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.     

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.     

Plasmids from two morphologically distinct cyanobacterial strains share a novel replication origin.

A 2.9-kbp replication origin from a plasmid endogenous to the filamentous cyanobacterium Fremyella diplosiphon UTEX 481 was genetically characterized and sequenced. Deletion analysis of the 2.9-kbp DNA fragment delimited the minimum region necessary for replication in F. diplosiphon Fd33 to approximately 2.5 kbp. DNA sequence analysis revealed that the F. diplosiphon plasmid replication origin is structurally very similar to and shares significant identity with the 1.75-kbp replication origin reported for plasmid pDU1, isolated from the morphologically distinct cyanobacterium Nostoc sp. strain PCC 7524. Each cyanobacterial plasmid replication origin includes a large open reading frame that predicts a conserved protein of unknown function; the predicted proteins of the replication origins are of similar sizes and 30% identical in amino acid sequence. Each cyanobacterial plasmid replication origin also possesses a region of dyad symmetry approximately 300 bp upstream of the conserved open reading frame.     

链霉菌质粒pSGL1最小复制子序列测定及分析

质粒ｐＳＧＬ１（７．４ｋｂ）是从球孢链霉菌（Ｓｔｒｅｐｔｏｍｙｃｅｓｇｌｏｂｉｓｐｏｒｕｓ）中分离得到的一个高拷贝质粒,已证明其最小复制子位于Ｓａｕ３ＡＩ酶切的２０ｋｂ片段上。对该片段进行亚克隆,测序后数据表明该片段是一个新序列。仅有一个开放阅读框架（ＯＲＦＲ）位于最小复制子中,推测其编码的蛋白质含有滚环复制质粒复制酶的特定序列。     

Isolation and sequencing of the replication region of Mycobacterium avium plasmid pLR7.

The Mycobacterium avium plasmid pLR7 is representative of a group of small plasmids that are common in isolates from AIDS patients with disseminated M. avium infections. Determination of the functions of these and other plasmids has been hampered by the lack of methods for genetic manipulation of M. avium. In this study, the region of pLR7 capable of replication was identified and sequenced. Fragments of pLR7 were cloned into a pUC18 derivative carrying a kanamycin resistance marker and introduced into a plasmid-free M. avium strain by electroporation. The origin of replication was located on a 1.8-kb PvuII-to-SmaI fragment. An open reading frame encoding a putative Rep protein was identified. Two other open reading frames were identified in this region. A shuttle vector, pMB351, was constructed with the pLR7 origin of replication, pUC18, and the kanamycin resistance gene from Tn5. This vector was successfully transformed into M. avium, Mycobacterium tuberculosis, and Mycobacterium bovis.     

Functional mapping of Autographa california nuclear polyhedrosis virus genes required for late gene expression.

A plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of an Autographa california nuclear polyhedrosis virus (AcNPV) late gene promoter was constructed. This plasmid (pL2cat) also contained the AcNPV hr5 enhancer element. Transient-expression assay experiments indicated that the late promoter was active in Spodoptera frugiperda cells cotransfected with pL2cat and AcNPV DNA but not when pL2cat was transfected alone. Low levels of CAT activity were observed in cells cotransfected with pL2cat and pIE-1 DNAs. However, CAT activity was not induced in a similar plasmid which lacked the cis-linked enhancer element, indicating that the enhancer was required for expression of the late gene. Cotransfection mapping of pPstI clones of AcNPV DNA indicated that the pPstI-G clone of viral DNA contained a factor which further stimulated late gene expression 3- to 10-fold. Transient-expression assay analysis of subclones of pPstI-G localized the trans-active factor to a 3.0-kilobase XbaI fragment. The nucleotide sequence of this fragment was determined and found to contain three potential open reading frames. A computer-assisted search of a protein database revealed no closely related proteins. One of the predicted amino acid sequences contained potential metal-binding domains similar to those found in nucleic acid-binding proteins. Subcloning and subsequent CAT assay indicated that two of the open reading frames were required for the activation of pL2cat. Nuclease S1 mapping of infected and transfected RNAs indicated that the two open reading frames were transcribed as delayed-early genes. Quantitative nuclease S1 analysis and differential DNA digestion of recovered plasmids indicated that the activation of pL2cat was not due to an increase in steady-state levels of mRNA replication of the viral DNA.     

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.     

Genetic analysis of the minimal replicon of the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid

Using a combination of mutagenesis with the transposon and polymerase chain reaction subcloning, the essential elements of the replication region of the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid have been identified. An open reading frame, coding for a protein with homology to Rep proteins from other Lactococcus plasmids, is essential. This protein is trans-acting and could not be replaced by the Rep protein from another Lactococcus plasmid. A second open reading frame immediately downstream from the first could be removed or inactivated with no apparent effect on plasmid replication. A region containing two 10 by direct repeats and three tandem repeats of a 22 by sequence, immediately upstream of the essential open reading frame, is also essential and probably includes the origin of replication. A 181-bp DNA fragment containing this region was sufficient to allow replication in Lactococcus if the trans-acting protein was provided on another replicon. Single-stranded replication intermediates could not be detected, suggesting that the citrate plasmid uses theta replication rather than rolling-circle replication.     

Cloning and DNA sequence of a plasmid-determined citrate utilization system in Escherichia coli.

The citrate utilization determinant from a large 200-kilobase (kb) naturally occurring plasmid was previously cloned into the PstI site of plasmid vector pBR325 creating the Cit+ tetracycline resistance plasmid pWR61 (15 kb). Tn5 insertion mutagenesis analysis of plasmid pWR61 limited the segment responsible for citrate utilization to a 4.8-kb region bordered by EcoRI and PstI restriction nuclease sites. The 4.8-kb fragment was cloned into phage M13, and the DNA sequence was determined by the dideoxyribonucleotide method. Within this sequence was a 1,296-base-pair open reading frame with a preceding ribosomal binding site. The 431-amino-acid polypeptide that could be translated from this open reading frame would be highly hydrophobic. A second long open reading frame with the potential of encoding a 379-amino-acid polypeptide preceded the larger open reading frame. Portions of the 4.8-kb fragment were further subcloned with restriction endonucleases BglII and BamHI, reducing the minimum size needed for a citrate-positive phenotype to a 1.9-kb BamHI-BglII fragment (which includes the coding region for the 431-amino-acid polypeptide, but only the distal 2/3 of the reading frame for the 379-amino-acid polypeptide). Citrate utilization results from a citrate transport activity encoded by the plasmid. With the 4.8-kb fragment (as with larger fragments) the citrate transport activity was inducible by growth on citrate. On transfer from glucose, succinate, malate, or glycerol medium to citrate medium, the Cit+ Escherichia coli strains showed a delay of 36 to 48 h before growth.     

Construction of a new Streptococcus pneumoniae-Escherichia coli shuttle vector based on the replicon of an indigenous pneumococcal cryptic plasmid.

The nucleotide sequence of a cryptic plasmid (pRMG1) isolated from a type 1 Streptococcus pneumoniae has been determined and two recombinant plasmids, pRMGE1 and pRMGE2, bearing the pRMG1 replicon have been constructed. pRMGE2 is a shuttle vector for Escherichia coli and S. pneumoniae. The important characteristics of this cloning vector are: a size of 5.5 kb including a 1.4 kb fragment of pRMG1 (containing a double-stranded replication origin and an open reading frame encoding a putative replication initiation protein), a multicloning site, two antibiotic resistance markers for selection of plasmid containing cells, and blue-white colony screening in E. coli for identification of insert-containing plasmids.