Characterization of a small cryptic plasmid isolated from a methicillin-resistant strain of Staphylococcus aureus

The nucleotide sequence of a small (1613 bp) plasmid, pOX2000, isolated from a methicillin-resistant strain of Staphylococcus aureus has been determined. The sequence contains only one large ORF and the predicted amino acid sequence shows homology to the REP proteins of some other small staphylococcal plasmids. In addition there are two palindromic sequences, palA and palJ, that are similar to but not identical with the palindromes known from other staphylococcal plasmids to be involved in lagging strand initiation and possibly leading strand termination, respectively. Preliminary functional analysis of pOX2000 has been carried out by assessing the effect of interrupting the sequence at three unique restriction endonuclease sites. The plasmid pOX2000, and its relationship to other small staphylococcal plasmids, is discussed.     

Characterization, localization, and sequence of F transfer region products: the pilus assembly gene product TraW and a new product, TrbI.

The traW gene of the Escherichia coli K-12 sex factor, F, encodes one of the numerous proteins required for conjugative transfer of this plasmid. We have found that the nucleotide sequence of traW encodes a 210-amino-acid, 23,610-Da polypeptide with a characteristic amino-terminal signal peptide sequence; in DNA from the F lac traW546 amber mutant, the traW open reading frame is interrupted at codon 141. Studies of traW expression in maxicells in the presence and absence of ethanol demonstrate that the traW product does undergo signal sequence processing. Cell fractionation experiments additionally demonstrated that mature TraW is a periplasmic protein. Electron microscopy also showed that F lac traW546 hosts do not express F pili, confirming that TraW is required for F-pilus assembly. Our nucleotide sequence also revealed the existence of an additional gene, trbI, located between traC and traW. The trbI gene encodes a 128-amino-acid polypeptide which could be identified as a 14-kDa protein product. Fractionation experiments demonstrated that TrbI is an intrinsic inner-membrane protein. Hosts carrying the pOX38-trbI::kan insertion mutant plasmids that we constructed remained quite transfer proficient but exhibited increased resistance to F-pilus-specific phages. Mutant plasmids pOX38-trbI472 and pOX38-trbI473 expressed very long F pili, suggestive of a pilus retraction deficiency. Expression of an excess of TrbI in hosts carrying a wild-type pOX38 plasmid also caused F-pilus-specific phage resistance. The possibility that TrbI influences the kinetics of pilus outgrowth and/or retraction is discussed.     

A cryptic miniplasmid from the hyperthermophilic bacterium Thermotoga sp. strain RQ7.

An 846-bp cryptic plasmid has been discovered in the hyperthermophilic bacterium Thermotoga sp. strain RQ7. This is the first plasmid described for an organism from this ancient bacterial lineage and the smallest plasmid described to date for any organism. Nucleotide sequencing revealed a single open reading frame possibly encoding a 25,460-Da basic protein (212 amino acids). Upstream of the putative promoter lie five 11-bp direct repeats, each separated by 1 to 4 bp, while between the promoter and the open reading frame lies an 11-bp palindromic sequence. Its mode of replication is unknown, but its sequence bears similarities to those of plasmids which replicate by a rolling-circle mechanism.     

Cloning and nucleotide sequences of the linear DNA killer plasmids from yeast.

The linear DNA killer plasmids (pGKL1 and pGKL2) isolated from a Kluyveromyces lactis killer strain are also maintained and expressed its killer character in Saccharomyces cerevisiae. After these killer plasmid DNAs isolated from S. cerevisiae were treated with alkali, four terminal fragments from each plasmid DNAs were cloned separately. Using these and other cloned DNA fragments, the terminal nucleotide sequences of pGKL2 and the complete nucleotide sequence of pGKL1 were determined. The inverted terminal repetitions of 202 bp and 182 bp were found in pGKL1 and pGKL2, respectively. The pGKL1 sequence showed an extremely high A + T content of 73.2% and it contained five large open reading frames. The largest of these open reading frame was suggested to code for a membrane-bound precursor of glycoprotein subunit of the killer toxin.     

A functional origin of transfer (oriT) on the conjugative transposon Tn916.

The origin of transfer (oriT) of the 18-kb conjugative transposon Tn916 has been localized to a 466-bp region which spans nucleotides 15215 to 15681 on the transposon map. The oriT lies within an intercistronic region between open reading frames ORF20 and ORF21 that contains six sets of inverted repeats ranging from 10 to 20 bp in size. The segment contains three sequences showing identity in 9 of 12 bp to the consensus nicking site (nic) of the IncP family of conjugative plasmids found in gram-negative bacteria. Overlapping one of these sequences is a region similar to the nic site of the F plasmid. Functionality was based on the ability of the oriT-containing sequence to provide a cis-acting mobilization of chimeras involving the shuttle vector pWM401 in response to activation in trans by an intact chromosome-borne transposon Tn916 delta E. Cloned segments of 466 or 376 nucleotides resulted in unselected cotransfer of the plasmid at levels of about 40% when selection was for Tn916 delta E, whereas a 110-bp segment resulted in cotransfer at a frequency of about 7%. Mobilization was specific in that gram-positive plasmids, such as pAD1 and pAM beta 1, and the gram-negative plasmids pOX38 (a derivative of F) and RP1 did not mobilize oriT-containing chimeras.     

Identification of a new insertion element, similar to gram-negative IS26, on the lactose plasmid of Streptococcus lactis ML3.

In Streptococcus lactis ML3, the lactose plasmid (pSK08) forms cointegrates with a conjugal plasmid (pRS01). It has been proposed that cointegration is mediated by insertion sequences (IS) present on pSK08 (D. G. Anderson and L.L. McKay, J. Bacteriol. 158:954-962, 1984). We examined the junction regions of the cointegrate pPW2 and the corresponding regions of pSK08 (donor) and pRS01 (target) and identified a new IS element on pSK08 (ISS1S) which was involved in and duplicated during formation of pPW2. ISS1S was 808 base pairs (bp) in size, had 18-bp inverted repeats (GGTTCTGTTGCAAAGTTT) at its ends, contained a single long open reading frame encoding a putative protein of 226 amino acids, and generated 8-bp direct repeats of target DNA during cointegrate formation. An iso-IS element, ISS1T, which is duplicated in some other cointegrate plasmids, was also found on pSK08. ISS1T was also 808 bp in size and was identical to ISS1S in sequence except for 4 bp, none of which altered the inverted repeats or amino acid sequence of the open reading frame. Comparison of ISS1 with gram-negative IS26 revealed strong homologies in size (820 bp), sequence of inverted repeats (GGCACTGTTGCAAA), size of direct repeats generated after cointegration (8 bp), and number, size, and amino acid sequence (44.5% identical) of the open reading of frame.     

Characterization of traX, the F plasmid locus required for acetylation of F-pilin subunits.

Acetylation of F-pilin subunits has previously been shown to depend upon expression of the F plasmid transfer operon gene traX. To assess the requirement for pilin acetylation in conjugative transfer of F, we constructed traX::kan insertion mutations and crossed them onto the transmissible F derivative pOX38. Under standard conditions, the function of traX seemed to be dispensable. Although pilin synthesized by mutant plasmids pOX38-traX482 and pOX38-traX483 was not acetylated, F-pilus production and F-pilus-specific phage infection appeared to be normal and transfer occurred at wild-type frequency. Analysis of labeled products showed that TraX+ plasmids expressed two approximately 24- (TraX1) and 22-kDa (TraX2) polypeptides that localized in the cytoplasmic membranes of cells. No product that was similar in size to the product predicted from the traX open reading frame (27.5 kDa) was detected. Therefore, we used site-directed mutagenesis, stop codon linker insertions, and phoA fusion analysis to investigate traX expression. Both TraX1 and TraX2 appeared to be encoded by the traX open reading frame. Insertion of a stop codon linker into the traX C-terminal coding region led to synthesis of two correspondingly truncated products, and fusions to phoA indicated that only the traX reading frame was translated. Expression was also very dependent on the traX M1 start codon; when this was altered, no protein products were observed. However, pilin acetylation activity was still detectable, indicating that some other in-frame start codon(s) can also be used. All sequences that are essential for activity are contained between traX codons 29 and 225. Sequence analysis indicated that traX mRNA is capable of forming a variety of base-paired structures. We suggest that traX expression is translationally controlled and that F-pilin acetylation activity may be regulated by physiological conditions in cells.     

Complete nucleotide sequence of a cryptic plasmid, pbaw301, from the ruminai anaerobe Ruminococcus flavefaciens R13e2

Abstract The complete nucleotide sequence of a cryptic plasmid designated pBAW301, from the Gram-positive ruminai bacterium Ruminococcus flavefaciens R13e2, has been determined. This plasmid is 1768 bp in size and has an overall G+C content of 43.5%. Computer analysis of the sequence data revealed an open reading frame, ORF1 (256 amino acids), which is similar to the Rep protein of the Bacillus borstelensis plasmid pHT926. ORF1 is preceded by Shine-Dalgarno and Escherichia coli —10 and —35 like sequences. Nine smaller open reading frames showed no significant homologies to known protein sequences. Analysis of replication intermediates and the nucleotide sequence indicate that the plasmid does not replicate by a rolling-circle mode of replication similar to other plasmids from Gram-positive bacteria. Moreover, sequences typical of theta replication origins were not found in the nucleotide sequence of pBAW301. These data suggest that this plasmid either replicates by an as yet undescribed mechanism, or represents a new class of theta replicating plasmids.     

A small plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC 6803 encodes a rep protein and replicates by a rolling circle mechanism.

Different cryptic plasmids are widely distributed in many strains of cyanobacteria. A small cryptic plasmid, pCA2.4, from Synechocystis strain PCC 6803 was completely sequenced, and its replication mode was determined. pCA2.4 contained 2,378 bp and encoded a replication (Rep) protein, designated RepA. An analysis of the deduced amino acid sequence revealed that RepA of pCA2.4 has significant homology with Rep proteins of pKYM from Shigella sonnei, a pUB110 plasmid family from gram-positive bacteria, and with a protein corresponding to an open reading frame in a Nostoc plasmid and open reading frame C of Plectonema plasmid pRF1. pKYM and pUB110 family plasmids replicate by a rolling circle mechanism in which a Rep protein nicks the origin of replication to allow the generation of a single-stranded plasmid as a replication intermediate. RepA encoded by pC2.4 was expressed in Escherichia coli cells harboring a vector, pCRP336, containing the entire repA gene. The observed molecular weight of RepA was consistent with the value of 39,200 calculated from its deduced amino acid sequence, as was the N-terminal sequence analysis done through the 12th residue. Single-stranded plasmid DNA of pCA2.4 that was specifically degraded by S1 nuclease was detected in Synechocystis cells by Southern hybridization. These observations suggest that pCA2.4 replicates by a rolling circle mechanism in Synechocystis cells.     

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage phi X174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5' region has been identified and is functional in Bacillus subtilis.     

The complete nucleotide sequence of a small cryptic plasmid from Lactobacillus plantarum

The complete nucleotide sequence of a cryptic plasmid isolated from a Lactobacillus plantarum strain has been determined. The plasmid, designated pC30i1, has a molecular size of 2140 bp and a GC content of 37%. The sequence contains one major open reading frame (ORF R) of 951 bp, encoding a basic polypeptide of 317 amino acids, and a molecular weight of 36,956. ORF R shows extensive sequence similarity with genes coding for replication-associated proteins in a group of gram-positive plasmids known to replicate via single-stranded intermediates (ssDNA plasmids), and a stretch of 9 amino acids in the translation of ORF R closely matches a conserved region in these proteins, as well as the active site of the phi X174 Rep protein. Sequences similar to the ssDNA plasmid origins of replication are also present in the pC30i1 sequence, strengthening the hypothesis that pC30i1 belongs to the ssDNA plasmid family. The other main feature of the pC30i1 sequence is a noncoding region consisting of 14 direct, imperfect repeats of a 17-bp sequence, which may have an incompatibility function.     

Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile.

The plasmid pGT5 (3,444 bp) from the hyperthermophilic archaeon Pyrococcus abyssi GE5 has been completely sequenced. Two major open reading frames with a good coding probability are located on the same strand and cover 85% of the total sequence. The larger open reading frame encodes a putative polypeptide which exhibits sequence similarity with Rep proteins of plasmids using the rolling-circle mechanism for replication. Upstream of this open reading frame, we have detected an 11-bp motif identical to the double-stranded origin of several bacterial plasmids that replicate via the rolling-circle mechanism. A putative single-stranded origin exhibits similarities both to bacterial primosome-dependent single-stranded initiation sites and to bacterial primase (dnaG) start sites. A single-stranded form of pGT5 corresponding to the plus strand was detected in cells of P. abyssi. These data indicate that pGT5 replicates via the rolling-circle mechanism and suggest that members of the domain Archaea contain homologs of several bacterial proteins involved in chromosomal DNA replication. Phylogenetic analysis of Rep proteins from rolling-circle replicons suggest that diverse families diverged before the separation of the domains Archaea, Bacteria, and Eucarya.     

Identification and nucleotide sequence analysis of a cryptic plasmid,pRM21, from Rhodothermus marinus

Here we report the identification and nucleotide sequence analysis of pRM21, a plasmid isolated from the thermophilic eubacterium Rhodothermus marinus. pRM21 consists of 2935 bp, has a G+C content of 58.2% and one major open reading frame whose deduced product shows significant similarities to RepA proteins from several plasmids, the highest being to the RepA of pSa from Escherichia coli. A region with the characteristics of iteron-containing replicons, three 19 bp repeats, DnaA boxes, an A+T rich region and GATC sequences, was identified. Of 40 additional R. marinus strains screened for plasmids, six (15%) were found to harbour plasmids with the same size and restriction pattern as pRM21.     

An open reading frame upstream from the nifH gene of Klebsiella pneumoniae

An open reading frame upstream from nifHDK operon of Klebsiella pneumoniae had been described. The orientation of this open reading frame is opposite to that of nifHDK and sequence homology was found between the open reading frame promoter and the promoter of nifHDK operon. A recombinant plasmid carrying the promoter region of the open reading frame fused to the beta-galactosidase gene was constructed. Strains of E.coli were transformed with the plasmid containing this open reading frame promoter-lacZ fusion or co-transformed with it and a plasmid carrying the nifA gene. An appreciable activity of beta-galactosidase was found in strains which received both plasmids, indicating that the promoter of the open reading frame can be activated by the product of nifA gene. Thus, the open reading frame found between nifHDK operon and nifJ behaves just like other nif genes of K.pneumoniae in requiring the product of nifA as the positive effector for expression.     

Plasmid pGA1 from Corynebacterium glutamicum codes for a gene product that positively influences plasmid copy number.

The complete nucleotide sequence (4,826 bp) of the cryptic plasmid pGA1 from Corynebacterium glutamicum was determined. DNA sequence analysis revealed four putative coding regions (open reading frame A [ORFA], ORFA2, ORFB, and ORFC). ORFC was identified as a rep gene coding for an initiator of plasmid replication (Rep) according to the high level of homology of its deduced amino acid sequence with the Rep proteins of plasmids pSR1 (from C. glutamicum) and pNG2 (from Corynebacterium diphtheriae). This function was confirmed by deletion mapping of the minimal replicon of pGA1 (1.7 kb) which contains only ORFC. Deletion derivatives of pGA1 devoid of ORFA exhibited significant decreases in the copy number in C. glutamicum cells and displayed segregational instability. Introduction of ORFA in trans into the cells harboring these deletion plasmids dramatically increased their copy number and segregational stability. The ORFA gene product thus positively influences plasmid copy number. This is the first report on such activity associated with a nonintegrating bacterial plasmid. The related plasmids pGA1, pSR1, and pNG2 lacking significant homology with any other plasmid seem to be representatives of a new group of plasmids replicating in the rolling-circle mode.     

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage φX174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5′ region has been identified and is functional in Bacillus subtilis.     

Isolation and sequencing of the replication region of plasmid pBFp1 isolated from a marine biofilm

A 24 kb plasmid, pBFp1, encoding mercury resistance was previously isolated from a marine biofilm. Isolation and sequencing of a 4280 bp DNA fragment containing the plasmid replicon (rep-pBFp1) revealed a putative open reading frame encoding a RepA protein and an oriV-like region containing an A+T rich sub-region, iterons, and DnaA boxes. Sequence comparisons showed significant similarities to the incW plasmid pSa both for the RepA amino acid sequence and in the iteron DNA sequence. Plasmid pBFp1 was also shown to be incompatible with pSa in standard incompatibility testing. A probe from the repA gene of pBFp1 was further made and tested on a collection of plasmids exogenously isolated from marine habitats in a previous study.     

Analysis of the entire nucleotide sequence of the cryptic plasmid of Chlamydia trachomatis serovar L1. Evidence for involvement in DNA replication.

Chlamydia trachomatis serovar L1/440/LN possesses a 7498bp plasmid which was designated pLGV440. The plasmid was cloned at the BamH1 site of pAT153 into Escherichia coli and the recombinant plasmid was designated pCTL1. A detailed restriction endonuclease map of pCTL1 was constructed. A fragment of the chlamydial plasmid was shown to function as a promoter in E. coli when placed upstream of the lacZ gene. The entire plasmid was sequenced by the chain termination method. Open reading frames were identified from the resulting consensus sequence together with a candidate for the plasmid origin of replication consisting of four perfect tandem repeats of a 22bp sequence, an A:T rich sequence and an open reading frame which could generate a 34.8kdal product. The predicted polypeptide products of the open reading frames were compared by computer with all reported protein sequences. Homology of the predicted polypeptide product of an open reading frame to the E. coli dnaB protein and the analogous product of gene 12 of bacteriophage P22 is described.