Two groups of phenylalanine biosynthetic operon leader peptides genes: a high level of apparently incidental frameshifting in decoding Escherichia coli pheL

The bacterial pheL gene encodes the leader peptide for the phenylalanine biosynthetic operon. Translation of pheL mRNA controls transcription attenuation and, consequently, expression of the downstream pheA gene. Fifty-three unique pheL genes have been identified in sequenced genomes of the gamma subdivision. There are two groups of pheL genes, both of which are short and contain a run(s) of phenylalanine codons at an internal position. One group is somewhat diverse and features different termination and 5′-flanking codons. The other group, mostly restricted to Enterobacteria and including Escherichia coli pheL, has a conserved nucleotide sequence that ends with UUC_CCC_UGA. When these three codons in E. coli pheL mRNA are in the ribosomal E-, P- and A-sites, there is an unusually high level, 15%, of +1 ribosomal frameshifting due to features of the nascent peptide sequence that include the penultimate phenylalanine. This level increases to 60% with a natural, heterologous, nascent peptide stimulator. Nevertheless, studies with different tRNAPro mutants in Salmonella enterica suggest that frameshifting at the end of pheL does not influence expression of the downstream pheA. This finding of incidental, rather than utilized, frameshifting is cautionary for other studies of programmed frameshifting

Wobble modifications and other features in transfer RNA important for decoding and reading frame maintenance

Transfer RNA (tRNA) is the adaptor molecule responsible for bringing the correct amino acid to the ribosome during protein synthesis. tRNA contains a number of modified nucleosides, which are derivatives of the four normal nucleosides. A great variety of modifications are found in the anticodon loop, especially at the first (wobble) position of the anticodon. According to Crick’s wobble hypothesis, a uridine at the wobble position of tRNA recognize codons ending with A and G. Uridine-5-oxyacetic acid (cmo5U34), found at the wobble position of six species of tRNA in Salmonella enterica, have been predicted to expand the codon recognition of uridine to include U-ending, but not C-ending codons. To study the function of cmo5U34 we have identified two genes, cmoA and cmoB, which are required for the synthesis of cmo5U34 in tRNA. We have shown that the proline, alanine and valine tRNAs containing cmo5U34 are capable of reading codons ending with any of the four nucleotides, while the threonine tRNA is not, and the importance of having cmo5U is different for the different tRNAs. In addition, we found that cmo5U is important for efficient reading of G-ending codons, which is surprising considering the wobble hypothesis, which states that uridine should read G-ending codons. The dominant +1 frameshift suppressor sufY suppresses the hisC3737 +1 frameshift mutation. We have demonstrated that sufY induces frameshifting at CCC-CAA (Pro-Gln), when tRNAPro[cmo5UGG] occupies the P-site. sufY mutants accumulate novel modified nucleosides at the wobble position of tRNAs that should normally have (c)mnm5s2U34. The presence of an extra sidechain (C10H17) on the wobble nucleoside of tRNAGln[(c)mnm5s2U] leads to slow decoding of CAA codons, inducing a translational pause that allows the P-site peptidyl-tRNAPro[cmo5UGG] to slip into the +1 frame. We have characterized 108 independent frameshift suppressor mutants in the gene encoding tRNAPro[cmo5UGG]. The altered tRNAs are still able to read all four proline codons in the A-site, but induce frameshifts after translocation into the P-site. Some of the mutations are in regions of the tRNA that are involved in interactions with components of the P-site. We hypothesize that the ribosomal P-site keeps a “grip” of the peptidyl-tRNA to prevent loss of the reading frame

Direct and Inverted Repeat stimulated excision (DIRex) : Simple, single-step, and scar-free mutagenesis of bacterial genes

The need for generating precisely designed mutations is common in genetics, biochemistry, and molecular biology. Here, I describe a new λ Red recombineering method (Direct and Inverted Repeat stimulated excision; DIRex) for fast and easy generation of single point mutations, small insertions or replacements as well as deletions of any size, in bacterial genes. The method does not leave any resistance marker or scar sequence and requires only one transformation to generate a semi-stable intermediate insertion mutant. Spontaneous excision of the intermediate efficiently and accurately generates the final mutant. In addition, the intermediate is transferable between strains by generalized transductions, enabling transfer of the mutation into multiple strains without repeating the recombineering step. Existing methods that can be used to accomplish similar results are either (i) more complicated to design, (ii) more limited in what mutation types can be made, or (iii) require expression of extrinsic factors in addition to λ Red. I demonstrate the utility of the method by generating several deletions, small insertions/replacements, and single nucleotide exchanges in Escherichia coli and Salmonella enterica. Furthermore, the design parameters that influence the excision frequency and the success rate of generating desired point mutations have been examined to determine design guidelines for optimal efficiency

Use of DIRex to generate deletions and replacements.

<p>Dark blue and green areas indicate the recombinogenic homology arms and light blue arrows indicate the DRs used for excision. For each construct, the top line shows the wild-type arrangement, the middle line the DIRex intermediate and the bottom line the final mutant. (A) Deletion of the <i>S</i>. <i>enterica ssrA</i> gene, encoding tmRNA. (B) Deletion of the <i>S</i>. <i>enterica gal</i> operon promoter region. (C) Deletion of the <i>S</i>. <i>enterica araC</i>, <i>B</i>, <i>A</i> and <i>D</i> genes. (D) Deletion of the <i>E</i>. <i>coli araFGH</i> operon, encoding a high affinity L-arabinose transporter. (E) Replacement of the native <i>araE</i> promoter with a synthetic promoter (<i>P</i>_{<i>J23106</i>}) in both <i>S</i>. <i>enterica</i> and <i>E</i>. <i>coli</i>. (F) Replacement of most of the <i>S</i>. <i>enterica galE</i> coding sequence with a <i>lux</i> transcriptional terminator.</p

Outline of the method.

<p>(A) Generation of a designed mutation in three days using DIRex. (Day 1) A culture expressing λ Red is transformed with two PCR products to generate a semi-stable DIRex intermediate containing a selectable and counter selectable cassette. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184126#pone.0184126.g002" target="_blank">Fig 2</a> for more details. (Day 2) Transformants are isolated and colony-purified on selective medium. (Day 3) Colonies growing on selective medium are picked and streaked on counter-selective medium. (Day 4) Nearly 100% of colonies growing on counter selective media contain the designed mutation. (B) Transferring a previously constructed mutation into another strain by generalized transduction. A phage lysate grown on a strain containing a DIRex intermediate is used as donor in the transduction. The steps involved are the same as in (A) except for a transduction instead of a recombineering step on day 1.</p

Excision frequencies increase with increasing DR size.

<p>(A) Assay for precise excision. The three DIRex constructs in <i>hisA</i> were identical except for having 20, 25 or 30 bp DRs (light blue arrows). When the DIRex intermediate is present the <i>hisA</i> gene is interrupted, and the cell is unable to grow in medium lacking histidine (His^-). AmilCP confers blue color and SacB causes sensitivity to sucrose. Selection on sucrose selection plates allows only cells lacking a functional <i>sacB</i> gene to grow. If the cassette is excised a functional wild-type copy of <i>hisA</i> is restored and the cells lose the blue color and becomes sucrose resistant. For a more detailed view of the primers used in this experiment, see panel B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184126#pone.0184126.s001" target="_blank">S1 Fig</a>. (B) Frequencies of segregants (white) and false positives (blue) in six independent cultures of each of the three constructs.</p

Primer design for DIRex.

<p>Light blue arrows indicate the location and orientation of DR sequences between which recombination is expected to occur. Sequence segments on the upper strand are labeled with lower case letters a–i, and on the lower strand the complementary sequences are labeled a’–i’. (A) Using DIRex for deleting a gene. (A, i) Two 40 nt regions of recombineering homology (a-b, dark blue; c-d, green) are chosen on either side of the sequence to delete. (A, ii) The “left” oligo (upper strand) is designed with a 40 nt homology extension (composed of segment a–b) followed by 15 nts from the other side of the sequence to delete (segment c), and a 20 nt 3’ primer (P1). (A, iii) The “right” oligo (lower strand) is designed with a 40 nt homology extension (composed of segment d’–c’) and 15 nts from the other side of the sequence to delete (segment b’), and a 20 nt 3’ primer (P1). (A, iv) The resulting DIRex intermediate, with two identical 30 nt DR sequences (b–c), each containing the designed deletion junction. (v) The designed deletion after excision of the DIRex intermediate. (B) Using DIRex for replacing a native sequence with a designed sequence (in this example replacing the promoter <i>P</i>_<i>geneE</i> with another promoter, <i>P</i>_<i>x</i>). (B, i) Two 40 nt regions of recombineering homology (e, dark blue; f, green) are chosen on either side of the sequence to replace. (B, ii) The “left” oligo (upper strand) is designed with a 40 nt homology extension (composed of segment e) followed by the sequence to replace it with (in this example a ~30 nt sequence containing a promoter, <i>P</i>_<i>x</i>), and a 20 nt 3’ primer (P1). (B, iii) The “right” oligo (lower strand) is designed with a 40 nt homology extension (composed of segment f’) followed by the sequence to replace it with (the reverse complement of <i>P</i>_<i>x</i>), and a 20 nt 3’ primer (P1). (B, iv) The resulting DIRex intermediate, with two identical 30 nt DR sequences, each composed of the replacing sequence (<i>P</i>_<i>x</i>). (B, v) The designed replacement after excision of the DIRex intermediate. (C) Using DIRex for introducing a point mutation. The desired mutation is marked with an asterisk. (C, i) Two regions of recombineering homology (g-h and h-i) are chosen on either side of the point mutation. (C, ii) The “left” oligo (upper strand) is designed with a 45–50 nt homology extension (composed of segment g, the desired point, and segment h), and a 20 nt 3’ primer (P1). (C, iii) The “right” oligo (lower strand) is designed with a 40 nt homology extension (composed of segment i’–h’), and a 20 nt 3’ primer (P1). The homology extension ends just next to the nucleotide(s) to be changed (C, iv) The resulting DIRex intermediate, with two identical 25–30 nt DR sequences, with the mutation next to the “left” DR sequence. (C, v) The designed mutant after excision of the DIRex intermediate. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184126#pone.0184126.s001" target="_blank">S1 Fig</a> for specific examples.</p

Overview of the method.

<p>The method is illustrated with an example for generating a precise deletion of a hypothetical gene. (A) Two overlapping “half-cassettes” are generated in separate PCR reactions (which can be run in parallel in the same PCR cycler) using one locus specific long primer “Fp1” or “Rp1” in combination with the cassette specific primers “<i>cat</i>-midR2” or “<i>cat</i>-midF”, respectively. Each PCR fragment contain one copy of the IR (yellow arrow) and DR (light blue arrrow), as well as one of the recombinogenic 5’-homology extensions. The templates (<i>Acatsac1</i> and <i>Acatsac3</i>) differ in the location and orientation of the IR sequence, which contains the gene encoding the blue chromoprotein AmilCP. (B) The two “half-cassettes” are mixed in equimolar amounts and electroporated into λ Red induced cells. For formation of a functional <i>cat</i> gene recombination has to occur between the recombinogenic ends and the chromosome, as well as in the sequence overlap between the two “half-cassettes”. (C) The structure of the semi-stable DIRex intermediate. (D) The structure of the final deletion after spontaneous excision of the DIRex intermediate (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184126#pone.0184126.s003" target="_blank">S3 Fig</a> for a possible mechanism of excision).</p

Evolution of antibiotic resistance without antibiotic exposure

Antibiotic use is the main driver in the emergence of antibiotic resistance. Another unexplored possibility is that resistance evolves coincidentally in response to other selective pressures. We show that selection in the absence of antibiotics can co-select for decreased susceptibility to several antibiotics. Thus, genetic adaptation of bacteria to natural environments may drive resistance evolution by generating a pool of resistance mutations that selection could act on to enrich resistant mutants when antibiotic exposure occurs