pC194 replication functions

et al. 1989

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The hybrid plasmid pJS37 is composed of the streptococcal plasmid pLS1, which confers tetracycline resistance, and the staphylococcal plasmid pC194, which confers chloramphenicol resistance. When grampositive bacteria containing pJS37 were grown in the presence of chloramphenicol, four different deleted derivatives accumulated. The deletions in the plasmid enhanced resistance to chloramphenicol by placing the cat gene of pC194 near promoters of pLS1. All four deletions shared a common endpoint that corresponded to the putative target site for DNA strand nicking by the pC194 replication protein, RepH. At the other, variable endpoint, the DNA sequence was similar to the putative RepH target sequence. Alteration of the RepH protein, by in vitro modification of the gene encoding it, eliminated this class of deletions. By extending a previously proposed model for the generation of a different but related class of deletions (B. Michel and S. D. Ehrlich, EMBO J. 5:3691-3696, 1986), a comprehensive model that could generate both classes of deletions is suggested.It proposes that a nicking-closing activity of the plasmid replication protein at its normal target site and, aberrantly, at sites with similar sequence can generate deletions either proximal or distal to the aberrant site during rolling-circle replication of the plasmid.The generation of deletions in recombinant plasmids by intramolecular rearrangement is a common phenomenon. Many of these deletions involve short direct repeats. Although some of them may arise by typical homologous recombination between the direct repeats, which in Escherichia coli requires recA function, others occur in recA mutants apparently as a result of other mechanisms for recombining DNA (1). Two kinds of intramolecular recombination events have been reported. In the first kind, the endpoints of deletions occur within short direct repeats (1,3,11,20); in the second, deletions occur between nonidentical sequences. The latter kind has been observed in Bacillus subtilis (20) In addition to these mechanisms, the plasmid replication machinery may be responsible for generating deletions (11,24). In B. subtilis, a hybrid plasmid composed of pUB110 and pSA2100 (itself composed of pC194 and pSA0501) showed a systematic rec-independent deletion between 18-base-pair (bp) directly repeated sequences (11) located at the plasmid origins of replication (9,21 suggested that a nick introduced in the postulated replication origin of pC194 by the plasmid replication protein served as a deletion endpoint (24). Since accumulation of singlestranded DNA during the replication of pC194 indicates that this plasmid replicates by a rolling-circle mechanism (30), there appears to be a correlation between deletion formation and replication through single-stranded intermediates.We have previously shown that the hybrid plasmid pJS37, composed of plasmids pLS1 and pC194, which carries two origins of replication instead of three as in the examples cited above, undergoes systematic deletions in Streptococcus pneumoniae...

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Section: Resultsmentioning

confidence: 54%

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confidence: 99%

Plasmid structural instability associated with pC194 replication functions

et al. 1989

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“…The downstream ORF is 64 amino acids long and is homologous to EPUA protein, a possible membrane-bound nuclease required for DNA trans- 555 bp were amplified by PCR and used to make constructs in which they flanked an antibiotic resistance gene. In the case of murA1, the constitutively expressed erythromycin resistance gene cassette ermAM from pAM␤1 was used (17), while the chloramphenicol resistance cat gene from pJS3 was used in the murA2 construct (2). To minimize the potential polar effects of gene replacement, the PCR primers were chosen so that flanking genes and potential promoters would remain intact in the deletion mutant.…”

Section: Methodsmentioning

confidence: 99%

Two Active Forms of UDP- N -Acetylglucosamine Enolpyruvyl Transferase in Gram-Positive Bacteria

Du¹,

Brown²,

Sylvester³

et al. 2000

J Bacteriol

124

121

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Gene sequences encoding the enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from many bacterial sources were analyzed. It was shown that whereas gram-negative bacteria have only one murA gene, gram-positive bacteria have two distinct genes encoding these enzymes which have possibly arisen from gene duplication. The two murA genes of the gram-positive organism Streptococcus pneumoniae were studied further. Each of the murA genes was individually inactivated by allelic replacement. In each case, the organism was viable despite losing one of its murA genes. However, when attempts were made to construct a doubledeletion strain, no mutants were obtained. This indicates that both genes encode active enzymes that can substitute for each other, but that the presence of a MurA function is essential to the organism. The two genes were further cloned and overexpressed, and the enzymes they encode were purified. Both enzymes catalyzed the transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine, confirming they are both active UDP-N-acetylglucosamine enolpyruvyl transferases. The catalytic parameters of the two enzymes were similar, and they were both inhibited by the antibiotic fosfomycin.

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“…Plasmids pMV158 (5), pLS1 (33), and the derivatives made in the present work were transferred into strain 1131(pIP501) by transformation. The source of the DpnIl fragment containing the cat gene was pJS3 (3) grown in S. pneuimoniae 708. Growth, transformation, and conjugation of bacteria. Cultures of S. pneiumoniie were grown with sucrose in semisynthetic medium (19) and transformed as previously de- For conjugal plasmid transfer, filter matings were carried out in CAT medium as described by Smith and Guild (31).…”

Section: Methodsmentioning

confidence: 99%

Region of the streptococcal plasmid pMV158 required for conjugative mobilization

Priebe

Lacks

1989

J Bacteriol

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The nonconjugative streptococcal plasmid pMV158 can be mobilized by the conjugative streptococcal plasmid pIP501. We determined the sequence of the 1.1-kilobase EcoRI fragment of pMV158 to complete the DNA sequence of the plasmid. We showed that an open reading frame, mob (able to encode a polypeptide of 58,020 daltons), is required for mobilization of pMV158. An intergenic region present in the EcoRI fragment contains four lengthy palindromes that are found also in one or more of the staphylococcal plasmids pT181, pE194, and pUBIIO. One palindromic sequence, palD, which is common to all four plasmids, also appeared to be necessary for mobilization. Circumstantial evidence indicates that this sequence contains both an oriT site and the mob promoter. The Mob protein is homologous in its amino-terminal half to Pre proteins encoded by pT181 and pE194 that were shown by others to be essential for site-specific cointegrative plasmid recombination; their main biological function may be plasmid mobilization.A variety of conjugative and nonconjugative plasmids are found in streptococcal species (for a review, see reference 6). The conjugative plasmid pIP501 confers erythromycin resistance (Emr) and chloramphenicol resistance (Cm'-). This 30-kilobase (kb) plasmid was found by Horodniceanu and co-workers in a strain of Streptococcus ag(llactiae (15) and shown to pass conjugatively into various streptococcal species (13). The 5.5-kb nonconjugative plasmid pMV158 confers tetracycline resistance (Tcr); it was found by Burdett in another strain of S. agalactiae (5). Guild and co-workers transferred the two plasmids into Str eptococcils pneitnoniae by transformation (32). They showed that when both plasmids were present in the same cell, pIP501 was able to mobilize pMV158 for conjugative transfer. In this work, we inquired into the molecular basis of such mobilization.A derivative of pMV158 called pLS1, which was constructed by removal of a 1.1-kb EcoRI fragment, replicates normally in S. pneirmoniae (33). Both pMV158 and pLS1 also replicate in other gram-positive bacteria, such as Bacillus sibtilis (11), and in gram-negative bacteria, such as Escherichia coli (20 Novick and co-workers found that recombination between two staphylococcal plasmids, pT181 and pE194, to give a cointegrate product occurred within closely related 70-basepair (bp) sequences (called RSA) in these plasmids (12,25). This site-specific recombination was independent of a )ecA mutation, but it required a protein called Pre (for plasmid recombination), encoded by a plasmid gene, pi-e (12). The * Corresponding author. pre gene product of either pT181 or pE194 could promote recombination, and the pT181 protein was shown to act in tr'atis, that is, when the active pre gene was present on a plasmid not participating in the cointegrate formation (12). The Pre proteins predicted from the DNA sequence of pT181 and pE194 appeared to be homologous, and it was pointed out that another staphylococcal plasmid, pUB110, contains a sequence identifical to part of RSA and ...

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Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids

Cited by 57 publications

References 21 publications

Plasmid structural instability associated with pC194 replication functions

Plasmid structural instability associated with pC194 replication functions

Two Active Forms of UDP- N -Acetylglucosamine Enolpyruvyl Transferase in Gram-Positive Bacteria

Region of the streptococcal plasmid pMV158 required for conjugative mobilization

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