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Caulobacter crescentus is a bacterium with a distinctive life cycle and so it is studied as a cell development model. In addition, we have adapted this bacterium for recombinant protein production and display based on the crystalline surface protein (S)-layer and its C-terminal secretion signal. We report here the development of small, high-copy-number plasmid vectors and methods for producing an...
Conditional expression systems were utilized for the ectopic induction of essential genes in Staphylococcus aureus. Resulting strains were then subjected to allelic-replacement mutagenesis of the native allele under inducing conditions for expression of the ectopic copy of the gene. This strategy produced test strains whereby cellular viability was uniquely dependent on the presence of inducer and...
Large conjugative transposons (CTn's) are widespread among Bacteroides spp. and they are responsible for the high rates of Bacteroides tetracycline resistance, which is mediated by the tetQ gene. These elements are self-transmissible and conjugation can be induced up to 1000-fold by the addition of tetracycline to cultures prior to mating. In addition to self-transfer, the Bacteroides CTn's, such...
We describe features of the basic replicon of the 10.6-kb medium-copy-number plasmid pAM10.6. pAM10.6 was able to replicate in various Pseudomonas strains but was maintained in Escherichia coli only after the p15A origin of replication was inserted. Deletion analysis suggests that the pAM10.6 origin of replication is located in a 0.5-kb region that includes inverted and direct repeats upstream of...
Sequence analysis of the nifA gene, including its 5′ flanking region, of Mesorhizobium huakuii revealed the presence of a novel IS-like element called ISMh2. It is 828 bp in length and possesses two imperfect terminal inverted repeats of 14 bp with only one mismatch. The putative transposase encoded by ISMh2 is composed of 204 amino acids. In comparison with other insertion sequences ISMh2 likely...
Several plasmids from Pasteurella multocida have been shown to carry antibiotic resistance genes but no other genes possibly related to the organism's pathogenesis. We report here that sequence from the plasmid pLEM from a fowl isolate of P. multocida, strain 1059, contained one open reading frame that had significant identity with a predicted protein from pVT745, a plasmid that was isolated from...
Plasmids carrying the ytl2 gene from the large resident plasmid pSLT of Salmonella typhimurium were stabilized >10 5 -fold (compared to control ytl2-free plasmids) in S. typhimurium cells. Purified Ytl2 protein was localized in the cell cytosol and bound to DNA in a sequence-independent manner to form a high-molecular-weight complex, suggesting cooperative binding to the DNA. A mutant ytl2...
DNA sequence polymorphism was determined for the microcin V gene cluster encoded on the microcin V plasmids of 12 natural isolates of Escherichia coli. These microcin V gene clusters are similar in DNA sequence, with only 10 of the 683 bp polymorphic. Further, the levels and patterns of microcin V gene cluster polymorphism differ from those of a chromosomal region, trpORF2, sequenced from each of...
We have developed Escherichia coli and Pseudomonas expression vectors based on the alkane-responsive Pseudomonas putida (oleovorans) GPo1 promoter PalkB. The expression vectors were tested in several E. coli strains, P. putida GPo12 and P. fluorescens KOB2Δ1 with catechol-2,3-dioxygenase (XylE). Induction factors ranged between 100 and 2700 for pKKPalk in E. coli and pCom8 in Pseudomonas strains,...
The complete sequencing of the Saccharomyces cerevisiae genome provides a powerful tool for studying and elucidating essential cellular processes. To aid in the application of this resource to investigations into the molecular mechanisms of endoplasmic reticulum-associated protein degradation, a simple procedure was designed to generate a unique 2-μm LEU2-selectable yeast expression vector. Putative...
The complete 4312-bp sequence of the pEC156 plasmid from Escherichia coli E1585-68, which carries genes encoding the EcoVIII restriction-modification (R-M) system, an isoschizomer of HindIII from Haemophilus influenzae, has been determined. Two clustered and convergently oriented open reading frames, large enough to encode genes of the EcoVIII R-M system, were found. The transcriptional start points...
pAD1 is a 59.3-kb plasmid in Enterococcus faecalis that has been the subject of intense investigation with regard to its pheromone-inducible conjugation behavior as well as its contribution to virulence. Approximately two-thirds of the pAD1 nucleotide sequence has been previously reported. Here we report on an analysis of the final ∼22 kb, a significant portion of which is believed to encode structural...
pT181 is a small rolling-circle plasmid from Staphylococcus aureus whose initiator protein, RepC, melts the plasmid's double-strand origin (DSO) and extrudes a cruciform involving IR II, a palindrome flanking the initiation nick site. We have hypothesized that the cruciform is required for initiation, providing a single-stranded region for the assembly of the replisome (R. Jin et al., 1997, EMBOJ...
A new collection of shuttle cloning vectors has been constructed that can be used in a broad host range, because they carry replication origins which are functional in Escherichia coli (p15A, pWV01, ColE1), Lactococcus lactis, lactobacilli, and Bacillus subtilis (pAMβ1, pWV01). These plasmids contain the lacZ-T1T2 cassette from pJDC9, which allows the X-gal selection and cloning of DNA fragments that...
Approximately 5% of strains of Streptococcus mutans contain plasmid DNA. Strain UA140 harbors a 5.6-kb cryptic plasmid, pUA140, with an overall G+C content of 32.7%. Five open reading frames (ORF), encoding peptides of larger than 100 amino acid residues, were initially designated as ORF1 to ORF5. These five ORFs were located on the same strand of pUA140. ORF1 (258 amino acids) resembled a replication...
pOM1 is a recombinant 4442-bp plasmid that includes the replicon of the Francisella novicida-like strain F6168 cryptic plasmid pFNL10 and the tetracycline resistance gene (tetC) of plasmid pBR328. pOM1 can stably replicate and is maintained in Francisella tularensis biovars tularensis, palaearctica, and palaearctica var. japonica. The replicon of pOM1 includes the ori region and the repA gene. The...
Three thermosensitive (Ts) suicide vectors, pSET4s, pSET5s, and pSET6s, have been constructed for gene replacement in Streptococcus suis. Each vector contains an antibiotic-resistance gene (spc or cat), a Ts replication origin of pWV01 lineage, multiple cloning sites, lacZ′, and the ColE1 replication origin of pUC19. These vectors could be propagated at 37°C in Escherichia coli, but their replication...
Nucleotide sequence analysis of two cryptic plasmids, pRS2 (2544 bp) and pRS3 (3948 bp), from Oenococcus oeni revealed the presence in both of three major open reading frames with significant similarity to other small cryptic plasmids from O. oeni. The results suggest that those plasmids could be separated into two subfamilies, one represented by pLo13 and pRS3, the other represented by pOg32, pRS1,...
We report here the molecular characterization of pFNL10, a 3990-bp cryptic plasmid of Francisella novicida-like F6168. The plasmid was maintained in F. novicida Utah 112 and F. tularensis LVS strains. We sequenced the entire plasmid and found six open reading frames (ORFs)—ORF1, ORF2, ORF3, ORF4, ORF5, and ORFm. ORF3, ORF4, ORF5, and ORFm are located on the same strand, and we designated it the plus...
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