A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis

Plasmid transformation of chlamydiae has created new opportunities to investigate host–microbe interactions during chlamydial infections; however, there are still limitations. Plasmid transformation requires a replicon derived from the native Chlamydia plasmid, and these transformations are species-specific. We explored the utility of a broad host-range plasmid, pBBR1MCS-4, to transform chlamydiae, with a goal of simplifying the transformation process. The plasmid was modified to contain chromosomal DNA from C. trachomatis to facilitate homologous recombination. Sequences flanking incA were cloned into the pBBR1MCS-4 vector along with the GFP:CAT cassette from the pSW2-GFP chlamydial shuttle vector. The final plasmid construct, pBVR2, was successfully transformed into C. trachomatis strain L2-434. Chlamydial transformants were analyzed by immunofluorescence microscopy and positive clones were sequentially purified using limiting dilution. PCR and PacBio-based whole genome sequencing were used to determine if the plasmid was maintained within the chromosome or as an episome. PacBio sequencing of the cloned transformants revealed allelic exchange events between the chromosome and plasmid pBVR2 that replaced chromosomal incA with the plasmid GFP:CAT cassette. The data also showed evidence of full integration of the plasmid into the bacterial chromosome. While some plasmids were fully integrated, some were maintained as episomes and could be purified and retransformed into E. coli. Thus, the plasmid can be successfully transformed into chlamydia without a chlamydial origin of replication and can exist in multiple states within a transformed population.

High-efficiency multi-site genomic editing (HEMSE) of Pseudomonas putida through thermoinducible ssDNA recombineering

SUMMARYWhile single-stranded DNA recombineering is a powerful strategy for higher-scale genome editing, its application to species other than enterobacteria is typically limited by the efficiency of the recombinase and the action of native mismatch repair (MMR) systems. By building on [i] availability of the Erf-like single-stranded DNA-annealing protein Rec2, [ii] adoption of tightly-regulated thermoinducible device and [iii] transient expression of a MMR-supressing mutL allele, we have set up a coherent genetic platform for entering multiple changes in the chromosome of Pseudomononas putida with an unprecedented efficacy and reliability. The key genetic construct to this end is a broad host range plasmid encoding co-transcription of rec2 and P. putida’s mutLE36KPP at high levels under the control of the PL/cI857 system. Cycles of short thermal shifts of P. putida cells followed by transformation with a suite of mutagenic oligos delivered different types of high-fidelity genomic changes at frequencies up to 10% per single change—that can be handled without selection. The same approach was instrumental to super-diversify short chromosomal portions for creating libraries of functional genomic segments—as shown in this case with ribosomal binding sites. These results enable the multiplexing of genome engineering of P. putida, as required for metabolic engineering of this important biotechnological chassis.

ArdC protein overpasses the recipient hsdRMS restriction system broadening conjugation host range

Plasmids, when transferred by conjugation, must overpass restriction-modification systems of the recipient cell. We demonstrate that protein ArdC, encoded by broad host range plasmid R388, was required for conjugation from Escherichia coli to Pseudomonas putida, but not from E. coli to E. coli. Surprisingly, expression of ardC was required in the recipient cells, but not in the donor cells. Besides, ardC was not required for conjugation if the hsdRMS system was deleted in P. putida recipient cells. Thus, ArdC has antirestriction activity against HsdRMS system, and consequently broadens R388 plasmid host range. The crystal structure of ArdC was solved both in the absence and in the presence of Mn2+. ArdC is composed of a non-specific ssDNA binding N-terminal domain and a C-terminal metalloprotease domain, although the metalloprotease activity is not needed for antirestriction function. We also observed by RNA-seq that ArdC-dependent conjugation triggers an SOS response in the P. putida recipient cells. Our findings give new insights, and open new questions, into the antirestriction strategies developed by plasmids to counteract bacterial restriction strategies.

Lactic Acid Containing Polymers Produced in EngineeredSinorhizobium melilotiandPseudomonas putida

This study demonstrates that novel polymer production can be achieved by introducing pTAM, a broad-host-range plasmid expressing codon-optimized genes encodingClostridium propionicumpropionate CoA transferase (PctCp) and a modifiedPseudomonassp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps6-19), intophaCmutant strains of the native polymer producersSinorhizobium melilotiandPseudomonas putida. Both phenotypic analysis and gas chromatography analysis indicated the synthesis and accumulation of biopolymers inS. melilotiandP. putidastrains. Expression inS. melilotiresulted in the production of PLA homopolymer up to 3.2% dried cell weight (DCW). The quaterpolymer P(3HB-co-LA-co-3HHx-co-3HO) was produced by expression inP. putida. TheP. putida phaCmutant strain produced this type of polymer the most efficiently with polymer content of 42% DCW when cultured in defined media with the addition of sodium octanoate, while the greatest LA fraction (4% mol) was achieved in the same strain cultured in LB with the addition of lactic acid. This is the first report, to our knowledge, of the production of a range of different biopolymers using the same plasmid-based system in different backgrounds. In addition, it is the first time that the novel polymer (P(3HB-co-LA-co-3HHx-co-3HO)), has been reported being produced in bacteria.

Short-term evolution under copper stress increases probability of plasmid uptake

Understanding plasmid transfer dynamics remains a key knowledge gap in the mitigation of antibiotic resistance gene spread. Direct effects of exposure to stressors on plasmid uptake are well monitored. However, it remains untested whether evolution of strains under stress conditions modulates subsequent plasmid uptake. Here, we evolved a compost derived microbial community for six weeks under copper stress and non-exposed control conditions. We then tested the ability of isolated clones from both treatments to take up the broad host range plasmid pKJK5 from anE.colidonor strain. Clones pre-adapted to copper displayed a significantly increased probability to be permissive towards the plasmid compared to those isolated from the control treatment. Further, increased phylogenetic distance to the donor strain was significantly and negatively correlated with plasmid uptake probabilities across both treatments.

Expanding the toolbox of broad host-range transcriptional terminators for Proteobacteria through metagenomics

As the field of synthetic biology moves towards the utilization of novel bacterial chassis, there is a growing need for biological parts with enhanced performance in a wide number of hosts. Is not unusual that biological parts (such as promoters and terminators), initially characterized in the model bacteria Escherichia coli, do not perform well when implemented in alternative hosts, such as Pseudomonas, therefore limiting the construction of synthetic circuits in industrially relevant bacteria. In order to address this limitation, we present here the mining of transcriptional terminators through functional metagenomics to identify novel parts with broad host-range activity. Using a GFP-based terminator trap strategy and a broad host-range plasmid, we identified 20 clones with potential terminator activity in Pseudomonas putida. Further characterization allowed the identification of 4 unique sequences between 58 bp and 181 bp long that efficiently terminates transcription in P. putida, E. coli, Burkholderia phymatum and two Pseudomonas strains isolated from Antarctica. Therefore, this work presents a new set of biological parts useful for the engineering of synthetic circuits in Proteobacteria.

Chromatic bacteria – A broad host-range plasmid and chromosomal insertion toolbox for fluorescent protein expression in bacteria

Differential fluorescent labelling of bacteria has become instrumental for many aspects of microbiological research, such as the study of biofilm formation, bacterial individuality, evolution, and bacterial behaviour in complex environments. We designed a variety of plasmids, each bearing one of eight unique, constitutively expressed fluorescent protein genes in conjunction with one of four different antibiotic resistance combinations. The fluorophores mTagBFP2, mTurquoise2, sGFP2, mClover3, sYFP2, mOrange2, mScarlet-I, and mCardinal, encoding for blue, cyan, green, green-yellow, yellow, orange, red, and far-red fluorescent proteins, respectively, were combined with selectable markers conferring tetracycline, gentamicin, kanamycin, and/or chloramphenicol resistance. These constructs were cloned into three different plasmid backbones: a broad host-range plasmid, a Tn5transposon delivery plasmid, and a Tn7transposon delivery plasmid. The utility of the plasmids and transposons was tested in bacteria from the phyla Actinobacteria, Proteobacteria, and Bacteroidetes. We were able to tag representatives from the phylum Proteobacteria at least via our Tn5transposon delivery system. The here constructed plasmids are available to the community and provide a valuable tool to investigate bacteria-bacteria, bacteria-host, and bacteria-environmental interactions.