Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones

Study of the molecular basis for Legionella pneumophila pathogenicity would be facilitated with an efficient mutagen that can not only mark genomic mutations, but can also be used to reflect gene expression during macrophage infection. A derivative of Jn903, Tn903dlllacZ, is shown to transpose with high efficiency in L. pneumophila. Tn903dlllacZ encodes resistance to kanamycin (Km^R) and carries a 5’truncated lacZ gene that can form translational fusions to L. pneumophila genes upon transposition. The cls-acting Tn903 transposase is supplied outside Tn903dlllacZ, and hence chromosomally integrated copies are stable. Km^R LacZ⁺ insertion mutants of L. pneumophila were isolated and shown by DNA hybridization to carry a single Tn903dlllacZ inserted within their chromosomes at various locations. One particular Km^R LacZ⁺ mutant, AB1156, does not produce the brown pigment (Pig) characteristic of Legionella species. Tn903dlllacZ is responsible for this phenotype since reintroduction of the transposonlinked mutation into a wild-type background results in a Pig phenotype. L. pneumophila pigment production is normally observed in stationary-phase growth of cells in culture, and β-galactosidase activity measured from the pig::lacZ fusion increased during the logarithmic-phase growth and peaked at the onset of stationary phase. Interestingly, pig::lacZ expression also increased during macrophage infection. The pigment itself, however, does not appear to be required for L. pneumophila to grow within or kill host macrophages.     

Improved cytotoxic effects of Salmonella‐producing cytosine deaminase in tumour cells

In order to increase the cytotoxic activity of a Salmonella strain carrying a salicylate‐inducible expression system that controls cytosine deaminase production, we have modified both, the vector and the producer bacterium. First, the translation rates of the expression module containing the Escherichia coli codA gene cloned under the control of the Pm promoter have been improved by using the T7 phage gene 10 ribosome binding site sequence and replacing the original GUG start codon by AUG. Second, to increase the time span in which cytosine deaminase may be produced by the bacteria in the presence of 5‐fluorocytosine, a 5‐fluorouracyl resistant Salmonella strain has been constructed by deleting its upp gene sequence. This new Salmonella strain shows increased cytosine deaminase activity and, after infecting tumour cell cultures, increased cytotoxic and bystander effects under standard induction conditions. In addition, we have generated a purD mutation in the producer strain to control its intracellular proliferation by the presence of adenine and avoid the intrinsic Salmonella cell death induction. This strategy allows the analysis and comparison of the cytotoxic effects of cytosine deaminase produced by different Salmonella strains in tumour cell cultures.     

Tn5/7-lux: a versatile tool for the identification and capture of promoters in Gram-negative bacteria

Background

The combination of imaging technologies and luciferase-based bioluminescent bacterial reporter strains provide a sensitive and simple non-invasive detection method (photonic bioimaging) for the study of diverse biological processes, as well as efficacy of therapeutic interventions, in live animal models of disease. The engineering of bioluminescent bacteria required for photonic bioimaging is frequently hampered by lack of promoters suitable for strong, yet stable luciferase gene expression.

Results

We devised a novel method for identification of constitutive native promoters in Gram-negative bacteria. The method is based on a Tn5/7 transposon that exploits the unique features of Tn5 (random transposition) and Tn7 (site-specific transposition). The transposons are designed such that Tn5 transposition will allow insertion of a promoter-less bacterial luxCDABE operon downstream of a bacterial gene promoter. Cloning of DNA fragments from luminescent isolates results in a plasmid that replicates in pir⁺ hosts. Sequencing of the lux-chromosomal DNA junctions on the plasmid reveals transposon insertion sites within genes or operons. The plasmid is also a mini-Tn7-lux delivery vector that can be used to introduce the promoter-lux operon fusion into other derivatives of the bacterium of interest in an isogenic fashion. Alternatively, promoter-containing sequences can be PCR-amplified from plasmid or chromosomal DNA and cloned into a series of accompanying mini-Tn7-lux vectors. The mini-Tn5/7-lux and mini-Tn7-lux vectors are equipped with diverse selection markers and thus applicable in numerous Gram-negative bacteria. Various mini-Tn5/7-lux vectors were successfully tested for transposition and promoter identification by imaging in Acinetobacter baumannii, Escherichia coli, and Burkholderia pseudomallei. Strong promoters were captured for lux expression in E. coli and A. baumannii. Some mini-Tn7-lux vectors are also equipped with attB sites for swapping of the lux operon with other reporter genes using Gateway technology.

Conclusions

Although mini-Tn5-lux and mini-Tn7-lux elements have previously been developed and used for bacterial promoter identification and chromosomal insertion of promoter-lux gene fusions, respectively, the newly developed mini-Tn5/7-lux and accompanying accessory plasmids streamline and accelerate the promoter discovery and bioluminescent strain engineering processes. Availability of vectors with diverse selection markers greatly extend the host-range of promoter probe and lux gene fusion vectors.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0354-3) contains supplementary material, which is available to authorized users.     

A pBRINT family of plasmids for integration of cloned DNA into the Escherichia coli chromosome

Plasmid pBRINT is an efficient vector for chromosomal integration of cloned DNA into the lacZ gene of Escherichia coli [Balbás et al., Gene 136(1993) 211–213]. A family of related plasmids containing different antibiotic-resistance markers (Cm^R or Gm^R or Km^R and a larger multiple cloning site (MCS) has been constructed. This set of plasmids, whose integration efficiencies are as good as those obtained with the prototype plasmid pBRINT, constitutes a collection of tools that allow rapid and easy integration of cloned DNA, at the chromosomal level. Their functionality as integration vectors has been ascertained by integrating the Vitreoscilla sp. hemoglobin-encoding gene and the Photobacterium leiognathi lux genes. To evaluate the level of expression obtained after chromosomal integration, we constructed strains carrying one or two copies of the cat gene integrated in the chromosome, and compared their enzymatic activities with those obtained from a strain carrying cat on a multicopy plasmid.     

Tn554: Isolation and characterization of plasmid insertions

Tn554, a transposon in Staphylococcus aureus that carries determinants of spectinomycin resistance and inducible macrolide-lincosamide resistance, is characterized by a highly efficient transposition, exceptional site specificity for insertion, and inhibition of transposition by a copy of the transposon inserted at its preferred chromosomal site. In this communication we describe the characteristics of a number of rare, secondary-site insertions of Tn554 into several related penicillinase plasmids. These plasmid insertions display considerable variation in the frequencies with which they can act as transposon donors, as well as in the frequencies at which they undergo apparently precise excision. Transposition from the plasmid transposon donors is ordinarily a duplicative process and these subsequent transposition events always return Tn554 to its preferred site in the S. aureus chromosome; such derivatives are indistinguishable from the primary chromosomal insertion from which they were originally derived. We also report an unusual relationship between Tn554 and the transducing phage, φ11, in which Tn554 is frequently transferred independently of its plasmid carrier. We suggest that the bacteriophage may play an important role in the mobility of Tn554, in addition to the usual transduction mechanism, in a process that we have referred to as “hitchhiking.”     

Protocols for yTREX/Tn5-based gene cluster expression in Pseudomonas putida

Bacterial gene clusters, which represent a genetic treasure trove for secondary metabolite pathways, often need to be activated in a heterologous host to access the valuable biosynthetic products. We provide here a detailed protocol for the application of the yTREX ‘gene cluster transplantation tool’: Via yeast recombinational cloning, a gene cluster of interest can be cloned in the yTREX vector, which enables the robust conjugational transfer of the gene cluster to bacteria like Pseudomonas putida, and their subsequent transposon Tn5-based insertion into the host chromosome. Depending on the gene cluster architecture and chromosomal insertion site, the respective pathway genes can be transcribed effectively from a chromosomal promoter, thereby enabling the biosynthesis of a natural product. We describe workflows for the design of a gene cluster expression cassette, cloning of the cassette in the yTREX vector by yeast recombineering, and subsequent transfer and expression in P. putida. As an example for yTREX-based transplantation of a natural product biosynthesis, we provide details on the cloning and activation of the phenazine-1-carboxylic acid biosynthetic genes from Pseudomonas aeruginosa in P. putidaKT2440 as well as the use of β-galactosidase-encoding lacZ as a reporter of production levels.     

Targeted transposition with Tn7 elements: safe sites,mobile plasmids,CRISPR/Cas and beyond

Transposon Tn7 is notable for the control it exercises over where transposition events are directed. One Tn7 integration pathways recognizes a highly conserved attachment (att) site in the chromosome, while a second pathway specifically recognizes mobile plasmids that facilitate transfer of the element to new hosts. In this review, I discuss newly discovered families of Tn7‐like elements with different targeting pathways. Perhaps the most exciting examples are multiple instances where Tn7‐like elements have repurposed CRISPR/Cas systems. In these cases, the CRISPR/Cas systems have lost their canonical defensive function to destroy incoming mobile elements; instead, the systems have been naturally adapted to use guide RNAs to specifically direct transposition into these mobile elements. The new families of Tn7‐like elements also include a variety of novel att sites in bacterial chromosomes where genome islands can form. Interesting families have also been revealed where proteins described in the prototypic Tn7 element are fused or otherwise repurposed for the new dual activities. This expanded understanding of Tn7‐like elements broadens our view of how genetic systems are repurposed and provides potentially exciting new tools for genome modification and genomics. Future opportunities and challenges to understanding the impact of the new families of Tn7‐like elements are discussed.     

Insertion vectors for construction of recombinant conjugative transposons in Bacillus subtilis and Enterococcus faecalis

The broad-host range of conjugal transfer and the chromosomal location make conjugative transposons (CT) attractive candidates as tools for genetic manipulation of a large variety of bacteria. In this paper we describe insertion vectors capable of integrating into Tn916, the prototype of CT in Gram-positive bacteria. The integration of vectors into a single chromosomal copy of Tn916 was studied both after natural transformation of Bacillus subtilis, and after electroporation in Enterococcus faecalis. Integration occurred either by double or by single crossover, and the integrated DNA segment was shown to be highly stable. All recombinant CT (rCT) were still able to excise from the chromosome to form circular intermediates, the first step of both transposition and conjugal transfer. All classes of rCT generated by insertion vector pSMB47 were capable of conjugal transfer, while using pVMB11 it was possible to generate non-conjugative rCT.     

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

The sugar nucleotide dTDP‐L‐rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A Streptococcus (GAS). The final step of the four‐step dTDP‐L‐rhamnose biosynthesis pathway is catalyzed by dTDP‐4‐dehydrorhamnose reductases (RmlD). RmlD from the Gram‐negative bacterium Salmonella is the only structurally characterized family member and requires metal‐dependent homo‐dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram‐negative and Gram‐positive RmlD homologues predicts that enzymes from all Gram‐positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gacA in a S. mutans rmlD knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn‐sequencing and generation of a conditional‐expression mutant identified gacA as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram‐positive bacteria and a subset of Gram‐negative bacteria. These results will help future screens for novel inhibitors of dTDP‐L‐rhamnose biosynthesis.     

An improved Tn7-based system for the single-copy insertion of cloned genes into chromosomes of gram-negative bacteria.

A system is described for the single-copy, stable insertion of cloned DNA sequences into the chromosomes of Gram- bacteria. Two narrow-host-range plasmids form the basis of this system: the 'carrier' plasmid contains the mini Tn7-Km transposon, into which foreign DNA can be cloned; the 'helper' plasmid provides the Tn7 transposition functions in trans. Both plasmids are readily transferred into Gram- bacteria by conjugation. The functionality of this system has been demonstrated in Rhodospirillum rubrum.     

Tn7 transposition as a probe of cis interactions between widely separated (190 kilobases apart) DNA sites in the Escherichia coli chromosome.

We have used the bacterial transposon Tn7 to examine communication between widely separated DNA sites in the Escherichia coli chromosome. Using Tn7 target immunity, a regulatory feature of transposition which influences target selection, we have evaluated (i) how the presence of Tn7 sequences at one DNA site affects Tn7 insertion into another site in the same DNA molecule and (ii) the nucleotide distances over which the two sites are able to communicate. We demonstrate that Tn7 sequences at one chromosomal site act at a distance to inhibit insertion of Tn7 elsewhere in that DNA as far away as 190 kb, reflecting effective long-range cis interactions. We have found that while target immunity is effective over a substantial region of the chromosome, insertion of Tn7 into a more distant site 1.9 Mb away in the same DNA is not inhibited; this observation provides evidence that target immunity relies on DNA spacing. We also find that within the region of the chromosome affected by target immunity, the magnitude of the immune effect is greater at close DNA sites than DNA sites farther away, suggesting that target immunity is distance dependent. We also extend the characterization of the Tn7 end-sequences involved in transposition and target immunity and describe how Tn7 target immunity can be used as a tool for probing bacterial chromosome structure.     

Characterisation of transfer-deficient mutants of the R100-1TcS plasmid pDU202, caused by insertion of Tn10

Summary The transposition of Tn10 from the E. coli chromosome to pDU202 (a Tc^S deletion mutant of R100-1) was selected by a mating technique: it took place at a frequency of 10^-7 in both rec ⁺ and recA hosts. Seventeen transfer deficient mutants of pDU202 caused by the insertion of Tn10 into the R factor's transfer genes were analysed. Insertion of Tn10 occurred at at least eight sites, with some apparent clustering in the second part of the transfer operon, and with equal numbers in each orientation. Both strongly and weakly polar insertions were observed, and the strength of the polarity was correlated with the Tn10 orientation. The map order of the second part of the transfer operon was confirmed as traC traF traH traG traS traD, analogous to that of F. The strongly polar insertion mutants still expressed traI at wild-type levels, providing further evidence that this gene does not belong to the transfer operon of R100.     

Control of Tn7 transposition

Summary Our isolate of Tn7 (named Tn7S) contains an IS1 insertion, and this IS1 can be converted into Tn9. In vitro and in vivo deletions of Tn7S and Tn7S:: Tn9 define regions of the transposon required for antibiotic resistance and transposition. Complementation of deletion mutants by cloned Tn7 fragments indicates the existence of two regions, denoted tnp7A and tnp7B, required for all transposition events. Another region, denoted tnp7C, is required for transposition from the chromosome to RP1 but not for transposition from a small IncP-1 replicon to the chromosome. The presence of Tn7S terminal sequences in an RP1 replicon reduces the transposition of a second Tn7S derivative from the chromosome by about one order of magnitude. The measured frequency of Tn7S transpositions from a small IncP-1 replicon to the chromosome depends on the particular incompatibility system used to eliminate that replicon. Genetic and physical data indicate that high frequencies of Tn7S transposition to the chromosome (40%) are triggered by the IncP-1 incompatibility reaction, thus suggesting the existence of a Tn7 mechanism for sensing the state of the carrier replicon.     

Take the tube: remodelling of the endosomal system by intracellular Salmonella enterica

Salmonella enterica is a facultative intracellular pathogen residing in a unique host cell‐derived membrane compartment, termed Salmonella‐containing vacuole or SCV. By the activity of effector proteins translocated by the SPI2‐endoced type III secretion system (T3SS), the biogenesis of the SCV is manipulated to generate a habitat permissive for intracellular proliferation. By taking control of the host cell vesicle fusion machinery, intracellular Salmonella creates an extensive interconnected system of tubular membranes arising from vesicles of various origins, collectively termed Salmonella‐induced tubules (SIT). Recent work investigated the dynamic properties of these manipulations. New host cell targets of SPI2‐T3SS effector proteins were identified. By applying combinations of live cell imaging and ultrastructural analyses, the detailed organization of membrane compartments inhabited and modified by intracellular Salmonella is now available. These studies provided unexpected new details on the intracellular environments of Salmonella. For example, one kind of SIT, the LAMP1‐positive Salmonella‐induced filaments (SIF), are composed of double‐membrane tubules, with an inner lumen containing host cell cytosol and cytoskeletal filaments, and an outer lumen containing endocytosed cargo. The novel findings call for new models for the biogenesis of SCV and SIT and give raise to many open questions we discuss in this review.     

A high efficient method of constructing recombinant Bombyx mori (silkworm) multiple nucleopolyhedrovirus based on zero‐background Tn7‐mediated transposition in Escherichia coli

A high efficient way for generation of recombinant Bombyx mori (silkworm) multiple nucleopolyhedrovirus by Tn7‐mediated transposition in Escherichia coli was performed. The new system consists of a conditional replication donor vector pRCDM and an attTn7 site blocked E. coli containing BmNPV‐Bacmid. The donor vector contains a replication origin derived from R6Kγ, which propagated only in host cells with pir gene expression decreased in the transposition background greatly. Compared with original vector derived from pUC, the transposition efficiency increased from 5.7 to 66% (≈10 fold) when using conditional replication vector pRCDM transposition into original BmDH10Bac. A further effort to decrease the transposition background was made by blocking the attTn7 site in host E. coli genome. The resulting attTn7 occupied BmDH10BacΔTn7 resulted in a significant increase from 5.7 to 23% (≈4 fold) in the efficacy of generate recombinant BmNPV Bacmid by transposition. Furthermore, the transposition of BmDH10BacΔTn7 with pRCDM resulted typically in 100% white colonies, and it indicated that a zero transposition background was accomplished. This high efficient and zero background transposition system provides a new simple and rapid method for construction of recombinant BmNPV used to express target genes or produce gene‐delivery virus particles in silkworm. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa

Broad host-range mini-Tn7 vectors facilitate integration of single-copy genes into bacterial chromosomes at a neutral, naturally evolved site. Here we present a protocol for employing the mini-Tn7 system in bacteria with single attTn7 sites, using the example Pseudomonas aeruginosa. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into P. aeruginosa by either transformation or conjugation, followed by selection of insertion-containing strains; third, PCR verification of mini-Tn7 insertions; and last, optional Flp-mediated excision of the antibiotic-resistance selection marker present on the chromosomally integrated mini-Tn7 element. From start to verification of the insertion events, the procedure takes as little as 4 d and is very efficient, yielding several thousand transformants per microgram of input DNA or conjugation mixture. In contrast to existing chromosome integration systems, which are mostly based on species-specific phage or more-or-less randomly integrating transposons, the mini-Tn7 system is characterized by its ready adaptability to various bacterial hosts, its site specificity and its efficiency. Vectors have been developed for gene complementation, construction of gene fusions, regulated gene expression and reporter gene tagging.     

The COPII complex and lysosomal VAMP7 determine intracellular Salmonella localization and growth

Salmonella invades epithelial cells and survives within a membrane‐bound compartment, the Salmonella‐containing vacuole (SCV). We isolated and determined the host protein composition of the SCV at 30 min and 3 h of infection to identify and characterize novel regulators of intracellular bacterial localization and growth. Quantitation of the SCV protein content revealed 392 host proteins specifically enriched at SCVs, out of which 173 associated exclusively with early SCVs, 124 with maturing SCV and 95 proteins during both time‐points. Vacuole interactions with endoplasmic reticulum‐derived coat protein complex II vesicles modulate early steps of SCV maturation, promoting SCV rupture and bacterial hyper‐replication within the host cytosol. On the other hand, SCV interactions with VAMP7‐positive lysosome‐like vesicles promote Salmonella‐induced filament formation and bacterial growth within the late SCV. Our results reveal that the dynamic communication between the SCV and distinct host organelles affects both intracellular Salmonella localization and growth at successive steps of host cell invasion.     

Unconventional Integration of the <Emphasis Type="Italic">bla</Emphasis> Gene from Plasmid pIT2 During IS<Emphasis Type="Italic">lacZ</Emphasis>/hah Transposon Mutagenesis in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1

The ISlacZ/hah transposon carried by pIT2 and derived originally from Tn5 has been a popular system in the generation of random insertion mutants of Pseudomonas aeruginosa. Using this system in the current study, two transconjugants were identified as conferring high levels of carbenicillin resistance. Analyses by gene complementation tests and site-specific gene knockout experiments support the conclusion that carbenicillin resistance in these two mutants is not due to the insertion of ISlacZ/hah transposon into the affected genes. Instead, the production of a TEM β-lactamase was detected, and integration of the bla gene from pIT2 to the chromosome of the recipient strain was confirmed by polymerase chain reaction. This surprising event was reproducible, with an estimated frequency among the transconjugants of 4% to 10%, and it may cause a potential complication in the interpretation of mutant phenotypes without notice.     

Site specific transposition of Tn7 into a Rhizobium meliloti megaplasmid

Summary Transposon Tn7 was shown to insert specifically into the megaplasmid of different Rhizobium meliloti strains. Tn7 transposition could not be detected in other Rhizobium strains such as R. trifolii, R. leguminosarum, R. phaseoli and R. japonicum. In R. meliloti strains, two unique sites in the megaplasmid were observed into which Tn7 can transpose at different frequencies. Only one copy of Tn7 could be detected in the megaplasmid and the insertion sites for Tn7 are outside the nif and nod region. Tn7 transposition in R. meliloti showed a marked preference for sites on plasmid RP4 compared to the megaplasmid sites. Attempts to cure Tn7 from the megaplasmid were unsuccessful. This site specific transposition of Tn7 in R. meliloti provides an additional genetic tool to further manipulate this important plasmid in symbiotic nitrogen fixation.