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Tuberculosis (TB) remains a major global health problem, and successful genetic manipulation of mycobacteria is crucial for developing new approaches to study the mechanism of pathogenesis of Mycobacterium tuberculosis (M.tb) and to combat TB. In this study, a series of M.tb furA gene operator/promoter (pfurA) mutants were generated aiming at optimization of the promoter activities in mycobacterial...
Polyethylenimines (PEIs) and cationic liposomes are widely used for nonviral gene delivery. When PEIs have been used alone, the transfection efficiency has been higher for larger or linear than smaller or branched PEIs. We have reported previously that a combination of small PEIs and liposomes results in a potentiation of transfection efficiency in vitro. Here, the role of PEI size and structure in...
To facilitate the study of the structure-function relationship of human hemoglobin (Hb A), we have developed a new hemoglobin expression vector, pGEX6P-α-[SD]-β. This vector allows the co-expression of α-Hb as a fusion protein with Glutathione S-Transferase (GST-α-Hb) and β-Hb with an additional methionine at the N-terminal extremity (rβ-Hb). These proteins were solubilized as GST-α-Hb/rβ-Hb complex...
Many conjugative or mobilizable plasmids from Gram-positive bacteria can be transferred mainly on filter-based surfaces. Such is the case of the streptococcal promiscuous plasmid pMV158 which can be mobilized by functions provided by auxiliary plasmids of the Inc18 family. We have developed a new large scale method consisting of a 96-well microtiter plate coupled with a filter so that we could perform...
A new cryptic plasmid pAP3.9 was discovered in symbiotic alpha-proteobacteria present in the cytoplasm of Amoeba proteus. The plasmid is 3869bp with a GC content of 34.66% and contains replication origins for both double-strand (dso) and single-strand (sso). It has three putative ORFs encoding Mob, Rep and phosphoglycolate phosphatase (PGPase). The pAP3.9 plasmid appears to propagate by the conjugative...
The complete nucleotide sequences of two plasmids from Exiguobacterium arabatum sp. nov. RFL1109, pEspA (4563bp) and pEspB (38,945bp), have been determined. Five ORFs were identified in the pEspA plasmid, and putative functions were assigned to two of them. Using deletion mapping approach, the Rep-independent replication region of pEspA, which functions in Bacillus subtilis, was localized within a...
Although plasmid DNA vectors have been extensively applied in biotechnology, there is still a lack of standard plasmid vector classification. Here, we propose a classification method for commonly used plasmid vectors. Plasmid vectors were classified into different classes based on their replication origin, selection marker and promoter information. The replication origins of plasmid vectors were classified...
The bacterial transposon Tn7 maintains two distinct lifestyles, one in horizontally transferred DNA and the other in bacterial chromosomes. Access to these two DNA pools is mediated by two separate target selection pathways. The proteins involved in these pathways have evolved to specifically activate transposition into their cognate target-sites using entirely different recognition mechanisms, but...
A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a different selectable marker (URA, TRP, or LEU), and the system provides high expression levels of three different...
The pheromone-responsive conjugative plasmids of Enterococcus faecalis and the multiresistance plasmids pSK1 and pSK41 of Staphylococcus aureus are among the best studied plasmids native to Gram-positive bacteria. Although these plasmids seem largely restricted to their native hosts, protein sequence comparison of their replication initiator proteins indicates that they are clearly related. Homology...
The nucleotide sequence of pRS5 (10153bp) is reported. Through sequence analysis, 9 open reading frames (ORFs) were identified and the following features observed: a region likely involved in replication whose structural features indicate that pRS5 belongs to the pUCL287 group of theta-type replicons, and hypothetical proteins putatively involved in plasmid copy number control, restriction–modification...
The tetracycline-inducible expression vector pALC2073 allowed high level expression of the cloned sasG gene but repression by uninduced cells was leaky. The −10 box of the tetR promoter was mutated to the Bacillus subtitlis consensus, which resulted in complete repression of SasG protein expression. Anhydrotetracycline at 1.28μgml −1 gave the same high level of induction that was obtained...
A plasmid containing multidrug resistance genes has been discovered from a clinical Actinobacillus pleuropneumoniae strain isolated in China. The complete 15079kb sequence of this plasmid, designated pHB0503, was analyzed with regard to the organization and evolution of multidrug resistance genes. The deduced amino acid sequences from seven open reading frames (sul2 catA3, aacC2, strA, truncated strB...
A new set of extrachromosomal Dictyostelium expression vectors is presented that can be modified according to the experimental needs with minimal cloning efforts. To achieve this, the vector consists of four functional regions that are separated by unique restriction sites, (1) an Escherichia coli replication region, and regions for (2) replication, (3) selection and (4) protein expression in Dictyostelium...
Inducible expression systems are essential for the expression of toxic proteins and are very convenient for proteins that induce strong side effects such as retardation of growth or development. Currently available systems for use in Dictyostelium either do not have a very tight control over expression levels or use a combination of an integrating and an extrachromosomal vector. We designed a new...
A meeting of the EMBO Conference Series on Replication and Segregation of Chromosomes was held in Geilo, Norway, 16–20 June, 2008, under a scenic backdrop of high mountains. The meeting focused on the mechanistic details of replication and segregation primarily from well-characterized systems. Because the same basic principles govern chromosome maintenance in all three domains of life, participants...
The Escherichia coli SeqA protein contributes to regulation of chromosome replication by preventing re-initiation at newly replicated origins. SeqA protein binds to new DNA which is hemimethylated at the adenine of GATC sequences. Most of the cellular SeqA is found complexed with the new DNA at the replication forks. In vitro the SeqA protein binds as a dimer to two GATC sites and is capable of forming...
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