Generation of bright autobioluminescent bacteria by chromosomal integration of the improved lux operon ilux2

The bacterial bioluminescence system enables light production in living cells without an external luciferin. Due to its relatively low levels of light emission, many applications of bioluminescence imaging would benefit from an increase in brightness of this system. In this report a new approach of mutagenesis and screening of the involved proteins is described that is based on the identification of mutants with improved properties under rate-limiting reaction conditions. Multiple rounds of screening in Escherichia coli resulted in the operon ilux2 that contains 26 new mutations in the fatty acid reductase complex which provides the aldehyde substrate for the bioluminescence reaction. Chromosomal integration of ilux2 yielded an autonomously bioluminescent E. coli strain with 7-fold increased brightness compared to the previously described ilux operon. The ilux2 strain produces sufficient signal for the robust detection of individual cells and enables highly sensitive long-term imaging of bacterial propagation without a selection marker.

Chromosomal integration of Tn5253 occurs downstream of a conserved 11-bp sequence of the rbgA gene in Streptococcus pneumoniae and in all the other known hosts of this integrative conjugative element (ICE)

Background Tn5253, a composite Integrative Conjugative Element (ICE) of Streptococcus pneumoniae carrying tet(M) and cat resistance determinants, was found to (i) integrate at specific 83-bp integration site (attB), (ii) produce circular forms joined by a 84-bp sequence (attTn), and (iii) restore the chromosomal integration site. The purpose of this study is to functionally characterize the attB in S. pneumoniae strains with different genetic backgrounds and in other bacterial species, and to investigate the presence of Tn5253 attB site into bacterial genomes. Results Analysis of representative Tn5253-carryng transconjugants obtained in S. pneumoniae strains with different genetic backgrounds and in other bacterial species, namely Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes, and Enterococcus faecalis showed that: (i) Tn5253 integrates in rbgA of S. pneumoniae and in orthologous rbgA genes of other bacterial species, (ii) integration occurs always downstream of a 11-bp sequence conserved among streptococcal and enterococcal hosts, (iii) length of the attB site corresponds to length of the duplication after Tn5253 integration, (iv) attB duplication restores rbgA CDS, (v) Tn5253 produced circular forms containing the attTn site at a concentration ranging between 2.0 × 10−5 to 1.2 × 10−2 copies per chromosome depending on bacterial species and strain, (vi) reconstitution of attB sites occurred at 3.7 × 10−5 to 1.7 × 10−2 copies per chromosome. A database search of complete microbial genomes using Tn5253 attB as a probe showed that (i) thirteen attB variants were present in the 85 complete pneumococcal genomes, (ii) in 75 pneumococcal genomes (88.3 %), the attB site was 83 or 84 nucleotides in length, while in 10 (11.7 %) it was 41 nucleotides, (iii) in other 19 bacterial species attB was located in orthologous rbgA genes and its size ranged between 17 and 84 nucleotides, (iv) the 11-bp sequence, which correspond to the last 11 nucleotides of attB sites, is conserved among the different bacterial species and can be considered the core of the Tn5253 integration site. Conclusions A functional characterization of the Tn5253 attB integration site combined with genome analysis contributed to elucidating the potential of Tn5253 horizontal gene transfer among different bacterial species.

Cre/lox-mediated chromosomal integration of biosynthetic gene clusters for heterologous expression in Aspergillus nidulans

Building strains for stable long-term heterologous expression of large biosynthetic pathways in filamentous fungi is limited by the low transformation efficiency or genetic stability of current methods. Here, we developed a system for targeted chromosomal integration of large biosynthetic gene clusters in Aspergillus nidulans based on site-specific recombinase mediated cassette exchange. We built A. nidulans strains harbouring a chromosomal landing pad for Cre/lox-mediated recombination and demonstrated efficient targeted integration of a 21.5 kb heterologous region in a single step. We further evaluated the integration at two loci by analysing the expression of a fluorescent reporter and the production of a heterologous polyketide. We compared chromosomal expression at those landing loci to episomal AMA1-based expression, which also shed light on uncharacterised aspects of episomal expression in filamentous fungi. This is the first demonstration of site-specific recombinase-mediated integration in filamentous fungi, setting the foundations for the further development of this tool.

Genome-wide patterns of bracovirus chromosomal integration into multiple host tissues during parasitism

Bracoviruses are domesticated viruses found in parasitic wasp genomes. They are composed of genes of nudiviral origin involved in particle production and proviral segments encoding virulence genes necessary for parasitism success. During particle production, proviral segments are amplified and individually packaged as DNA circles in nucleocapsids. These particles are injected by parasitic waspstogether with their eggs into host larvae. Bracovirus circles of two wasp species were reported toundergo chromosomal integration in parasitized host hemocytes, through a conserved sequence named Host Integration Motif (HIM). Here, we used bulk Illumina sequencing to survey integrations of Cotesia typhae bracovirus circles in the DNA of its host, the maize corn borer ( Sesamia nonagrioides ) seven days after parasitism. First, assembly and annotation of a high-quality genome for C. typhae enabled us to characterize 27 proviral segments clustered in proviral loci. Using these data, we characterized large numbers of chromosomal integrations (from 12 to 85 events per host haploid genome) for all 16 bracovirus circles containing a HIM. Integrations were found in four S. nonagrioides tissues and in the body of a caterpillar in which parasitism had failed. The 12 remaining circles do not integrate but are maintained at high levels in host tissues. Surprisingly, we found that HIM-mediated chromosomal integration has occurred at least six times accidentally in thewasp germline during evolution. Overall, our study furthers our understanding of wasp-host genome interactions and supports HIM-mediated chromosomal integration as a possible mechanism ofhorizontal transfer from wasps to their hosts. Importance Bracoviruses are endogenous domesticated viruses of parasitoid wasps that are injected together with wasp eggs into wasp host larvae during parasitism. Several studies have shown that some DNA circles packaged into bracovirus particles become integrated into host somatic genomes during parasitism, but the phenomenon has never been studied using non-targeted approaches. Here we use bulk Illumina sequencing to systematically characterize and quantify bracovirus circle integrations that occur in four tissues of the Mediterranean corn borer ( Sesamia nonagrioides ) during parasitism by the Cotesia typhae wasp. Our analysis reveals that all circles containing a host integration motif (HIM) integrate at substantial levels (from 12 to 85 integrations per host cell in total) in all tissues while other circles do not integrate. In addition to shedding new light on wasp-bracovirus-host interaction, our study supports HIM-mediated chromosomal integration of bracovirus as a possible source of wasp-to-host horizontal transfer with long term evolutionary consequences.

Chromosomal Integration of Huge and Complex blaNDM-Carrying Genetic Elements in Enterobacteriaceae

In this study, a detailed genetic dissection of the huge and complex blaNDM-carrying genetic elements and their related mobile genetic elements was performed in Enterobacteriaceae. An extensive comparison was applied to 12 chromosomal genetic elements, including six sequenced in this study and the other six from GenBank. These 12 genetic elements were divided into five groups: a novel IME Tn6588; two related IMEs Tn6523 (SGI1) and Tn6589; four related ICEs Tn6512 (R391), Tn6575 (ICEPvuChnBC22), Tn6576, and Tn6577; Tn7 and its derivatives Tn6726 and 40.7-kb Tn7-related element; and two related IMEs Tn6591 (GIsul2) and Tn6590. At least 51 resistance genes, involved in resistance to 18 different categories of antibiotics and heavy metals, were found in these 12 genetic elements. Notably, Tn6576 carried another ICE Tn6582. In particular, the six blaNDM-carrying genetic elements Tn6588, Tn6589, Tn6575, Tn6576, Tn6726, and 40.7-kb Tn7-related element contained large accessory multidrug resistance (MDR) regions, each of which had a very complex mosaic structure that comprised intact or residual mobile genetic elements including insertion sequences, unit or composite transposons, integrons, and putative resistance units. Core blaNDM genetic environments manifested as four different Tn125 derivatives and, notably, two or more copies of relevant Tn125 derivatives were found in each of Tn6576, Tn6588, Tn6589, and 40.7-kb Tn7-related element. The huge and complex blaNDM-carrying genetic elements were assembled from complex transposition and homolog recombination. Firstly identified were eight novel mobile elements, including three ICEs Tn6576, Tn6577, and Tn6582, two IMEs, Tn6588 and Tn6589, two composite transposons Tn6580a and Tn6580b, and one integron In1718.

CLINICAL CASES OF DETECTION OF CHROMOSOMALLY INTEGRATED HUMAN HERPESVIRUS TYPE 6 IN TWO CHILDREN

The article describes two clinical observations of the detection of chromosomally integrated human herpes virus type 6 (HHV-6). In both cases, the children were referred to an immunologist with a diagnosis of chronic infection caused by the human herpesvirus type 6, in order to exclude an immunodeficiency state and determine the strategies of treating this infection. However, during the molecular biological examination of children, a chromosomal integration of HHV-6 was determined, which led to a revision of the diagnostic search and abandoning massive antiviral therapy.

Overexpression of thermophilic a -amylase in Bacillus licheniformis using a high efficiency chromosomal integration and amplification strategy

Background: The production of industrially important enzymes depends on the development of genetically stable and high-yielding microorganisms. In order to simplify the development of strains able to efficiently overproduce enzymes, a new strategy based on chromosomal integration and amplification in Bacillus sp. was developed.Results: A thermosensitive replicable plasmid pUB-MazF and an integrated expression plasmid pUB'-Ex1 were constructed. pUB-MazF with a thermo-sensitive replicase RepF encoded by gene repF can self-replicate in Bacillus sp. if the cultivation temperature is no more than 35 oC and if the MazF encoded by mazF is not expressed. mazF is controlled by an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducing promoter and MazF is toxic to the cell. When IPTG is present and the MazF encoded by mazF in pUB-MazF is expressed, the host cell should lose the plasmid to survive. At a cultivation temperature over 37 oC, pUB-MazF will not well replicate and is not stable. pUB'-Ex1 has a disrupted repF and cannot self-replicate but can replicate when pUB-MazF is present. By using this approach, pUB'-Ex1 and constructs can replicate and will be forced to integrate into the host chromosome at 37oC or over when a selectable marker is present. The host was cured by MazF expression after induction with IPTG. Bacillus licheniformis BL-UBM integrated with pUB-MazF was transformed with pUB'-amyS derived from pUB'-Ex1 by in-frame cloning of amyS encoding a thermophilic α-amylase from Geobacillus stearothermophilus ATCC 31195. B. licheniformis BL-UBM transformed with pUB'-amyS was cultivated at 42oC with 1 mmol/l IPTG and 500 μg/ml kanamycin and the recombinants expressing high α-amylase activities were selected. All recombinants evaluated were genetically stable. The highest yield of α-amylase of 50 753 U/ml was produced by recombinant BLiS-002 carrying five copies of amyS in a 50-l fermenter. Conclusion: The strategy developed in this study has potential application for convenient and rapid development of strains overexpressing industrially important enzymes.