Examination of Rickettsial Host Range for Shuttle Vectors Based on dnaA and parA Genes from the pRM Plasmid of Rickettsia monacensis.

The genus Rickettsia encompasses a diverse group of obligate intracellular bacteria that are highly virulent disease agents of mankind as well as symbionts of arthropods. Native plasmids of Rickettsia amblyommatis (AaR/SC) have been used as models to construct shuttle vectors for genetic manipulation of several Rickettsia species. Here, we report on the isolation of the complete plasmid (pRM658B) from Rickettsia monacensis IrR/Munich mutant Rmona658B and the construction of shuttle vectors based on pRM. To identify regions essential for replication, we made vectors containing the dnaA and parA genes of pRM with various portions of the region surrounding these genes and a selection reporter cassette conferring resistance to spectinomycin and expression of green fluorescent protein. Rickettsia amblyommatis (AaR/SC), R. monacensis (IrR/Munich), Rickettsia bellii (RML 369-C), Rickettsia parkeri (Tate's Hell), and Rickettsia montanensis (M5/6) were successfully transformed with shuttle vectors containing pRM parA and dnaA. PCR assays targeting pRM regions not included in the vectors revealed that native pRM was retained in R. monacensis transformants. Determination of native pRM copy number using a plasmid-carried gene (RM_p5) in comparison to chromosomally carried gltA indicated reduced copy numbers in R. monacensis transformants. In transformed R. monacensis strains, native pRM and shuttle vectors with homologous parA and dnaA formed native plasmid-shuttle vector complexes. These studies provide insight on the maintenance of plasmids and shuttle vectors in rickettsiae. IMPORTANCERickettsia spp. are found in a diverse array of organisms, from ticks, mites, and fleas to leeches and insects. Many are not pathogenic, but others, such as Rickettsia rickettsii and Rickettsia prowazeckii, can cause severe illness or death. Plasmids are found in a large percentage of nonpathogenic rickettsiae, but not in species that cause severe disease. Studying these plasmids can reveal their role in the biology of these bacteria, as well as the molecular mechanism whereby they are maintained and replicate in rickettsiae. Here, we describe a new series of shuttle plasmids for the transformation of rickettsiae based on parA and dnaA sequences of plasmid pRM from Rickettsia monacensis. These shuttle vectors support transformation of diverse rickettsiae, including the native host of pRM, and are useful for investigating genetic determinants that govern rickettsial virulence or their ability to function as symbionts.

Generating a Small Shuttle Vector for Effective Genetic Engineering of Methanosarcina mazei Allowed First Insights in Plasmid Replication Mechanism in the Methanoarchaeon.

Due to their role in methane production, methanoarchaea are of high ecological relevance and genetic systems have been ever more established in the last two decades. The system for protein expression in Methanosarcina using a comprehensive shuttle vector is established; however, details about its replication mechanism in methanoarchaea remain unknown. Here, we report on a significant optimisation of the rather large shuttle vector pWM321 (8.9 kbp) generated by Metcalf through a decrease in its size by about 35% by means of the deletion of several non-coding regions and the ssrA gene. The resulting plasmid (pRS1595) still stably replicates in M. mazei and-most likely due to its reduced size-shows a significantly higher transformation efficiency compared to pWM321. In addition, we investigate the essential gene repA, coding for a rep type protein. RepA was heterologously expressed in Escherichia coli, purified and characterised, demonstrating the significant binding and nicking activity of supercoiled plasmid DNA. Based on our findings we propose that the optimised shuttle vector replicates via a rolling circle mechanism with RepA as the initial replication protein in Methanosarcina. On the basis of bioinformatic comparisons, we propose the presence and location of a double-strand and a single-strand origin, which need to be further verified.

Simultaneous transformation of five vectors in Gluconobacter oxydans.

Gluconobacter oxydans is an obligate Gram-negative bacterium that belongs to the family Acetobacteraceae. It is one of the most frequently used microorganisms in industrial biotechnology to produce chemicals related to incomplete oxidation. However, the fine-tuning of G. oxydans is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. Thus, a series of shuttle vectors for G. oxydans inspired by a series of wild-type plasmids in different G. oxydans strains were constructed. Fifteen shuttle vectors were employed to express mCherry in G. oxydans WSH-003 using the replication origin of these wild-type plasmids. Among them, the intensity of fluorescent proteins expressed by p15-K-mCherry was about 10 times that of fluorescent proteins expressed by p5-K-mCherry. Quantitative real-time polymerase chain reaction showed that the relative copy number of p15-K-mCherry reached 19 and had high stability. In contrast, some of the plasmids had a relative copy number of less than 10. The co-expression of multiple shuttle vectors revealed five shuttle vectors that could be transformed into G. oxydans WSH-003 and could express five different fluorescent proteins. The shuttle vectors will facilitate genetic operations for Gluconobacter strains to produce useful compounds more efficiently.

Enhanced Activity by Genetic Complementarity: Heterologous Secretion of Clostridial Cellulases by Bacillus licheniformis and Bacillus velezensis.

To adapt to various ecological niches, the members of genus Bacillus display a wide spectrum of glycoside hydrolases (GH) responsible for the hydrolysis of cellulose and lignocellulose. Being abundant and renewable, cellulose-containing plant biomass may be applied as a substrate in second-generation biotechnologies for the production of platform chemicals. The present study aims to enhance the natural cellulase activity of two promising 2,3-butanediol (2,3-BD) producers, Bacillus licheniformis 24 and B. velezensis 5RB, by cloning and heterologous expression of cel8A and cel48S genes of Acetivibrio thermocellus. In B. licheniformis, the endocellulase Cel8A (GH8) was cloned to supplement the action of CelA (GH9), while in B. velezensis, the cellobiohydrolase Cel48S (GH48) successfully complemented the activity of endo-cellulase EglS (GH5). The expression of the natural and heterologous cellulase genes in both hosts was demonstrated by reverse-transcription PCR. The secretion of clostridial cellulases was additionally enhanced by enzyme fusion to the subtilisin-like signal peptide, reaching a significant increase in the cellulase activity of the cell-free supernatants. The results presented are the first to reveal the possibility of genetic complementation for enhancement of cellulase activity in bacilli, thus opening the prospect for genetic improvement of strains with an important biotechnological application.

Cloning of pAhX22, a small cryptic plasmid from Aeromonas hydrophila, and construction of a pAhX22-derived shuttle vector.

In this study, a cryptic plasmid from Aeromonas hydrophila (pAhX22) was cloned and characterized. pAhX22 was 2523 bp long, had a GC content of 59.9%, and contained two putative open reading frames (ORFs). orf1 and orf2 encoded putative proteins of 458 amino acids and 88 amino acids, respectively; these putative proteins might be involved in plasmid replication. An Escherichia coli-A. hydrophila shuttle vector, pAEsv-1 (4587 bp, KanR), was constructed using in-fusion cloning, combining pAhX22 with the kanamycin-resistance gene and the origin of replication from E. coli expression vector pET-28a. The transformation efficiency of pAEsv-1 in A. hydrophila strains ranged from 2.2 × 106 to 1.0 × 107 CFU/µg DNA, while transformation efficiency in E. coli DH5α was about 1.6 × 106 CFU/µg DNA. pAEsv-1 was segregationally and structurally stable in A. hydrophila in the absence of selective pressure. A green fluorescent protein gene (gfp) from pHT315-gfp was successfully cloned and expressed in A. hydrophila strain X2 using pAEsv-1, and 82.3% ± 2.5% of cells maintained the recombinant plasmid after one week in liquid culture without kanamycin. These results suggested that pAEsv-1 might potentially be used as a stable cloning vector for A. hydrophila, which might facilitate genetic studies of A. hydrophila.

Development of a supF-based mutation-detection system in the extreme thermophile Thermus thermophilus HB27.

Thermus thermophilus (T. thermophilus) HB27 is an extreme thermophile that grows optimally at 65-72 °C. Heat-induced DNA lesions are expected to occur at a higher frequency in the genome of T. thermophilus than in those of mesophiles; however, the mechanisms underlying the maintenance of genome integrity at high temperatures remain poorly understood. The study of mutation spectra has become a powerful approach to understanding the molecular mechanisms responsible for DNA repair and mutagenesis in mesophilic species. Therefore, we developed a supF-based system to detect a broad spectrum of mutations in T. thermophilus. This system was validated by measuring spontaneous mutations in the wild type and a udgA, B double mutant deficient in uracil-DNA glycosylase (UDG) activity. We found that the mutation frequency of the udgA, B strain was 4.7-fold higher than that of the wild type and G:C→A:T transitions dominated, which was the most reasonable for the mutator phenotype associated with the loss of UDG function in T. thermophilus. These results show that this system allowed for the rapid analysis of mutations in T. thermophilus, and may be useful for studying the molecular mechanisms responsible for DNA repair and mutagenesis in this extreme thermophile.

Transmating: conjugative transfer of a new broad host range expression vector to various Bacillus species using a single protocol.

BACKGROUND: The genus Bacillus includes a great variety of species with potential applications in biotechnology. While species such as B. subtilis or B. licheniformis are well-known and used to provide various products at industrial scale, other Bacillus species are less characterized and are not yet used in commercial processes. One reason for this is the fact that genetic manipulation of new isolates is usually complicated with conventional techniques which have to be adapted to each new strain. Even in well-established strains, the available transformation protocols often suffer from low efficiencies. RESULTS: In this paper, we provide a new broad host range E. coli/Bacillus shuttle vector, named pBACOV (Bacillus conjugation vector), that can be efficiently transferred to various Bacillus species using a single protocol. A variant of pBACOV carrying the sfGFP gene was successfully transferred to eight different species from the genus Bacillus and to one Paenibacillus species using triparental conjugation ("transmating"). This was achieved using a single protocol and worked for nine out of eleven tested acceptor species. The transmating procedure was used to test expression of the heterologous reporter gene sfGFP under control of the PaprE-promoter from B. subtilis in several Bacillus species in parallel. Expression of sfGFP was found in eight out of nine transmates. For several of the tested species, this is the first report of a method for genetic modification and heterologous gene expression. The expression level, analyzed by measuring the relative sfGFP-fluorescence normalized to the cell density of the cultures, was highest in B. mojavensis. CONCLUSIONS: The new shuttle vector pBACOV can be transferred to many different Bacillus and Paenibacillus species using a simple and efficient transmating protocol. It is a versatile tool facilitating the application of recombinant DNA technology in new as well as established strains, or selection of an ideal host for heterologous gene expression from a multitude of strains. This paves the way for the genetic modification and biotechnological exploitation of the broad diversity of species of Bacillus and related genera as well as different strains from these species.

Construction of Rhodococcus expression vectors and expression of the aminoalcohol dehydrogenase gene in Rhodococcus erythropolis.

NADP+-dependent aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154 produces double chiral aminoalcohols, which are used as pharmaceuticals. However, the genetic manipulation of Rhodococcus strains to increase their production of such industrially important enzymes is not well studied. Therefore, I aimed to construct Rhodococcus expression vectors, derived from the Rhodococcus-Escherichia coli shuttle vector pRET1102, to express aadh. The plasmid pRET1102 could be transformed into many actinomycete strains, including R. erythropolis. The transformation efficiency for a species closely related to R. erythropolis was higher than that for other actinomycete strains. Promoters of various strengths, hsp, 1200rep, and TRR, were obtained from Gram-positive bacteria. The activity of TRR was stronger than that of hsp and 1200rep. The aadh-expressing plasmid pRET1172 with TRR could be transformed into many actinomycete strains to increase their AADH production. The Rhodococcus expression vector, pRET11100, constructed by removing aadh from the pRET1172 plasmid may be useful for bioconversion.

Plasmid copy number mutation in repA gene encoding RepA replication initiator of cryptic plasmid pHM1519 in Corynebacterium glutamicum.

Cryptic plasmid pHM1519 is a rolling-circular replication mode plasmid of the pCG1 plasmid family in coryneform bacteria. The derived shuttle vector pPK4 is maintained at about 40-50 copies per chromosome in Corynebacterium glutamicum 2256 (ATCC 13869). We found that a mutation (designated copA1) within the repA gene encoding essential initiator protein RepA of the pHM1519-replicon increased the copy number of the mutant plasmid to about 800 copies per chromosome. The mutation was a single G to A base transition, which changed Gly to Glu at position 429 of the amino acid sequence of RepA. In silico secondary structure prediction of RepA suggested that Gly429 is situated in a disordered region in a helix-turn-helix motif, which is a typical DNA-binding domain. This study shows the first example of a high copy number of a C. glutamicum cryptic plasmid caused by an altered replication initiator protein.

Characterization of a cryptic plasmid isolated from Lactobacillus casei CP002616 and construction of shuttle vectors based on its replicon.

The cryptic plasmid pLC2W was isolated from Lactobacillus casei CP002616. Nucleotide sequence analysis revealed that 4 putative open reading frames (ORF) were responsible for DNA replication. Four Escherichia coli-Lactobacillus shuttle vectors were constructed using different lengths of the pLC2W replicon to identify the shortest functional replicon. The length of the pLC2W replicon did not affect the stability of the plasmids. Green fluorescent protein (GFP) as a reporter was expressed successfully in several lactobacilli using our constructed vectors. The results suggested that the expression vectors pUE-F0GFP and pUE-F1GFP are potential molecular tools for heterologous gene cloning and expression in lactobacilli. Moreover, 2 plasmid-curing methods were used to eliminate pLC2W from L. casei. We detected no difference between L. casei CP002616 and L. casei CP002616 pLC2WΔ-IC (mutant strain cured by plasmid incompatibility method) in production of exopolysaccharide (EPS) or acid. However, EPS and acid production were both reduced in L. casei CP002616 pLC2WΔ-HT (mutant strain cured by high-temperature heat treatment method), demonstrating a difference between these 2 curing methods. Sequence analysis of pLC2W and plasmid curing data suggest that plasmid pLC2W is not involved in EPS synthesis.

Characterization of the cryptic plasmid pOfk55 from Legionella pneumophila and construction of a pOfk55-derived shuttle vector.

In this study, a cryptic plasmid pOfk55 from Legionella pneumophila was isolated and characterized. pOfk55 comprised 2584bp with a GC content of 37.3% and contained three putative open reading frames (ORFs). orf1 encoded a protein of 195 amino acids and the putative protein shared 39% sequence identity with a putative plasmid replication protein RepL. ORF1 was needed for replication in L. pneumophila but pOfk55 did not replicate in Escherichia coli. orf2 and orf3 encoded putative hypothetical proteins of 114 amino acids and 78 amino acids, respectively, but the functions of the putative proteins ORF2 and OFR3 are not clear. The transfer mechanism for pOfk55 was independent on the type IVB secretion system in the original host. A L. pneumophila-E. coli shuttle vector, pNT562 (5058bp, KmR), was constructed by In-Fusion Cloning of pOfk55 with a kanamycin-resistance gene from pUTmini-Tn5Km and the origin of replication from pBluescript SK(+) (pNT561). Multiple cloning sites from pBluescript SK(+) as well as the tac promoter region and lacI gene from pAM239-GFP were inserted into pNT561 to construct pNT562. The transformation efficiency of pNT562 in L. pneumophila strains ranged from 1.6×101 to 1.0×105CFU/ng. The relative number of pNT562 was estimated at 5.7±1.0 copies and 73.6% of cells maintained the plasmid after 1week in liquid culture without kanamycin. A green fluorescent protein (GFP) expression vector, pNT563, was constructed by ligating pNT562 with the gfpmut3 gene from pAM239-GFP. pNT563 was introduced into L. pneumophila Lp02 and E. coli DH5α, and both strains expressed GFP successfully. These results suggest that the shuttle vector is useful for genetic studies in L. pneumophila.

Construction of pDUO: A bicistronic shuttle vector series for dual expression of recombinant proteins.

Our ability to genetically manipulate microbial systems is often hampered by the availability of genetic tools. Thus, there is a need for the continued expansion of our molecular tool box. In support of this expansion, this study reports the design, construction, and validation of a new bicistronic shuttle vector series, pDUO, for the dual expression of genes in different hosts. Each vector was designed and constructed to contain two araC-pBAD inducible promoter systems for tight control over gene expression. Each araC-pBAD promoter precedes a ribosomal binding site and a multicloning site (MCS). The 5' end of MCS1 contains a sequence encoding an affinity HIS-tag N-terminus and MCS2 terminates with a sequence encoding an affinity S-tag C-terminus for one-step purification of recombinant proteins encoded by the inserted genes. Both MCS are followed by an rrnBT1 and T2 transcriptional terminator sequence. Each vector in this series also contains a PBR322 and pRO1600-derived replicon to support replication in different host bacteria along with one of four different selectable markers. The functionality of the pDUO vector series was validated through the dual expression of oxalate biosynthetic component (obc) 1 and mrfp in Escherichia coli and Pseudomonas fluorescens. It is anticipated that this new vector series will facilitate functional studies as well as the engineering of bacterial strains for biotechnological purposes.

Construction, properties, and application of the pCB5 plasmid, a novel conjugative shuttle vector with a Cupriavidus basilensis origin of replication.

Cupriavidus basilensis is a species with diverse metabolic capabilities, including degradation of xenobiotics and heavy metal resistance. Although the genomes of several strains of this species have been sequenced, no plasmid has yet been constructed for genetic engineering in this species. In this study, we identified a novel plasmid, designated pWS, from C. basilensis WS with a copy number of 1-3 per cell and a length of 2150 bp. pWS contained three protein-coding genes, among which only rep was required for plasmid replication. Rep showed no homology with known plasmid replication initiators. Unlike most plasmids, pWS did not have a cis-acting replication origin outside the region of rep. The minimal replicon of pWS was stable in C. basilensis WS without selection. A conjugative C. basilensis/Escherichia coli shuttle vector, pCB5, was constructed using the minimal replicon of pWS. Interestingly, the copy number of pCB5 was flexible and could be manipulated. Enhancing the expression level of Rep in pCB5 by either doubling the promoter or coding region of rep resulted in doubling of the plasmid copy number. Moreover, replacing the native promoter of rep with the lac promoter increased the copy number by over fivefold. Finally, using two different ß-galactosidase reporting systems constructed with pCB5, we successfully demonstrated the different regulatory patterns of bph and dmp operons during diphenyl ether (DE) degradation in C. basilensis WS. Thus, this shuttle vector provided an efficient tool for DNA cloning and metabolic engineering in C. basilensis.

Development of nitrilase promoter-derived inducible vectors for Streptomyces.

An inducible expression vector, pSH19, which harbors regulatory expression system PnitA-NitR, for streptomycetes was constructed previously. Here, we have modified pSH19 to obtain shuttle vectors for Streptomyces-E. coli by introducing the replication origin of a plasmid for E. coli (ColE1) and an antibiotic-resistant gene. Six inducible shuttle vectors, pESH19cF, pESH19cR, pESH19kF, pESH19kR, pESH19aF, and pESH19aR, for Streptomyces-E. coli, were successfully developed. The stability of these vectors was examined in five different E. coli strains and Streptomyces lividans TK24. The stability test showed that the pSH19-derived shuttle vectors were stable in E. coli Stbl2 and S. lividans TK24. Heterologous expression experiments involving each of the catechol 2,3-dioxygenase, nitrilase, and N-substituted formamide deformylase genes as a reporter gene showed that pESH19cF, pESH19kF, and pESH19aF possess inducible expression ability in S. lividans TK24. Thus, these vectors were found to be useful expression tools for experiments on both Gram-negative and Gram-positive bacterial genes.

Construction of heterologous gene expression cassettes for the development of recombinant Clostridium beijerinckii.

Gene-expression cassettes for the construction of recombinant Clostridium beijerinckii were developed as potential tools for metabolic engineering of C. beijerinckii. Gene expression cassettes containing ColE1 origin and pAMB origin along with the erythromycin resistance gene were constructed, in which promoters from Escherichia coli, Lactococcus lactis, Ralstonia eutropha, C. acetobutylicum, and C. beijerinckii are examined as potential promoters in C. beijerinckii. Zymogram analysis of the cell extracts and comparison of lipase activities of the recombinant C. beijerinckii strains expressing Pseudomonas fluorescens tliA gene suggested that the tliA gene was functionally expressed by all the examined promoters with different expression level. Also, recombinant C. beijerinckii expressing C. beijerinckii secondary alcohol dehydrogenase by the constructed expression cassettes successfully produced 2-propanol from glucose. The best promoter for TliA expression was the R. eutropha phaP promoter while that for 2-propanol production was the putative C. beijerinckii pta promoter. Gene expression cassettes developed in this study may be useful tools for the construction of recombinant C. beijerinckii strains as host strains for the valuable chemicals and fuels from renewable resources.

Construction of a shuttle vector based on the small cryptic plasmid pJY33 from Weissella cibaria 33.

A cryptic plasmid, pJY33, from Weissella cibaria 33 was characterized. pJY33 was 2365 bp in size with a GC content of 41.27% and contained two putative open reading frames (ORFs). orf1 encoded a putative hypothetical protein of 134 amino acids. orf2 was 849 bp in size, and its putative translation product exhibited 87% identity with a replication initiation factor from a plasmid from W. cibaria KLC140. A Weissella-Escherichia coli shuttle vector, pJY33E (6.5 kb, Em(r)), was constructed by ligation of pJY33 with pBluescript II SK(-) and an erythromycin resistance gene (Em(r)). pJY33E replicated in Lactococcus lactis, Leuconostoc citreum, Lactobacillus brevis, Lactobacillus plantarum, and Weissella confusa. A single-stranded DNA intermediate was detected from Lb. brevis 2.14 harbouring pJY33E, providing evidence for rolling-circle replication of pJY33. Most Lb. brevis 2.14 cells (85.9%) retained pJY33E after one week of daily culturing in MRS broth without Em. An aga gene encoding α-galactosidase (α-Gal) from Leuconostoc mesenteroides was successfully expressed in Lb. brevis 2.14 using pJY33E, and the highest level of α-Gal activity (36.13 U/mg protein) was observed when cells were grown on melibiose.

A simplified system for generating recombinant E3-deleted canine adenovirus-2.

Canine adenovirus type 2 (CAV-2) has been used extensively as a vector for studying gene therapy and vaccine applications. We describe a simple strategy for generating a replication-competent recombinant CAV-2 using a backbone vector and a shuttle vector. The backbone plasmid containing the full-length CAV-2 genome was constructed by homologous recombination in Escherichia coli strain BJ5183. The shuttle plasmid, which has a deletion of 1478 bp in the nonessential E3 viral genome region, was generated by subcloning a fusion fragment containing the flanking sequences of the CAV-2 E3 region and expression cassette sequences from pcDNA3.1(+) into modified pUC18. To determine system effectiveness, a gene for enhanced green fluorescent protein (EGFP) was inserted into the shuttle plasmid and cloned into the backbone plasmid using two unique NruI and SalI sites. Transfection of Madin-Darby canine kidney (MDCK) cells with the recombinant adenovirus genome containing the EGFP expression cassette resulted in infectious viral particles. This strategy provides a solid foundation for developing candidate vaccines using CAV-2 as a delivery vector.

Isolation of a novel plasmid from Couchioplanes caeruleus and construction of two plasmid vectors for gene expression in Actinoplanes missouriensis.

To date, no plasmid vector has been developed for the rare actinomycete Actinoplanes missouriensis. Moreover, no small circular plasmid has been reported to exist in the genus Actinoplanes. Here, a novel plasmid, designated pCAZ1, was isolated from Couchioplanes caeruleus subsp. azureus via screening for small circular plasmids in Actinoplanes (57 strains) and Couchioplanes (2 strains). Nucleotide sequencing revealed that pCAZ1 is a 5845-bp circular molecule with a G + C content of 67.5%. The pCAZ1 copy number was estimated at 30 per chromosome. pCAZ1 contains seven putative open reading frames, one of which encodes a protein containing three motifs conserved among plasmid-encoded replication proteins that are involved in the rolling-circle mechanism of replication. Detection of single-stranded DNA intermediates in C. caeruleus confirmed that pCAZ1 replicates by this mechanism. The ColE1 origin from pBluescript SK(+) and the oriT sequence with the apramycin resistance gene aac(3)IV from pIJ773 were inserted together into pCAZ1, to construct the Escherichia coli-A. missouriensis shuttle vectors, pCAM1 and pCAM2, in which the foreign DNA fragment was inserted into pCAZ1 in opposite directions. pCAM1 and pCAM2 were successfully transferred to A. missouriensis through the E. coli-mediated conjugative transfer system. The copy numbers of pCAM1 and pCAM2 in A. missouriensis were estimated to be one and four per chromosome, respectively. Thus, these vectors can be used as effective genetic tools for homologous and heterologous gene expression studies in A. missouriensis.

A series of conditional shuttle vectors for targeted genomic integration in budding yeast.

The capacity of Saccharomyces cerevisiae to repair exposed DNA ends by homologous recombination has long been used by experimentalists to assemble plasmids from DNA fragments in vivo. While this approach works well for engineering extrachromosomal vectors, it is not well suited to the generation, recovery and reuse of integrative vectors. Here, we describe the creation of a series of conditional centromeric shuttle vectors, termed pXR vectors, that can be used for both plasmid assembly in vivo and targeted genomic integration. The defining feature of pXR vectors is that the DNA segment bearing the centromere and origin of replication, termed CEN/ARS, is flanked by a pair of loxP sites. Passaging the vectors through bacteria that express Cre recombinase reduces the loxP-CEN/ARS-loxP module to a single loxP site, thereby eliminating the ability to replicate autonomously in yeast. Each vector also contains a selectable marker gene, as well as a fragment of the HO locus, which permits targeted integration at a neutral genomic site. The pXR vectors provide a convenient and robust method to assemble DNAs for targeted genomic modifications.

Conjugative reporter system for the use in Bacillus licheniformis and closely related Bacilli.

Bacillus wild-type strains are genetically difficult to manipulate, and thus, the options for rational strain investigation and design are limited. Here, we present a set of small conjugative shuttle vectors for the use in Bacillus licheniformis and related, genetically difficult accessible wild-type strains. The vector set comprises the modular general-purpose vector pV2 and its derivatives pV3SDlacZ and pV3lacZ. The pV3 vectors are designed for the investigation of transcriptional and translational activities of regulatory regions like promoters and ribosomal binding sites (RBS). The vector set has been tested for investigating gene regulation under aerobic and anaerobic conditions.