The use of mini-Gal plasmids for rapid incompatibility grouping of conjugative R plasmids

Davey, Ronald B.; Bird, Phillip I.; Nikoletti, S; Praszkier, Judyta; Pittard, J

doi:10.1016/0147-619x(84)90029-5

Cited by 36 publications

(25 citation statements)

References 18 publications

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“…Bases in the interior loops of the upper stem have been shown to be as critical to the interaction between the antisense molecules and their targets as bases in the hairpin loops (43), so it is not surprising that despite the similarity in their hairpin loops, the IncL/M and IncK plasmids are compatible. Similarly, the differences in the hairpin loops and the interior loops of the upper stems of the respective antisense RNAs are sufficient to account for the observation that pMU604 is also compatible with both IncB and FII plasmids (9). pMU604 was found to encode RepA, a protein of 351 amino acids which shares approximately 40% identity over most of its length with the replication initiator proteins of the IncI 1 plasmid ColIb-P9 (13), the IncB plasmid pMU720 (36), the Rep-FIC replicon P307 (23), and the IncK plasmid pMU2209 (36).…”

Section: Discussionmentioning

confidence: 96%

“…If transcribed, such an RNA molecule would be 75 to 77 bases long. To determine whether the region of DNA predicted to encode this small RNA is responsible for the observed incompatibility reactivity of the 805-bp PstI fragment (nt 1 to 805 [9]), a fragment spanning nucleotides 507 to 698 of pMU604 was cloned into pACYC177. Incompatibility tests performed with this derivative, pMU3265, revealed that it expressed strong incompatibility against pMU604.…”

Section: Resultsmentioning

confidence: 99%

“…To test for incompatibility reactivity between two plasmids, the host strain JP3438 containing the IncL/M mini-Gal plasmid, in which the minimal replicon of the IncL/M plasmid pMU407.1 is linked to the Gal operon as a marker (9), was transformed with derivatives of pACYC177, each of which contained the individual PstI fragments constituting the replicon of pMU604 (Fig. 1).…”

Section: Methodsmentioning

confidence: 99%

“…The minimal medium used was half-strength buffer 56 (27) supplemented with 0.2% (wt/vol) glucose or other carbon sources as indicated, thiamine (10 g/ml), and necessary growth factors. The indicator medium used to screen clones for their ability to forment galactose consisted of a galactose, tryptone, and agar basal medium with tetrazolium salts (T medium) and appropriate antibiotics (9) 32 P]ATP (3,000 Ci/mmol; 10 mCi/ml) were obtained from Amersham Corp. or NEN-Dupont. Kanamycin was used at a final concentration of 20 g/ml, rifampin was used at a final concentration of 100 g/ml, trimethoprim was used at final concentrations of 10 g/ml in minimal medium and 40 g/ml in nutrient medium, isopropyl-␤-D1-thiogalactopyranoside (IPTG) was used at 1 mM, and 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal) was used at 25 g/ml.…”

Section: Methodsmentioning

confidence: 99%

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The replication of an IncL/M plasmid is subject to antisense control

1995

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A 2,385-bp sequence that contains the information for the autonomous replication of the IncL/M plasmid pMU604 was characterized. Genetic analyses revealed that the replicon specifies at least four structural genes, designated repA, repB, repC, and rnaI. The repA gene encodes a protein with a molecular weight of 40,861 which probably functions as an initiator for replication. The functions of the proteins of the repB and repC genes are unclear; however, mutations in the start codon of repB reduced the expression of both repB and repA, indicating that these two genes are translationally coupled. The rnaI gene encodes a small antisense RNA of about 75 to 77 bases and is responsible for the incompatibility phenotype, thus implicating its role as the main copy number determinant. RNAI exerts its effect in trans to repress the expression of repA at the posttranscriptional level. Furthermore, two complementary sequences of 8 bases, with the potential to interact and form a putative pseudoknot structure, were identified in the leader region of the repA mRNA. Base-pairing between the two complementary sequences was shown to be critical for efficient repA expression. A model for the regulation of pMU604 replication involving both translational coupling and pseudoknot formation is proposed.Plasmids that employ a system of copy number control that involves inhibitor molecules binding to target sequences generally regulate their replication via a small antisense RNA molecule. The target of this inhibitory RNA is the complementary region of a large RNA transcript, whose product is essential for replication. Both antisense and target RNAs are highly structured molecules capable of assuming stable stem-andloop configurations. These structural motifs are just as important as their primary sequences for efficient and specific interaction between antisense and target molecules (6,10,32,34,43,47,52). Because the small RNAs act in trans, they are also able to affect the replication of other plasmids showing the same specificity of replication control. The ability to interact with the replication machinery of other plasmids means that antisense RNAs act as incompatibility determinants (29).Although a number of plasmids use antisense RNA to regulate the frequency with which they initiate replication, the particular molecular mechanism differs for each system. For example, replication of the ColE1 plasmids is regulated by an antisense RNA, which by binding to a preprimer RNA alters its folding and prevents its subsequent processing to form a primer for the initiation of DNA synthesis (19,24,49,50). The antisense RNAs of the pT181, FII, IncI 1 and IncB plasmids inhibit the expression of the genes coding for the essential replication initiation proteins (Rep) (13,30,31,35,42,46). In pT181, binding of the antisense alters the folding of the rep mRNA, resulting in transcriptional attenuation of the message (31). Control of copy number in the FII plasmids R1 and NR1 involves the hybridization of the antisense RNA with its complementary sequ...

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Section: Discussionmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The replication of an IncL/M plasmid is subject to antisense control

1995

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“…Most of the antibiotics, media, and chemicals have been described in detail by Davey et al (8). Galactose fermentation was monitored on galactose-tetrazolium agar (T medium) described by Davey et al (8).…”

Section: Methodsmentioning

confidence: 99%

Analysis of the incompatibility determinants of I-complex plasmids

et al. 1988

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The isolation and characterization of minireplicons corresponding to group B and I-complex plasmids are reported. The molecular structures of the small RNAs that may play a major role in the replication control and incompatibility reactions of the plasmids are compared. A mutant plasmid with changed copy number and incompatibility specificity is described. This mutant had a single-base-pair substitution in the DNA region that codes for the small RNA.Plasmid replication in bacteria is, in some cases, primarily controlled by a small RNA molecule whose sequence is complementary to a part of an RNA molecule essential for replication (rep) (5,6,16,17,30,(35)(36)(37). The rep RNA and the small countertranscript RNA are transcribed from complementary strands of the DNA double helix. The small RNAs act in trans to inhibit or control replication. In the case of ColEl, it is postulated that the rep RNA acts as a primer for the initiation of plasmid replication and that the interaction with the countertranscript RNA interferes with the processing of the primer (16,20,33,34,36). In the case of FII plasmids, although the function of the rep mRNA remains unclear, it has been shown that the countertranscript RNA interferes with its translation (18).Because they act in trans, these countertranscript RNAs also affect the replication of other plasmid molecules showing the same specificity of replication control. In this way, these RNAs appear to play a major role in defining the incompatibility reaction of these particular plasmids.The group B plasmids and plasmids previously grouped together in the I complex exhibit some cross-reactivity in their incompatibility phenotypes (7). Studies of a minireplicon derived from a group B plasmid have shown that a small RNA is synthesized from a region of DNA which specifies essential replication functions (4). More recent work has shown that this small RNA is a countertranscript RNA and that it interferes with the elongation, stability, or translation of the rep mRNA with which it shows complementarity (this paper; J. Praszkier, P. Bird, S. Nikoletti, and J. Pittard, manuscript in preparation).In this paper, we describe the isolation and characterization of minireplicons corresponding to group B, I1, and IY plasmids and compare the molecular structures of the small RNAs which appear to play a major role in both replication control and incompatibility reactions of these plasmids. We also describe a mutant plasmid which exhibited changes in both copy number and incompatibility specificity. This plasmid had a single-base-pair substitution in the region of DNA coding for the small RNA.

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Plasmid Incompatibility

Thomas¹

2018

Molecular Life Sciences

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The use of mini-Gal plasmids for rapid incompatibility grouping of conjugative R plasmids

Cited by 36 publications

References 18 publications

The replication of an IncL/M plasmid is subject to antisense control

The replication of an IncL/M plasmid is subject to antisense control

Analysis of the incompatibility determinants of I-complex plasmids

Plasmid Incompatibility

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