PBAD-based shuttle vectors for functional analysis of toxic and highly regulated genes in Pseudomonas and Burkholderia spp. and other bacteria.
Ontology highlight
ABSTRACT:
SUBMITTER: Qiu D
PROVIDER: S-EPMC2592904 | biostudies-literature | 2008 Dec
REPOSITORIES: biostudies-literature
Publications
PBAD-based shuttle vectors for functional analysis of toxic and highly regulated genes in Pseudomonas and Burkholderia spp. and other bacteria.
Qiu Dongru D Damron F Heath FH Mima Takehiko T Schweizer Herbert P HP Yu Hongwei D HD
Applied and environmental microbiology 20081010 23
We report the construction of a series of Escherichia-Pseudomonas broad-host-range expression vectors utilizing the P(BAD) promoter and the araC regulator for routine cloning, conditional expression, and analysis of tightly controlled and/or toxic genes in pseudomonads. ...[more]
Similar Datasets
Project description:Actinomycetales, such as the genus Streptomyces, are well-known cell factories employed to produce a wide variety of secondary metabolites for industrial use. However, not only is the genetic engineering of Streptomyces more complicated and tedious than other model laboratory species, such as Escherichia coli, there is also a considerable lack of genetic tools, hindering its adoption as a common chassis for synthetic biology. In this work, 23 novel shuttle vectors are presented that follow the canonical SEVA (Standard European Vector Architecture) common architecture with the goal of increasing the genetic toolbox repertoire for Streptomyces and other actinomycetes. The ORI module of these plasmids is composed of the combination of two origins of replication, one for Gram-negative bacteria and the other for Streptomyces, a Gram-positive bacteria. Origins of replication have been included in the collection for integrative, low-copy number, and medium-to-high-copy number vectors for Streptomyces. Also, a new selection marker has been developed that confers resistance to apramycin. The functionality of these plasmids was tested via the heterologous expression of GFP and the heterologous production of the plant flavonoid apigenin in Streptomyces albus J1074, with successful results in both cases, therefore expanding the current repertoire of genetic manipulation tools in Streptomyces species.
| S-EPMC7294301 | biostudies-literature
Project description:Single bacteria strains from Bacillus, Xanthomonas, Pseudomonas ans Burkholderia species grown in nutrient broth.
2022-08-08 | MSV000090093 | MassIVE
Project description:Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 x 10(6) and 7.1 x 10(6) transformants/mug DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.
| S-EPMC2042079 | biostudies-literature
Project description:BackgroundThe genus Burkholderia consists of species that occupy remarkably diverse ecological niches. Its best known members are important pathogens, B. mallei and B. pseudomallei, which cause glanders and melioidosis, respectively. Burkholderia genomes are unusual due to their multichromosomal organization, generally comprised of 2-3 chromosomes.ResultsWe performed integrated genomic analysis of 127 Burkholderia strains. The pan-genome is open with the saturation to be reached between 86,000 and 88,000 genes. The reconstructed rearrangements indicate a strong avoidance of intra-replichore inversions that is likely caused by selection against the transfer of large groups of genes between the leading and the lagging strands. Translocated genes also tend to retain their position in the leading or the lagging strand, and this selection is stronger for large syntenies. Integrated reconstruction of chromosome rearrangements in the context of strains phylogeny reveals parallel rearrangements that may indicate inversion-based phase variation and integration of new genomic islands. In particular, we detected parallel inversions in the second chromosomes of B. pseudomallei with breakpoints formed by genes encoding membrane components of multidrug resistance complex, that may be linked to a phase variation mechanism. Two genomic islands, spreading horizontally between chromosomes, were detected in the B. cepacia group.ConclusionsThis study demonstrates the power of integrated analysis of pan-genomes, chromosome rearrangements, and selection regimes. Non-random inversion patterns indicate selective pressure, inversions are particularly frequent in a recent pathogen B. mallei, and, together with periods of positive selection at other branches, may indicate adaptation to new niches. One such adaptation could be a possible phase variation mechanism in B. pseudomallei.
| S-EPMC6307245 | biostudies-literature
Project description:There is a lack of shuttle vectors to be needed for investigations into the genetics of Porphyromonas gingivalis and related species. To better understand the prevalence of candidates for such tools, we have examined multiple strains of black-pigmented anaerobes (clinical and laboratory isolates) for plasmids. As no plasmids were found in P. gingivalis strains, we have used the pYH420 plasmid, derived from P. asaccharolytica, as backbone to construct a shuttle vector in combination with pUC19 from Escherichia coli. Nucleotide sequence determination of the pYH420 plasmid revealed that it contained a gene with similarity to rep from plasmid pTS1 (isolated from Treponema denticola) as well as a homolog of mobA, a member of a gene family found on mobilizable genetic elements found in the genus Bacteroides. We constructed the pG106 and pG108 shuttle vectors using parts of the pUC19 and pYH420 vectors. This resulted in a vector with a multiple cloning site (MCS) in the lacZ gene enabling us to perform blue-white colony selection. The pG106 and pG108 shuttle vectors are electro-transformable into E. coli, P. gingivalis and B. thetaiotaomicron, where they are stable. We demonstrated that these vectors were suitable in these species for applications of molecular cloning including complementation and gene expression studies. Using the pG108 vector, we complement the hcpR mutant strain of P. gingivalis and rescued its NO2- -sensitive phenotype. We also performed a gene expression study using the P-glow BS2 fluorescent reporter gene and the ahpC promoter in B. thetaiotaomicron.
| S-EPMC7894024 | biostudies-literature
Project description:Several gene clusters that are responsible for toxin synthesis in bloom-forming cyanobacteria have been found to be associated with transposable elements (TEs). In particular, insertion sequence (IS) elements were shown to play a role in the inactivation or recombination of the genes responsible for cyanotoxin synthesis. Plasmids have been considered important vectors of IS element distribution to the host. In this study, we aimed to elucidate the IS elements propagated on the plasmids and the chromosome of the toxic cyanobacterium Planktothrix agardhii NIVA-CYA126/8 by means of high-throughput sequencing. In total, five plasmids (pPA5.5, pPA14, pPA50, pPA79, and pPA115, of 5, 6, 50, 79, and 120 kbp, respectively) were elucidated, and two plasmids (pPA5.5, pPA115) were found to propagate full IS element copies. Large stretches of shared DNA information between plasmids were constituted of TEs. Two plasmids (pPA5.5, pPA14) were used as candidates to engineer shuttle vectors (named pPA5.5SV and pPA14SV, respectively) in vitro by PCR amplification and the subsequent transposition of the Tn5 cat transposon containing the R6K? origin of replication of Escherichia coli. While pPA5.5SV was found to be fully segregated, pPA14SV consistently co-occurred with its wild-type plasmid even under the highest selective pressure. Interestingly, the Tn5 cat transposon became transferred by homologous recombination into another plasmid, pPA50. The availability of shuttle vectors is considered to be of relevance in investigating genome plasticity as a consequence of homologous recombination events. Combining the potential of high-throughput sequencing and in vitro production of shuttle vectors makes it simple to produce species-specific shuttle vectors for many cultivable prokaryotes.
| S-EPMC4135779 | biostudies-literature
Project description:Viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. Little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. We therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group streptococci that are activated by specific environmental stimuli in vitro or in vivo. A genomic library of Streptococcus gordonii strain CH1 was constructed in one of the new vectors, and this library was introduced into a homologous bacterium by using an optimized electroporation protocol for viridans group streptococci. Because viridans group streptococci entering the bloodstream from the oral cavity encounter an increase in pH, we selected promoters upregulated by this specific stimulus. One of the selected promoter sequences showed homology to the promoter region of the hydA gene from Clostridium acetobutylicum, the expression of which is known to be regulated by the environmental pH. The isolation of this pH-regulated promoter shows that S. gordonii can sense an increase in the environmental pH, which serves as a signal for bacterial gene activation. Furthermore, this demonstrates the usefulness of these new selection vectors in research on adaptive gene expression of viridans group streptococci and possibly also of other gram-positive bacteria.
| S-EPMC91860 | biostudies-literature
Project description:BackgroundInorganic polyphosphate (polyP), a polymer of tens or hundreds of phosphate residues linked by ATP-like bonds, is found in all organisms and performs a wide variety of functions. PolyP is synthesized in bacterial cells by the actions of polyphosphate kinases (PPK1 and PPK2) and degraded by an exopolyphosphatase (PPX). Bacterial cells with polyP deficiencies are impaired in many structural and important cellular functions such as motility, quorum sensing, biofilm formation and virulence. Knockout mutants of the ppk1 gene have been the most frequent strategy employed to generate polyP deficient cells.ResultsAs an alternative method to construct polyP-deficient bacteria we developed constitutive and regulated broad-host-range vectors for depleting the cellular polyP content. This was achieved by the overexpression of yeast exopolyphosphatase (PPX1). Using this approach in a polyphosphate accumulating bacteria (Pseudomonas sp. B4), we were able to eliminate most of the cellular polyP (>95%). Furthermore, the effect of overexpression of PPX1 resembled the functional defects found in motility and biofilm formation in a ppk1 mutant from Pseudomonas aeruginosa PAO1. The plasmids constructed were also successfully replicated in other bacteria such as Escherichia coli, Burkholderia and Salmonella.ConclusionTo deplete polyP contents in bacteria broad-host-range expression vectors can be used as an alternative and more efficient method compared with the deletion of ppk genes. It is of great importance to understand why polyP deficiency affects vital cellular processes in bacteria. The construction reported in this work will be of great relevance to study the role of polyP in microorganisms with non-sequenced genomes or those in which orthologs to ppk genes have not been identified.
| S-EPMC2667530 | biostudies-literature
Project description:Bacterial artificial chromosome (BAC) mediated transgenesis has proven to be a highly reliable way to obtain accurate transgene expression for in vivo studies of gene expression and function. A rate-limiting step in use of this technology to characterize large numbers of genes has been the process with which BACs can be modified by homologous recombination in Escherichia coli. We report here a highly efficient method for modifying BACs by using a novel set of shuttle vectors that contain the R6Kgamma origin for DNA replication, the E. coli RecA gene for recombination, and the SacB gene for negative selection. These new vectors greatly increased the ease with which one can clone the shuttle vectors, as well as screen for co-integrated and resolved clones. Furthermore, we simplify the shuttle vector cloning to one step by incorporation of a "built-in" resolution cassette for rapid removal of the unwanted vector sequences. This new system has been used to modify a dozen BACs. It is well suited for efficient production of modified BACs for use in a variety of in vivo studies.
| S-EPMC187570 | biostudies-literature
Project description:Toxic cyanobacterial blooms directly threaten both human safety and the ecosystem of surface waters. The widespread occurrence of these organisms, coupled with the tumor-promoting properties of the microcystin toxins that they produce, demands action to mitigate their potential impacts and, thus, a robust understanding of their ecological dynamics. In the present work, the abundance of toxic Microcystis spp. and microcystin (MC)-degrading bacteria in Dianchi Lake, located in Yunnan Province, China, was studied using quantitative PCR. Samples were taken at monthly intervals from June 2010 to December 2011 at three sampling stations within this freshwater lake. Results revealed that variation in the abundance of both total Microcystis spp. and toxic Microcystis spp. exhibited similar trends during the period of the algal bloom, including the reinvasion, pelagic growth, sedimentation, and overwintering periods, and that the proportion of toxic Microcystis was highest during the bloom and lowest in winter. Importantly, we observed that peaks in mlrA gene copy numbers of MC-degrading bacteria occurred in the months following observed peaks in MC concentrations. To understand this phenomenon, we added MCs to the MC-degrading bacteria (designated strains HW and SW in this study) and found that MCs significantly enhanced mlrA gene copy numbers over the number for the control by a factor of 5.2 for the microcystin-RR treatment and a factor of 3.7 for the microcystin-LR treatment. These results indicate that toxic Microcystis and MC-degrading bacteria exert both direct and indirect effects on each other and that MC-degrading bacteria also mediate a shift from toxic to nontoxic populations of Microcystis.
| S-EPMC3957655 | biostudies-literature