The nucleotide sequence of Staphylococcus aureus plasmid pT48 conferring inducible macrolide-lincosamide-streptogramin B resistance and comparison with similar plasmids expressing constitutive resistance

The complete nucleotide sequence of a naturally occurring Staphylococcus aureus plasmid, pT48 (from S. aureus strain T48), has been determined. The 2475 bp plasmid confers inducible resistance to macrolide-lincosamide-streptogramin B (MLS) type antibiotics. It is similar to the constitutive MLS resistance plasmid, pNE131, from Staphylococcus epidermidis and shows homology with S. aureus plasmids pSN2 and pE194. It contains a palA structure homologous to that on S. aureus plasmid pT181. The open reading frame, ORF B, within the pSN2 homologous region has a frameshifted C-terminus, relative to pNE131, resulting in a smaller, 158 amino acid putative polypeptide. The pE194 homologous region has the ermC resistance determinant and retains the leader region, deleted in pNE131, required for inducible expression of an adenine methylase. Another naturally occurring S. aureus strain, J74, shows constitutive resistance to erythromycin and contains a small plasmid, pJ74, which is similar to pNE131 but with a different deletion in the leader sequence. The results are consistent with the translational attenuation model for ermC expression.     

Characterization of a macrolide, lincosamide, and streptogramin resistance plasmid in Staphylococcus epidermidis.

A strain of Staphylococcus epidermidis was transduced to erythromycin resistance, and all of the transductants exhibited the macrolide, lincosamide, streptogramin B resistance phenotype. Curing and antibiotic disk studies also indicated that these resistances were controlled by a single plasmid determinant and were constitutive. Agarose gel electrophoresis of plasmid deoxyribonucleic acid (DNA) from donor, cured, and transduced strains showed that a single plasmid was responsible. This plasmid, designated pNE131, was examined for sequence homology to two other plasmids, pE194 and p1258, from Staphylococcus aureus, which also code for erythromycin resistance. DNA from plasmids pNE131 and pE194 hybridized with one another, but no extensive homology to pI258 with either pNE131 or pE194 was found. Restriction endonuclease digests of pNE131 and pE194 showed no common fragments. However, sequence homology was localized to the nucleotides in pE194 that code for the 29,000-dalton protein responsible for erythromycin resistance. pNE131 was calculated to have 2,220 base pairs and is the smallest naturally occurring plasmid with a known function yet reported in S. epidermidis.     

Naturally occurring Staphylococcus epidermidis plasmid expressing constitutive macrolide-lincosamide-streptogramin B resistance contains a deleted attenuator.

A naturally occurring constitutive macrolide-lincosamide-streptogramin B (MLS) resistance plasmid, pNE131, from Staphylococcus epidermidis was chosen to study the molecular basis of constitutive expression. Restriction and functional maps of pNE131 are presented along with the nucleotide sequence of ermM, the gene which mediates constitutive MLS resistance. Sharing 98% sequence homology within the 870-base-pair Sau3A-TaqI fragment, ermM appears to be almost identical to ermC, the inducible MLS resistance determinant from S. aureus (pE194). The two genes share nearly identical sequences, except in the 5' promoter region of ermM. Constitutive expression of ermM is due to the deletion of 107 base pairs relative to ermC; the deletion removes critical sequences for attenuation, resulting in constitutive methylase expression.     

Replication properties of pIM13, a naturally occurring plasmid found in Bacillus subtilis, and of its close relative pE5, a plasmid native to Staphylococcus aureus.

A naturally occurring plasmid from Bacillus subtilis, pIM13, codes for constitutively expressed macrolide-lincosamide-streptogramin B (MLS) resistance, is stably maintained at a high copy number, and exists as a series of covalent multimers. The complete sequence of pIM13 has been reported (M. Monod, C. Denoya, and D. Dubnau, J. Bacteriol. 167:138-147, 1986) and two long open reading frames have been identified, one of which (ermC') is greater than 90% homologous to the ermC MLS resistance determinant of the Staphylococcus aureus plasmid pE194. The second reading frame (repL) shares homology with the only long open reading frame of the cryptic S. aureus plasmid pSN2 and is probably involved in plasmid replication. The map of pIM13 is almost a precise match with that of pE5, a naturally occurring, stable, low-copy-number, inducible MLS resistance plasmid found in S. aureus. pIM13 is unstable in S. aureus but still multimerizes in that host, while pE5 is unstable in B. subtilis and does not form multimers in either host. The complete sequence of pE5 is presented, and comparison between pIM13 and pE5 revealed two stretches of sequence present in pE5 that were missing from pIM13. It is likely that a 107-base-pair segment in the ermC' leader region missing from pIM13 accounts for the constitutive nature of the pIM13 MLS resistance and that the lack of an additional 120-base-pair segment in pIM13 that is present on pE5 gives rise to the high copy number, stability, and multimerization in B. subtilis. The missing 120 base pairs occur at the carboxyl-terminal end of the putative replication protein coding sequence and results in truncation of that protein. It is suggested either that the missing segment contains a site involved in resolution of multimers into monomers or that the smaller replication protein causes defective termination of replication. It is concluded that pIM13 and pE5 are coancestral plasmids and it is probable that pIM13 arose from pE5.     

Similarity of minus origins of replication and flanking open reading frames of plasmids pUB110, pTB913 and pMV158.

Plasmids pMV158 and pTB913, originating from Streptococcus agalactiae and a thermophilic Bacillus respectively, were sequenced to completion. Both contained a BA3-type minus origin of replication and an RSA-site, believed to constitute a site-specific recombination site. These two regions were more than 99% homologous to the corresponding regions of the Staphylococcus aureus plasmid pUB110. Deleting the BA3-type minus origin resulted in the accumulation of a considerable amount of single-stranded DNA, both in L. lactis subsp. lactis and B. subtilis, indicating that this minus origin was functional in both bacterial species. Like pUB110, both plasmids contained an open reading frame encoding a putative plasmid recombination enzyme (Pre protein), which was located downstream of the RSA-site. On the basis of sequence comparisons between pUB110, pMV158, pTB913, pT181, pE194, pNE131 and pT48 two distinct families of RSA-sites and Pre proteins could be distinguished.     

The nucleotide sequence of a small cryptic plasmid found in Staphylococcus aureus and its relationship to other plasmids

The nucleotide sequence of a small (1273 bp) plasmid (pOX1000) of Staphylococcus aureus has been determined and compared with similar plasmids. The sequence includes a single open reading frame; two large palindromes and a 22 bp palindrome that is contiguously repeated three times upstream of the open reading frame. Composite plasmids of pE194ts and pOX1000 were constructed with pUC18 separately inserted into five different sites on pOX1000 and used to analyse the replication functions of the cryptic plasmid.     

Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies.

pE194 is a small plasmid (isolated originally in Staphylococcus aureus) which confers erythromycin-inducible resistance to macrolide, lincosamide, and streptogramin type B (MLS) antibiotics. The nucleotide sequence of pE194 contains 3,728 base pairs (bp), corresponding to a molecular mass of 2.4 million daltons. By means of site-specific cleavage with restriction endonucleases and cloning resultant fragments, determinants of the two major biological functions of p E194, i.e., inducible MLS resistance and replication, could be localized and assigned to specific sequences in the plasmid. Restriction endonuclease TaqI cut pE194 at three sites. TaqI fragment A (1,443 bp) contained the determinant for inducible MLS resistance, whereas TaqI fragment B (1,354 bp) contained a determinant necessary for plasmid replication. Regulatory mutations resulting in constitutive expression of MLS resistance mapped in TaqI fragment A, whereas a mutation associated with elevated plasmid copy number was mapped in TaqI fragment B. Also mapping in TaqI fragment B was a plasmid replication determinant comprising two sets of inverted complementary repeat sequences, one of which spanned 124 bp and was adjacent to a second smaller set which was rich in guanine and cytosine residues. pE194 contained six open reading frames which were theoretically capable of coding for proteins with maximum molecular masses as follows (in daltons): A, 48,300; B, 29,200; C, 14,000; D, 13,900; E, 12,600; and F, 2,700. Insertion of plasmid pBR322 into the single PstI site located in frame A of pE194 resulted in a composite plasmid which could replicate in both Bacillus subtilis and Escherichia coli, suggesting that an intact polypeptide A is dispensable for both replication of pE194 and for MLS resistance. Frame B specified inducible MLS resistance, whereas frame F specified the putative peptide associated with the proposed B determinant translational attenuator. The extent to which frames C, D, and E, all contained in TaqI fragment B, were translated into polypeptide products is not known; however, a base change in frame E was found in a comparison between the high-copy-number mutant, cop-6, and the wild-type strains.     

Determination of the complete nucleotide sequence of pNS1, a staphylococcal tetracycline-resistance plasmid propagated in Bacillus subtilis

Abstract The complete nucleotide sequence of pNS1 (3879 bp), a tetracycline-resistance (Tc^R) plasmid drived from staphylococcal plasmid pTP5, has been determined and compared with that of the staphylococcal Tc^R plasmid pT181 [6]. The nucleotide sequences of the 2 plasmids are in agreement, except for 18 nucleotides, but these differences are significant in that they give rise to new open reading frames (ORFs). A short ORF-D is found in the copy control region, and the Tc^R region contains a single large ORF-A, that encodes the Tet protein (50 kDa). The upstream region of ORF-A contains 3 inverted repeat sequences, which can generate structures very similar in conformation of the structure of the control region of the inducible erythromycin-resistance gene of pE194.     

Homology of mycoplasma plasmid pADB201 and staphylococcal plasmid pE194.

The complete nucleotide sequence of pADB201, a 1.7-kilobase cryptic plasmid from Mycoplasma mycoides subsp. mycoides, is reported. The sequence contains a single large open reading frame capable of coding for a polypeptide of up to 198 codons long. The sequence of the putative polypeptide shows significant similarity to that of the repF gene product of staphylococcal plasmid pE194.     

Region of the streptococcal plasmid pMV158 required for conjugative mobilization.

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 pUB110. 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.     

Nucleotide sequence of pS194, a streptomycin-resistance plasmid from Staphylococcus aureus

pS194 is a naturally occurring Staphylococcus aureus plasmid encoding streptomycin resistance. The plasmid has a copy number of about 25 per cell, and belongs to the inc5 incompatibility group. The nucleotide sequence of pS194 has been determined and consists of 4397 base pairs including four open reading frames potentially encoding proteins of greater than 100 amino acids. All four of these reading frames are on the same coding strand. The first reading frame, repE, encodes a 38 kd protein specifically required for pS194 replication. The second open reading frame, str, encodes a 34 kd polypeptide required for streptomycin resistance, probably a streptomycin adenylyltransferase. The third potential polypeptide, rlx, would be 37 kd and is probably required for relaxation complex formation and plasmid mobilization by conjugative plasmids. The fourth, orfD, overlapping the rlx reading frame, is potentially 27 kd, and may also be involved in mobilization.     

Sequence and properties of pIM13, a macrolide-lincosamide-streptogramin B resistance plasmid from Bacillus subtilis.

We initiated a study of pIM13, a multicopy, macrolide-lincosamide-streptogramin B (MLS) plasmid first isolated from a strain of Bacillus subtilis and described by Mahler and Halvorson (J. Gen. Microbiol. 120:259-263, 1980). The copy number of this plasmid was about 200 in B. subtilis and 30 in Staphylococcus aureus. The MLS resistance determinant of pIM13 was shown to be highly homologous to ermC, an inducible element on the S. aureus plasmid pE194. The product of the pIM13 determinant was similar in size to that of ermC and immunologically cross-reactive with it. The MLS resistance of pIM13 was expressed constitutively. The complete base sequence of pIM13 is presented. The plasmid consisted of 2,246 base pairs and contained two open reading frames that specified products identified in minicell extracts. One was a protein of 16,000 molecular weight, possibly required for replication. The second was the 29,000-molecular-weight MLS resistance methylase. The regulatory region responsible for ermC inducibility was missing from pIM13, explaining its constitutivity. The remainder of the pIM13 MLS determinant was nearly identical to ermC. The ends of the region of homology between pIM13 and pE194 were associated with hyphenated dyad symmetries. A segment partially homologous to one of these termini on pIM13 and also associated with a dyad was found in pUB110 near the end of a region of homology between that plasmid and pBC16. The entire sequence of pIM13 was highly homologous to that of pE5, an inducible MLS resistance plasmid from S. aureus that differs from pIM13 in copy control.     

Replication in yeasts of plasmid pE194 from Staphylococcus aureus

The ability of the plasmid pE194 from S. aureus to serve as an autonomously replicating sequence (ARS) in yeast was shown. The hybrid plasmid pLD744 that contains pE194 and the yeast LEU2 gene sequences is unstable in yeast like other YRp-vectors: the mitotic stability of the pLD744 was as much as 1%. The plasmid pLD712 that differs from pLD744 by the existence of a centromeric sequence from the chromosome III of yeast Saccharomyces cerevisiae reveals about one order greater stability. The observation that there are some sequences in the primary structure of the pE194 which strongly conform to the ARS consensus in yeast inclines us to infer that the existence of ARS consensus on pE194 DNA is not sufficient for its effective replication in yeast.     

pIH01, a small cryptic plasmid from Leuconostoc citreum IH3

A small cryptic plasmid pIH01 from Leuconostoc citreum IH3 was characterized. This 1.8-kb sized plasmid contains single open reading frame that encodes a RepC class protein (342 amino acids) and a conserved pT181-type double strand origin, suggesting a rolling circle replication mode. This putative replicase protein shows the highest similarity to a replicase from pFR18 plasmid of Leuconostoc mesenteroides FR52 (64% identity), one of the pT181-type rolling circle plasmid family and contains a strictly conserved RepC-type active site sequence of pT181 family. A shuttle vector that was developed on the basis of this cryptic plasmid by insertion of both erythromycin resistance gene (ermC) from pE194 and Escherichia coli ColE1 origin was able to transform Leuconostoc strains, Lactobacillus plantarum, and Lactococcus lactis. Therefore, pIH01 derivative plasmids might be useful for the manipulation of Leuconostoc strains.     

Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance.

The nucleotide sequence of pC194, a small plasmid from Staphylococcus aureus which is capable of replication in Bacillus subtilis, has been determined. The genetic determinant of chloramphenicol (CAM) resistance, which includes the chloramphenicol acetyl transferase (CAT) structural gene, the putative promoter and controlling element of this determinant, have been mapped functionally by subcloning a 1,035-nucleotide fragment which specifies the resistance phenotype using plasmid pBR322 as vector. Expression of CAM resistance is autogenously regulated since the 1,035-nucleotide fragment containing the CAT gene sequence and its promoter cloned into pBR322 expresses resistance inducibly in the Escherichia coli host. A presumed controlling element of CAT expression consists of a 37-nucleotide inverted complementary repeat sequence that is located between the -10 and ribosome-loading sequences of the CAT structural gene. Whereas the composite plasmid containing the minimal CAT determinant cloned in pBR322 could not replicate in B. subtilis, ability to replicate in B. subtilis was seen if the fragment cloned included an extension consisting of an additional 300 nucleotides beyond the 5' end of the single pC194 MspI site associated with replication. This 5' extension contained a 120-nucleotide inverted complementary repeat sequence similar to that found in pE194 TaqI fragment B which contains replication sequences of that plasmid. pC194 was found to contain four opening reading frames theoretically capable of coding for proteins with maximum molecular masses, as follows: A, 27,800 daltons; B, 26,200 daltons; C, 15,000 daltons; and D, 9,600 daltons. Interruption or deletion of either frame A or D does not entail loss of ability to replicate or to express CAM resistance, whereas frame B contains the CAT structural gene and frame C contains sequences associated with plasmid replication.     

Expression in Escherichia coli of a staphylococcal gene for resistance to macrolide, lincosamide, and streptogramin type B antibiotics.

Plasmid pBD9, which comprises two plasmids from Staphylococcus aureus, pE194 and pUB110, was joined to plasmid pBR322 by in vitro recombination to form plasmid pKH80. The ermC gene of plasmid pE194 confers inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics. When pKH80 was transferred to Escherichia coli K-12, the bacteria became resistant to several of these antibodies.     

Localization of the start sites of lagging-strand replication of rolling-circle plasmids from Gram-positive bacteria

A number of small, multicopy plasmids from Gram-positive bacteria replicate by an asymmetric rolling-circle mechanism. Previous studies with several of these plasmids have identified a palindromic sequence, SSO_A, that acts as the single-strand origin (SSO) for the replication of the lagging-strand DNA. Although not all the SSO_A sequences share ONA sequence homology, they are structurally very similar. We have used an in vitro system to study the lagging-strand replication of several plasmids from Gram-positive bacteria using the SSO_A sequences of pT181, pE194 and pSN2 as representative of three different groups of Staphylococcus aureus plasmids. In addition, we have investigated the lagging-strand replication of the pUB110 plasmid that contains an alternative single-strand origin, sso_u. Our results confirm that RNA polymerase is involved in lagging-strand synthesis from both SSO_A and ssou-type lagging-strand origins. Interestingly, while initiation of lagging-strand DNA synthesis of pUB110 occurred predominantly at a single position within sso_u, replication of pT181, pSN2 and pE194 plasmids initiated at multiple positions from SSO_A.