Gabriella Balikó

Reduced evolvability of Escherichia coli MDS42, an IS-less cellular chassis for molecular and synthetic biology applications

BackgroundEvolvability is an intrinsic feature of all living cells. However, newly emerging, evolved features can be undesirable when genetic circuits, designed and fabricated by rational, synthetic biological approaches, are installed in the cell. Streamlined-genome E. coli MDS42 is free of mutation-generating IS elements, and can serve as a host with reduced evolutionary potential.ResultsWe analyze an extreme case of toxic plasmid clone instability, and show that random host IS element hopping, causing inactivation of the toxic cloned sequences, followed by automatic selection of the fast-growing mutants, can prevent the maintenance of a clone developed for vaccine production. Analyzing the molecular details, we identify a hydrophobic protein as the toxic byproduct of the clone, and show that IS elements spontaneously landing in the cloned fragment relieve the cell from the stress by blocking transcription of the toxic gene. Bioinformatics analysis of sequence reads from early shotgun genome sequencing projects, where clone libraries were constructed and maintained in E. coli, suggests that such IS-mediated inactivation of ectopic genes inhibiting the growth of the E. coli cloning host might happen more frequently than generally anticipated, leading to genomic instability and selection of altered clones.ConclusionsDelayed genetic adaptation of clean-genome, IS-free MDS42 host improves maintenance of unstable genetic constructs, and is suggested to be beneficial in both laboratory and industrial settings.

Indispensability of horizontally transferred genes and its impact on bacterial genome streamlining

Why are certain bacterial genomes so small and compact? The adaptive genome streamlining hypothesis posits that selection acts to reduce genome size because of the metabolic burden of replicating DNA. To reveal the impact of genome streamlining on cellular traits, we reduced the Escherichia coli genome by up to 20% by deleting regions which have been repeatedly subjects of horizontal transfer in nature. Unexpectedly, horizontally transferred genes not only confer utilization of specific nutrients and elevate tolerance to stresses, but also allow efficient usage of resources to build new cells, and hence influence fitness in routine and stressful environments alike. Genome reduction affected fitness not only by gene loss, but also by induction of a general stress response. Finally, we failed to find evidence that the advantage of smaller genomes would be due to a reduced metabolic burden of replicating DNA or a link with smaller cell size. We conclude that as the potential energetic benefit gained by deletion of short genomic segments is vanishingly small compared with the deleterious side effects of these deletions, selection for reduced DNA synthesis costs is unlikely to shape the evolution of small genomes.

Expression vectors based on the rac fusion promoter.

The -35 region of the rrnB P2 promoter and the -10 region of the lacZpo promoter-operator were fused to form the strong and regulatable rac promoter. Vectors were constructed that allow the attachment of protein-coding sequences to the beta-galactosidase alpha-peptide (LacZ alpha) in any reading frame. By introducing a high-copy-number mutation, the synthesis of a LacZ alpha-chloramphenicol acetyltransferase fusion protein reached more than 60% of total cell protein in Escherichia coli.

A family of expression vectors based on the rrnBP2 promoter of Escherichia coli

We describe here the construction of a family of expression vectors, based on the P2 promoter of the Escherichia coli rrnB gene by removing regulatory sequences downstream of the Pribnow-box and replacing them with the lac operator. These vectors allow cloning of foreign genes in such a way that their products are synthesized either in the form of fusion proteins of different length, or without fusion partners, with or without the original translational initiation signals. One of the vectors contains a synthetic oligothreonine-coding sequence that helps to stabilize the product of the cloned gene. These vectors allow high-level regulated expression of foreign genes, even if their products are relatively short peptides.

Cloning and Characterization of the DNA Region Responsible for Megacin A-216 Production in Bacillus megaterium 216

Upon induction, Bacillus megaterium 216 produces the bacteriocin megacin A-216, which leads to lysis of the producer cell and kills B. megaterium and a few other bacterial species. The DNA region responsible for megacinogeny was cloned in B. megaterium. The nucleotide sequence of a 5,494-bp-long subfragment was determined, and the function of the genes on this fragment was studied by generating deletions and analyzing their effects on MegA phenotypes. An open reading frame (ORF) encoding a 293-amino-acid protein was identified as the gene (megA) coding for megacin A-216. BLAST searches detected sequence similarity between megacin A-216 and proteins with phospholipase A2 activity. Purified biologically active megacin A-216 preparations contained three proteins. Mass spectrometry analysis showed that the largest protein is the full-length translation product of the megA gene, whereas the two shorter proteins are fragments of the long protein created by cleavage between Gln-185 and Val-186. The molecular masses of the three polypeptides are 32,855, 21,018, and 11,855 Da, respectively. Comparison of different megacin preparations suggests that the intact chain as well as the two combined fragments can form biologically active megacin. An ORF located next to the megA gene and encoding a 91-amino-acid protein was shown to be responsible for the relative immunity displayed by the producer strain against megacin A-216. Besides the megA gene, at least two other genes, including a gene encoding a 188-amino-acid protein sharing high sequence similarity with RNA polymerase sigma factors, were shown to be required for induction of megacin A-216 expression.

New approaches to increase the expression and stability of cloned foreign genes in Escherichia coli

A family of expression plasmid vectors were constructed by fusing the strong P2 promoter of the rrnB gene of Escherichia coli (coding for ribosomal RNA) to the lac operator, thereby eliminating regulatory sequences from the rrnB gene and placing the expression under lac repressor control. This promoter proved to be stronger in vivo than the well-known consensus tac promoter, and its strength could be further increased by converting the sequence to consensus. The stability of the recombinant proteins could be increased by fusion to various lengths of the N-terminal end of beta-galactosidase, or by inserting a synthetic oligonucleotide, coding for heptathreonine. A new method was developed for the stabilization of recombinant plasmids without antibiotic selection, based on the presence of an essential gene on the plasmid and its absence from the chromosome. The application of this method is illustrated by the example of a plasmid expressing human proinsulin.

An Escherichia coli gene in search of a function

SummaryThe rrB gene of Escherichia coli is preceded by an open reading frame, which is contranscribed with rrnB both in vivo and in vitro. It has earlier been shown that a 289 amino acid protein corresponding to this gene is actually synthesized in E. coli. In this paper we show that: (1.) The transcription of this gene diminishes the stringent response of the P1 promoter of the linked rrB gene, but this is a cis effect and is not mediated by the protein product of the gene. (2.) The functional integrity of this gene seems to be essential, because efforts to replace it by a plasmidcoded copy mutagenized by Tn5 completely failed. (3.) The protein product of this gene was strongly overproduced by a recombinant plasmid, exploiting the principle of “translational coupling”. This overproduction did not change the phenotype of the host cell significantly. The protein was purified to apparent electrophoretic homogenity.