Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Natural DNA Uptake by Escherichia coli

Natural DNA Uptake by Escherichia coli

  • Sunita Sinha, 
  • Rosemary J. Redfield
PLOS
x

Abstract

Escherichia coli has homologues of the competence genes other species use for DNA uptake and processing, but natural competence and transformation have never been detected. Although we previously showed that these genes are induced by the competence regulator Sxy as in other gamma-proteobacteria, no conditions are known that naturally induce sxy expression. We have now tested whether the competence gene homologues encode a functional DNA uptake machinery and whether DNA uptake leads to recombination, by investigating the effects of plasmid-borne sxy expression on natural competence in a wide variety of E. coli strains. High- and low-level sxy expression alone did not induce transformation in any of the strains tested, despite varying the transforming DNA, its concentration, and the incubation conditions used. Direct measurements of uptake of radiolabelled DNA were below the limit of detection, however transformants were readily detected when recombination functions were provided by the lambda Red recombinase. This is the first demonstration that E. coli sxy expression can induce natural DNA uptake and that E. coli's competence genes do encode a functional uptake machinery. However, the amount of transformation cells undergo is limited both by low levels of DNA uptake and by inefficient DNA processing/recombination.

Introduction

Many bacteria can actively take up DNA from their environment, a genetically programmed ability called natural competence [1]. When this DNA recombines and changes the cell's genotype, the cell is said to be transformed. Natural competence has never been directly demonstrated in Escherichia coli, and most transformation instead relies on artificial permeabilisation to bring DNA into cells [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12]. Though this DNA can recombine with the host chromosome using the cell's recombination machinery if RecBCD is disabled, higher levels of recombination can be attained by controlled expression of phage recombination proteins (recombineering) [2], [6], [13], [14], [15]. Several studies have shown modest ‘natural’ uptake of plasmid DNA by E. coli under specific conditions, but this appears to bypass the classical DNA uptake machinery (the type IV pilus, TFP) so its relationship to natural competence is unclear [16], [17], [18], [19], [20], [21], [22], [23], [24], [25]. Palchevskiy and Finkel showed that E. coli can grow in medium containing DNA as the sole source of carbon and energy and that this growth depends on competence gene homologues, suggesting that E. coli can take up DNA, but they could not detect chromosomal transformation [26], [27].

Despite not being naturally transformable, E. coli and other Enterobacteriaceae have homologues of all but one of the competence genes (pilF2) common to their nearest transformable relatives, bacteria in the families Pasteurellaceae and Vibrionaceae [28], [29]. In the best-studied species, Haemophilus influenzae (Pasteurellaceae) and Vibrio cholerae (Vibrionaceae), expression of competence genes is controlled by two positive regulators, CRP and Sxy (also known as TfoX) [30], [31], [32], [33], [34], [35], [36], [37], [38]. The CRP regulator (cAMP receptor protein, also called catabolite activator protein (CAP)) responds to the availability of carbon and energy sources signaled by cAMP; it activates transcription by bending DNA to enhance contacts with RNA polymerase [39], [40], [41]. Much less is known about Sxy's function; it is required for competence-specific transcription of a subset of CRP-dependent promoters (CRP-S promoters) and is thought to make direct contacts with CRP [29].

In H. influenzae and V. cholerae, Sxy is absolutely required for competence development, and the combination of sxy induction and CRP activation are sufficient to induce high levels of competence and transformation [30], [33], [34], [37], [38], [42]. We have previously shown that expression of E. coli sxy induces all of its competence-gene homologues, but we were unable to detect natural transformation [29]. This raises the question of whether E. coli's CRP-S regulon is truly a competence regulon (i.e. one that encodes machinery used for DNA uptake). In this work, we investigate the ability of E. coli Sxy to induce natural competence in a wide variety of strains, using assays of transformation and DNA uptake. We show that although sxy expression alone is not sufficient for natural competence, transformation can occur when recombination functions are provided artificially.

Results

sxy expression in different E. coli strains does not induce transformation

We previously showed that expression of plasmid-borne sxy in strain BW25113 sxy::kan strongly induces all of E. coli's competence gene homologues, but we were unable to detect natural transformation with marked chromosomal DNA [29]. Because extensive strain-to-strain differences in competence and transformability are the norm in other species [43], [44], we investigated the effects of sxy expression on transformation in a wide range of E. coli strains.

K-12 strains.

The sxy plasmid used in our gene expression study (pEcsxy) [29] was transferred to the K-12 derivatives BW25113, C600 and W3110. IPTG was added to induce sxy expression in log phase, and cells were given marked chromosomal DNA before plating on selective and non-selective media. No transformants were ever recovered, despite varying (i) the duration of sxy induction/incubation with DNA (10, 30, 60, 90, 120, 180 minutes and overnight), (ii) the DNA concentration (1–20 ug/ml), (iii) the DNA type (chromosomal DNA or PCR product) and/or (iv) the DNA marker (purK::tet, crp::kan, fliC::kan, NalR point mutation). All combinations of assay conditions tested are listed in Table S2.

Sequencing confirmed that the plasmid-encoded sxy gene was free of mutations, and qRT-PCR confirmed that sxy had been strongly induced upon IPTG addition (Figure 1). The lack of transformation also could not be attributed to failure of Sxy and CRP to induce the CRP-S genes, as we confirmed transcription of ppdD and yrfD by qRT-PCR (Figure 1, black bars) and production of PpdD pilin by western blotting (Figure 2). Boosting CRP activity by addition of cAMP also had no effect.

thumbnail
Figure 1. Gene expression upon induction of plasmid-encoded sxy genes.

qRT-PCR quantification of changes in the expression of sxy, ppdD and yrfD upon sxy induction in strain BW25113 after 60 mins expression time. Each bar represents the average of two independent biological replicates for each gene, with error bars indicating standard deviation from the mean. Expression levels shown were normalised for each mRNA sample using 23S rRNA levels.

https://doi.org/10.1371/journal.pone.0035620.g001

thumbnail
Figure 2. Expression of PpdD pilin protein upon induction of plasmid-encoded sxy genes.

Western blotting with PpdD antiserum was performed on whole-cell extracts from broth-grown BW25113 60 mins after IPTG induction of sxy expression. Lane 1: pEcppdD (positive control, His-tagged ppdD); Lane 2: pEcsxy; Lane 3: pEcsxylow. The positions of native processed (15 kDa) and His-tagged (17 kDa) PpdD proteins are indicated with arrows.

https://doi.org/10.1371/journal.pone.0035620.g002

We also tested the effect of sxy expression in strain ZK126, which Palchevskiy and Finkel showed could use DNA for growth [26], [27]. As in other K-12 strains, sxy expression could not mediate natural transformation of ZK126 under any of the conditions described above (Table S2).

Other strains.

This screen was further extended to natural populations of E. coli by investigating the ECOR collection, which is representative of this species' genetic diversity [45], [46]. None of the 72 ECOR strains produced PpdD pilin in overnight or log-phase cultures (data not shown), showing that CRP-S expression (and therefore likely sxy expression) is normally low in these strains. Attempts to transform pools of eight strains in overnight culture were also unsuccessful (Table S2). We transferred the sxy plasmid to 15 strains chosen to represent diversity in host species, pathogenicity, geographical distribution, subspecific grouping and MSLT type [45], [46]. Again, Sxy expression was never able to mediate chromosomal transformation.

Moderate sxy expression does not induce transformation

Since we had previously observed toxicity with long-term sxy expression from the high copy number vector pEcsxy, we reduced expression levels by recloning E. coli sxy in the low copy number vector pSU20 to generate pEcsxylow. qRT-PCR confirmed sxy expression from this plasmid, and showed induction of the CRP-S genes ppdD and yrfD (Figure 1, grey bars). This expression was lower than from pEcsxy, but still significant. Processed PpdD pilin could be detected by western blotting albeit at reduced levels (Figure 2), suggesting that the competence machinery could still be assembled. All of the transformation conditions described above were retested with pEcsxylow, but again sxy induction never led to chromosomal transformation.

Co-expression of sxy with H. influenzae pilF2 does not induce transformation

Although several genes of the H. influenzae CRP-S regulon are absent from the E. coli genome, only pilF2 is required for DNA uptake and transformation [28], [29], [47]. This gene is also present in V. cholerae and all transformable Pasteurellaceae. Since Pasteurellaceae are the closest relatives of Enterobacteriaceae, we cloned the H. influenzae pilF2 gene downstream of sxy in pEcsxy and pEcsxylow and tested whether co-induction of sxy and pilF2 permitted transformation in strain ZK126 under the conditions described above. Despite detectable plasmid-borne expression of both sxy and pilF2 from both plasmids (Figure 3), no transformants were detected (Table S2).

thumbnail
Figure 3. Induction of plasmid-encoded sxy and pilF2 genes in strain ZK126.

PCR reactions on cDNA (lanes 1 to 4) confirm that both sxy (515 bp; top) and pilF2 (265 bp; bottom) are strongly expressed when IPTG is added. As a control for DNA contamination of RNA preparations, PCR reactions were also performed on the RNA samples before reverse transcription. As expected, these reactions generated no product (lanes 4 to 8). Lanes 1 and 5: pEcsxylow; Lanes 2 and 6: pEcsxylow with IPTG; Lanes 3 and 7: pEcsxy; Lanes 4 and 8: pEcsxy with IPTG.

https://doi.org/10.1371/journal.pone.0035620.g003

E. coli sxy expression does induce DNA uptake

The inability of diverse E. coli strains to be transformed could be due to a lack of DNA uptake across the outer membrane and/or to a defect in DNA processing. This was tested by direct measurement of uptake of radiolabelled chromosomal DNA fragments after sxy induction in BW25113 cells. For both pEcsxy or pEcsxylow, only 0.02% of the DNA added remained cell-associated after extensive washing (133 cpm per 109 induced cells). This baseline level was higher than background (17 cpm in tubes with no cells), but was not significantly higher than in uninduced cells (107 cpm per 109 cells). When 200 bp DNA fragments were used as donor DNA, levels of cell-associated cpm were not significantly above background (data not shown).

Since DNA uptake at our detection limit would give substantial numbers of transformants in known naturally transformable bacteria, we developed a more sensitive indirect test of DNA uptake. We measured sxy-inducible transformation when recombination functions were provided by the lambda Red recombinase system, which efficiently recombines mutations flanked by short homologous sequences into the chromosome [14]. These experiments used SW102 cells, which carry a chromosomally-encoded Red recombinase [48]. The results of these assays are shown in Figure 4.

thumbnail
Figure 4. Results of indirect DNA uptake assays.

SW102 cells with or without sxy plasmids were transformed with 1 µg fliC::kan or fliC::spec DNA. Cells without plasmids were electroporated. In other cells, sxy was induced with IPTG. Lambda Red expression was induced by heat shock. none = no plasmid, sxy = high-copy sxy plasmid, sxylow = low-copy sxy plasmid, sxy,pilF2 = high-copy sxy-pilF2 plasmid, sxy,pilF2low = low-copy sxy-pilF2 plasmid, vector = high-copy no insert plasmid, vectorlow = low-copy no insert plasmid.

https://doi.org/10.1371/journal.pone.0035620.g004

In control experiments, SW102 cells were first heat shocked to induce the recombinase and then electroporated with the test DNA fragment, a KanR cassette flanked by 50 bp segments of chromosomal homology to the fliC gene (fliC::kan). The transformation efficiency was 284 cfu/µg DNA±160 (Figure 4). To test whether sxy expression could induce DNA uptake, we repeated the experiment in SW102 cells carrying pEcsxy or pEcsxylow, replacing electroporation with IPTG-induction of sxy (Figure 4). Both plasmids gave small but reproducible numbers of KanR transformants: 6±3 transformants/ug DNA for pEcsxy and 23±5 transformants/ug DNA for pEcsxylow. Transformant genotypes were confirmed by PCR. Negative control cells carrying an empty plasmid (no sxy), or cells with no heat shock (no recombinase) never gave any transformants.

To confirm that the sxy-dependent transformation we observed uses the Sxy-regulated type IV pilus machinery for DNA uptake, we repeated the assay in a strain lacking hofQ, which is predicted to encode the outer membrane secretin through which DNA enters competent cells [1], [20], [26], [27], [29], [49], [50], [51]. Because the hofQ deletion contains a KanR cassette, the donor DNA was changed to fliC::spec. While SW102 hofQ::kan cells electroporated with fliC::spec DNA gave 215±60 transformants/ug DNA, cells expressing sxy gave none (Figure 4), confirming that the DNA uptake we observed used the competence machinery.

Since transformation following sxy-mediated natural DNA uptake was lower than transformation following electroporation, we also tested whether transformation efficiency could be improved by co-expression of H. influenzae pilF2, again using plasmids pEcsxy-pilF2 and pEcsxy-pilF2low. As shown in Figure 4, transformation efficiency was not improved.

These experiments confirm that E. coli can indeed take up small amounts of DNA using the DNA uptake machinery, and that this uptake is competence-specific and Sxy-inducible. Collectively, our experiments suggest that natural transformation is undetectable because artificial induction of sxy causes very little DNA to be taken up.

Discussion

E. coli can be transformed in the laboratory, but this requires artificial entry of DNA into cells. However E. coli should be naturally transformable because it has a set of inducible competence gene homologues in a functional regulon [29]. Here we show that sxy expression can lead to natural DNA uptake using the competence machinery. Nevertheless, the amount of DNA uptake is very low, and transformation only occurs when recombination functions are provided artificially, suggesting that recombination of incoming DNA itself is also inefficient. Recombination in E. coli may be limited by expression of the RecBCD exonuclease, though studies in H. influenzae have shown that transformation is not increased in recBCD mutants [6], [52], [53]. The combination of low uptake and limited recombination could explain why researchers have until now been unable to demonstrate natural competence in E. coli. A further hurdle is that none of the extensive conditions we and others have tested induce expression of sxy or the CRP-S genes it regulates [26], [29], [49], [50].

All competent species studied show extensive strain-to-strain variation in their ability to take up DNA and be transformed [43], [44]. This has caused some species initially classed as non-competent on the basis of one non-transformable isolate to be later re-classified when broader screening identified transformable strains. However none of the E. coli strains we tested could undergo chromosomal transformation upon sxy expression, so this is not a strain-specific problem.

Since E. coli's CRP-S genes do encode a functional DNA uptake machinery, natural conditions must exist under which higher levels of natural competence occur. Laboratory culture is a very unnatural condition, as is the IPTG induction of sxy expression, so it may be that other factors are required which are missing when Sxy is artificially induced. It is therefore important to find more natural conditions that induce sxy expression, which requires a better understanding of how it is regulated.

Insights into the regulation of sxy expression can be gained from studies in H. influenzae and V. cholerae, where sxy is controlled by both transcriptional and translational regulation [33], [36], [37], [54]. Transcriptional control is mediated by CRP. Like its homologues in H. influenzae and V. cholerae, E. coli sxy has a strong CRP binding site upstream of the transcriptional start site, strongly suggesting that CRP also induces sxy transcription in this species. Translational control in H. influenzae and V. cholerae occurs through the formation of a stem loop structure in sxy mRNA that limits the amount of translation [36], [37], [54]. This stem loop is thought to respond to competence-inducing signals, modulating the amount of Sxy protein cells make and thus controlling competence. We have mapped the transcriptional start site of E. coli sxy mRNA and found that transcription initiates at a G residue situated 115 bp upstream of the ATG start. Mfold simulations with E. coli sxy mRNA predict a stem loop structure very similar to those found in H. influenzae and V. cholerae. Moreover, Yamamoto et al. identified strong inverted repeats in the promoter region of V. cholerae sxy, and showed that these were essential for transcriptional and translational control of sxy expression [36], [37]. Such regions of strong base pairing are also found in the region upstream of H. influenzae and E. coli sxys, suggesting that similar mechanisms of sxy regulation exist in E. coli and perhaps all gamma-proteobacteria.

The signals that control sxy expression and induce competence in H. influenzae and V. cholerae are known (purine nucleotide depletion and chitin availability, respectively). Finding the signals that control sxy expression in E. coli is crucial to finding out how this species becomes competent in its natural environment. This signal is likely to be something that E. coli encounters in vivo, so it is conceivable that different classes of E. coli might respond to slightly different cues. Clearly these conditions are not being met in the laboratory setting.

Materials and Methods

Bacterial growth conditions, strains, and plasmids

Details on the strains used in this study are given in Table 1. E. coli was grown on Luria Bertani (LB) broth or LB agar (1.2%) at 30°C or 37°C. Plasmids were introduced by CaCl2–heat shock transformation. When required, antibiotics were used at the following concentrations: kanamycin 15 µg/ml, chloramphenicol 20 µg/ml, nalidixic acid 20 µg/ml, ampicillin 100 µg/ml, tetracycline 10 µg/ml. Where required, isopropyl β-D-1-thiogalactopyranoside (IPTG) and cyclic adenosine monophosphate (cAMP) were used at 1 mM.

Strain SW102 hofQ::kan was constructed using recombineering to transfer the mutation from the Keio collection strain JW3354 to the chromosome of strain SW102 [48]. Plasmid pEcsxy contains the coding sequence of E. coli sxy, cloned downstream of the p-T5-lac promoter in the high-copy number vector pCA24N [55]. To create plasmid pEcsxylow, an 800 bp segment flanking and including the coding sequence of E. coli sxy (b0959) was PCR-amplified from the genome of strain BW25113 using primers with integrated ClaI and EcoRI restriction sites (all PCR primers are listed in Table S1). The PCR product was digested with ClaI and EcoRI, and cloned into the low-copy number vector pSU20 digested with the same enzymes, putting sxy under control of the lac promoter [56].

To create plasmids pEcsxy-pilF2 and pEcsxylow-pilF2, the H. influenzae pilF2 gene (HI0366) was first PCR-amplified and cloned into pGEMT-Easy (Promega) to generate pHipilF2. The pilF2 insert was then released from pHipilF2 by NotI or EcoRI digestions, and cloned into the NotI site of pEcsxy to generate plasmid pEcsxy-pilF2, or the EcoRI site of pEcsxylow to generate plasmid pEcsxylow-pilF2. In both plasmids pilF2 is cloned in the forward orientation downstream of E. coli sxy and under control of the lac promoter.

Transformation assays

Overnight cultures of strains carrying sxy-expressing plasmids were diluted 1∶100 in LB-Cm and grown at 37°C. At OD600 0.3, sxy expression was induced with IPTG and 1–20 ug/ml donor DNA was added. The donor DNA was either E. coli chromosomal DNA or purified PCR product with one of the following genotypes: purK::tet, crp::kan, fliC::kan or BW25113-NalR (see Table 1). Where necessary, cAMP was added at the same time as IPTG. Cells were incubated with DNA for 10, 30, 60, 90, 120 or 180 minutes, or overnight, before being plated on selective and non-selective media. The full list of combinations tested is given in Table S2.

DNA uptake assays

BW25113 chromosomal DNA was digested with XbaI and EcoRI and end-labelled with Klenow using 33P-dATP. Cultures of strain BW25113 carrying pEcsxy or pEcsxylow were grown at 37°C and sxy expression was induced at OD600 0.3 for 30 minutes. Cells were then incubated with 3 µg/ml 33P-labelled DNA for 30 minutes at 37°C, pelleted and washed twice with LB, and finally resuspended in LB for scintillation counting.

Indirect DNA uptake assays

These assays used the recombineering strain SW102, which contains chromosomally encoded exo, bet and gam genes from phage λ, controlled by the inducible cI857 promoter [48]. In positive control assays, overnight cultures were diluted in 3 mL LB and grown at 30°C to OD600 0.6, when recombination functions were induced by transfer to 42°C for 15 mins. Cells were then pelleted, washed three times with ice-cold dH20, concentrated 100-fold and electroporated with 1 µg donor DNA. Electroporated cells were allowed to recover in 1 mL LB for 1 h at 30°C before plating.

Assays testing the effect of sxy on DNA uptake omitted the electroporation step. Instead, sxy expression was induced with IPTG at OD600 0.3 and cells were given 1 µg/ml DNA. When the OD600 reached 0.6, recombination functions were induced by heat shock as above. Cells were then returned to 30°C and allowed to grow for a further 1.5 h at 30°C before plating. The donor DNAs used in these experiments were ΔfliC PCR products amplified from either the chromosome of the Keio strain JW1908 (fliC::kan) (Table 1) [57] or plasmid pRSM2832 (fliC::spec) [58].

Gene and protein expression methods

Expression of plasmid-borne sxy and pilF2 genes was confirmed by RT-PCR (primer sequences are given in Table S1). For each sample, 1 µg RNA was reverse-transcribed with iScript™ (Biorad), and 1 µL cDNA was used in PCR. Quantitative PCR was performed as described in Cameron and Redfield [28], using primers specified in Sinha et al. [29] (listed in Table S1). Protein sample preparation and immunoblotting were performed as described in Sinha et al. [29].

Supporting Information

Table S2.

Conditions tested in transformation assays. None gave any transformants.

https://doi.org/10.1371/journal.pone.0035620.s002

(PDF)

Acknowledgments

We thank Ghazaleh Kiani for technical assistance and Prof. Julian Davies for providing us with the ECOR collection.

Author Contributions

Conceived and designed the experiments: SS RJR. Performed the experiments: SS. Analyzed the data: SS. Contributed reagents/materials/analysis tools: SS RJR. Wrote the paper: SS RJR.

References

  1. 1. Dubnau D (1999) DNA uptake in bacteria. Annu Rev Microbiol 53: 217–244.D. Dubnau1999DNA uptake in bacteria.Annu Rev Microbiol53217244
  2. 2. Cosloy SD, Oishi M (1973) Genetic transformation in Escherichia coli K12. Proc Natl Acad Sci U S A 70: 84–87.SD CosloyM. Oishi1973Genetic transformation in Escherichia coli K12.Proc Natl Acad Sci U S A708487
  3. 3. Dagert M, Ehrlich SD (1979) Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6: 23–28.M. DagertSD Ehrlich1979Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells.Gene62328
  4. 4. Bukau B, Brass JM, Boos W (1985) Ca2+-induced permeabilization of the Escherichia coli outer membrane: comparison of transformation and reconstitution of binding-protein-dependent transport. J Bacteriol 163: 61–68.B. BukauJM BrassW. Boos1985Ca2+-induced permeabilization of the Escherichia coli outer membrane: comparison of transformation and reconstitution of binding-protein-dependent transport.J Bacteriol1636168
  5. 5. van Die IM, Bergmans HE, Hoekstra WP (1983) Transformation in Escherichia coli: studies on the role of the heat shock in induction of competence. J Gen Microbiol 129: 663–670.IM van DieHE BergmansWP Hoekstra1983Transformation in Escherichia coli: studies on the role of the heat shock in induction of competence.J Gen Microbiol129663670
  6. 6. Wackernagel W (1973) Genetic transformation in E. coli: the inhibitory role of the recBC DNase. Biochem Biophys Res Commun 51: 306–311.W. Wackernagel1973Genetic transformation in E. coli: the inhibitory role of the recBC DNase.Biochem Biophys Res Commun51306311
  7. 7. Bergmans HE, van Die IM, Hoekstra WP (1981) Transformation in Escherichia coli: stages in the process. J Bacteriol 146: 564–570.HE BergmansIM van DieWP Hoekstra1981Transformation in Escherichia coli: stages in the process.J Bacteriol146564570
  8. 8. Weston A, Brown MG, Perkins HR, Saunders JR, Humphreys GO (1981) Transformation of Escherichia coli with plasmid deoxyribonucleic acid: calcium-induced binding of deoxyribonucleic acid to whole cells and to isolated membrane fractions. J Bacteriol 145: 780–787.A. WestonMG BrownHR PerkinsJR SaundersGO Humphreys1981Transformation of Escherichia coli with plasmid deoxyribonucleic acid: calcium-induced binding of deoxyribonucleic acid to whole cells and to isolated membrane fractions.J Bacteriol145780787
  9. 9. Sabelnikov AG, Domaradsky IV (1979) Proton conductor vs. cold in induction of Ca(2+)-dependent competence in Escherichia coli. Mol Gen Genet 172: 313–317.AG SabelnikovIV Domaradsky1979Proton conductor vs. cold in induction of Ca(2+)-dependent competence in Escherichia coli.Mol Gen Genet172313317
  10. 10. Sabelnikov AG, Avdeeva AV, Ilyashenko (1975) Enhanced uptake of donor DNA by Ca2+ treated Escherichia coli cells. Mol Gen Genet 138: 351–358.AG SabelnikovAV AvdeevaIlyashenko1975Enhanced uptake of donor DNA by Ca2+ treated Escherichia coli cells.Mol Gen Genet138351358
  11. 11. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166: 557–580.D. Hanahan1983Studies on transformation of Escherichia coli with plasmids.J Mol Biol166557580
  12. 12. Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96: 23–28.H. InoueH. NojimaH. Okayama1990High efficiency transformation of Escherichia coli with plasmids.Gene962328
  13. 13. Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, et al. (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 97: 5978–5983.D. YuHM EllisEC LeeNA JenkinsNG Copeland2000An efficient recombination system for chromosome engineering in Escherichia coli.Proc Natl Acad Sci U S A9759785983
  14. 14. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97: 6640–6645.KA DatsenkoBL Wanner2000One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.Proc Natl Acad Sci U S A9766406645
  15. 15. Gutterson NI, Koshland DE Jr (1983) Replacement and amplification of bacterial genes with sequences altered in vitro. Proc Natl Acad Sci U S A 80: 4894–4898.NI GuttersonDE Koshland Jr1983Replacement and amplification of bacterial genes with sequences altered in vitro.Proc Natl Acad Sci U S A8048944898
  16. 16. Maeda S, Sawamura A, Matsuda A (2004) Transformation of colonial Escherichia coli on solid media. FEMS Microbiol Lett 236: 61–64.S. MaedaA. SawamuraA. Matsuda2004Transformation of colonial Escherichia coli on solid media.FEMS Microbiol Lett2366164
  17. 17. Maeda S, Ito M, Ando T, Ishimoto Y, Fujisawa Y, et al. (2006) Horizontal transfer of nonconjugative plasmids in a colony biofilm of Escherichia coli. FEMS Microbiol Lett 255: 115–120.S. MaedaM. ItoT. AndoY. IshimotoY. Fujisawa2006Horizontal transfer of nonconjugative plasmids in a colony biofilm of Escherichia coli.FEMS Microbiol Lett255115120
  18. 18. Etchuuya R, Ito M, Kitano S, Shigi F, Sobue R, et al. (2011) Cell-to-cell transformation in Escherichia coli: a novel type of natural transformation involving cell-derived DNA and a putative promoting pheromone. PLoS One 6: e16355.R. EtchuuyaM. ItoS. KitanoF. ShigiR. Sobue2011Cell-to-cell transformation in Escherichia coli: a novel type of natural transformation involving cell-derived DNA and a putative promoting pheromone.PLoS One6e16355
  19. 19. Sobue R, Kurono N, Etchuya R, Maeda S (2011) Identification of a novel DNA element that promotes cell-to-cell transformation in Escherichia coli. FEBS Lett 585: 2223–2228.R. SobueN. KuronoR. EtchuyaS. Maeda2011Identification of a novel DNA element that promotes cell-to-cell transformation in Escherichia coli.FEBS Lett58522232228
  20. 20. Sun D, Zhang X, Wang L, Prudhomme M, Xie Z, et al. (2009) Transforming DNA uptake gene orthologs do not mediate spontaneous plasmid transformation in Escherichia coli. J Bacteriol 191: 713–719.D. SunX. ZhangL. WangM. PrudhommeZ. Xie2009Transforming DNA uptake gene orthologs do not mediate spontaneous plasmid transformation in Escherichia coli.J Bacteriol191713719
  21. 21. Sun D, Zhang Y, Mei Y, Jiang H, Xie Z, et al. (2006) Escherichia coli is naturally transformable in a novel transformation system. FEMS Microbiol Lett 265: 249–255.D. SunY. ZhangY. MeiH. JiangZ. Xie2006Escherichia coli is naturally transformable in a novel transformation system.FEMS Microbiol Lett265249255
  22. 22. Baur B, Hanselmann K, Schlimme W, Jenni B (1996) Genetic transformation in freshwater: Escherichia coli is able to develop natural competence. Appl Environ Microbiol 62: 3673–3678.B. BaurK. HanselmannW. SchlimmeB. Jenni1996Genetic transformation in freshwater: Escherichia coli is able to develop natural competence.Appl Environ Microbiol6236733678
  23. 23. Woegerbauer M, Jenni B, Thalhammer F, Graninger W, Burgmann H (2002) Natural genetic transformation of clinical isolates of Escherichia coli in urine and water. Appl Environ Microbiol 68: 440–443.M. WoegerbauerB. JenniF. ThalhammerW. GraningerH. Burgmann2002Natural genetic transformation of clinical isolates of Escherichia coli in urine and water.Appl Environ Microbiol68440443
  24. 24. Bauer F, Hertel C, Hammes WP (1999) Transformation of Escherichia coli in foodstuffs. Syst Appl Microbiol 22: 161–168.F. BauerC. HertelWP Hammes1999Transformation of Escherichia coli in foodstuffs.Syst Appl Microbiol22161168
  25. 25. Tsen SD, Fang SS, Chen MJ, Chien JY, Lee CC, et al. (2002) Natural plasmid transformation in Escherichia coli. J Biomed Sci 9: 246–252.SD TsenSS FangMJ ChenJY ChienCC Lee2002Natural plasmid transformation in Escherichia coli.J Biomed Sci9246252
  26. 26. Finkel SE, Kolter R (2001) DNA as a nutrient: novel role for bacterial competence gene homologs. J Bacteriol 183: 6288–6293.SE FinkelR. Kolter2001DNA as a nutrient: novel role for bacterial competence gene homologs.J Bacteriol18362886293
  27. 27. Palchevskiy V, Finkel SE (2006) Escherichia coli competence gene homologs are essential for competitive fitness and the use of DNA as a nutrient. J Bacteriol 188: 3902–3910.V. PalchevskiySE Finkel2006Escherichia coli competence gene homologs are essential for competitive fitness and the use of DNA as a nutrient.J Bacteriol18839023910
  28. 28. Cameron AD, Redfield RJ (2006) Non-canonical CRP sites control competence regulons in Escherichia coli and many other gamma-proteobacteria. Nucleic Acids Res 34: 6001–6014.AD CameronRJ Redfield2006Non-canonical CRP sites control competence regulons in Escherichia coli and many other gamma-proteobacteria.Nucleic Acids Res3460016014
  29. 29. Sinha S, Cameron AD, Redfield RJ (2009) Sxy induces a CRP-S regulon in Escherichia coli. J Bacteriol 191: 5180–5195.S. SinhaAD CameronRJ Redfield2009Sxy induces a CRP-S regulon in Escherichia coli.J Bacteriol19151805195
  30. 30. Redfield RJ (1991) sxy-1, a Haemophilus influenzae mutation causing greatly enhanced spontaneous competence. J Bacteriol 173: 5612–5618.RJ Redfield1991sxy-1, a Haemophilus influenzae mutation causing greatly enhanced spontaneous competence.J Bacteriol17356125618
  31. 31. Redfield RJ, Cameron AD, Qian Q, Hinds J, Ali TR, et al. (2005) A novel CRP-dependent regulon controls expression of competence genes in Haemophilus influenzae. J Mol Biol 347: 735–747.RJ RedfieldAD CameronQ. QianJ. HindsTR Ali2005A novel CRP-dependent regulon controls expression of competence genes in Haemophilus influenzae.J Mol Biol347735747
  32. 32. Blokesch M (2012) Chitin colonization, chitin degradation and chitin-induced natural competence of Vibrio cholerae are subject to catabolite repression. Environ Microbiol. M. Blokesch2012Chitin colonization, chitin degradation and chitin-induced natural competence of Vibrio cholerae are subject to catabolite repression.Environ Microbiol
  33. 33. Meibom KL, Blokesch M, Dolganov NA, Wu CY, Schoolnik GK (2005) Chitin induces natural competence in Vibrio cholerae. Science 310: 1824–1827.KL MeibomM. BlokeschNA DolganovCY WuGK Schoolnik2005Chitin induces natural competence in Vibrio cholerae.Science31018241827
  34. 34. Pollack-Berti A, Wollenberg MS, Ruby EG (2010) Natural transformation of Vibrio fischeri requires tfoX and tfoY. Environ Microbiol. A. Pollack-BertiMS WollenbergEG Ruby2010Natural transformation of Vibrio fischeri requires tfoX and tfoY.Environ Microbiol
  35. 35. Suckow G, Seitz P, Blokesch M (2011) Quorum sensing contributes to natural transformation of Vibrio cholerae in a species-specific manner. J Bacteriol 193: 4914–4924.G. SuckowP. SeitzM. Blokesch2011Quorum sensing contributes to natural transformation of Vibrio cholerae in a species-specific manner.J Bacteriol19349144924
  36. 36. Yamamoto S, Izumiya H, Mitobe J, Morita M, Arakawa E, et al. (2011) Identification of a chitin-induced small RNA that regulates translation of the tfoX gene, encoding a positive regulator of natural competence in Vibrio cholerae. J Bacteriol 193: 1953–1965.S. YamamotoH. IzumiyaJ. MitobeM. MoritaE. Arakawa2011Identification of a chitin-induced small RNA that regulates translation of the tfoX gene, encoding a positive regulator of natural competence in Vibrio cholerae.J Bacteriol19319531965
  37. 37. Yamamoto S, Morita M, Izumiya H, Watanabe H (2010) Chitin disaccharide (GlcNAc)2 induces natural competence in Vibrio cholerae through transcriptional and translational activation of a positive regulatory gene tfoXVC. Gene 457: 42–49.S. YamamotoM. MoritaH. IzumiyaH. Watanabe2010Chitin disaccharide (GlcNAc)2 induces natural competence in Vibrio cholerae through transcriptional and translational activation of a positive regulatory gene tfoXVC.Gene4574249
  38. 38. Zulty JJ, Barcak GJ (1995) Identification of a DNA transformation gene required for com101A+ expression and supertransformer phenotype in Haemophilus influenzae. Proc Natl Acad Sci U S A 92: 3616–3620.JJ ZultyGJ Barcak1995Identification of a DNA transformation gene required for com101A+ expression and supertransformer phenotype in Haemophilus influenzae.Proc Natl Acad Sci U S A9236163620
  39. 39. Lawson CL, Swigon D, Murakami KS, Darst SA, Berman HM, et al. (2004) Catabolite activator protein: DNA binding and transcription activation. Curr Opin Struct Biol 14: 10–20.CL LawsonD. SwigonKS MurakamiSA DarstHM Berman2004Catabolite activator protein: DNA binding and transcription activation.Curr Opin Struct Biol141020
  40. 40. Chen S, Gunasekera A, Zhang X, Kunkel TA, Ebright RH, et al. (2001) Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: alteration of DNA binding specificity through alteration of DNA kinking. J Mol Biol 314: 75–82.S. ChenA. GunasekeraX. ZhangTA KunkelRH Ebright2001Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: alteration of DNA binding specificity through alteration of DNA kinking.J Mol Biol3147582
  41. 41. Grainger DC, Hurd D, Harrison M, Holdstock J, Busby SJ (2005) Studies of the distribution of Escherichia coli cAMP-receptor protein and RNA polymerase along the E. coli chromosome. Proc Natl Acad Sci U S A 102: 17693–17698.DC GraingerD. HurdM. HarrisonJ. HoldstockSJ Busby2005Studies of the distribution of Escherichia coli cAMP-receptor protein and RNA polymerase along the E. coli chromosome.Proc Natl Acad Sci U S A1021769317698
  42. 42. Marvig RL, Blokesch M (2010) Natural transformation of Vibrio cholerae as a tool–optimizing the procedure. BMC Microbiol 10: 155.RL MarvigM. Blokesch2010Natural transformation of Vibrio cholerae as a tool–optimizing the procedure.BMC Microbiol10155
  43. 43. Bosse JT, Sinha S, Schippers T, Kroll JS, Redfield RJ, et al. (2009) Natural competence in strains of Actinobacillus pleuropneumoniae. FEMS Microbiol Lett 298: 124–130.JT BosseS. SinhaT. SchippersJS KrollRJ Redfield2009Natural competence in strains of Actinobacillus pleuropneumoniae.FEMS Microbiol Lett298124130
  44. 44. Maughan H, Redfield RJ (2009) Extensive variation in natural competence in Haemophilus influenzae. Evolution 63: 1852–1866.H. MaughanRJ Redfield2009Extensive variation in natural competence in Haemophilus influenzae.Evolution6318521866
  45. 45. Archer CT, Kim JF, Jeong H, Park JH, Vickers CE, et al. (2011) The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli. BMC Genomics 12: 9.CT ArcherJF KimH. JeongJH ParkCE Vickers2011The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli.BMC Genomics129
  46. 46. Ochman H, Selander RK (1984) Standard reference strains of Escherichia coli from natural populations. J Bacteriol 157: 690–693.H. OchmanRK Selander1984Standard reference strains of Escherichia coli from natural populations.J Bacteriol157690693
  47. 47. VanWagoner TM, Whitby PW, Morton DJ, Seale TW, Stull TL (2004) Characterization of three new competence-regulated operons in Haemophilus influenzae. J Bacteriol 186: 6409–6421.TM VanWagonerPW WhitbyDJ MortonTW SealeTL Stull2004Characterization of three new competence-regulated operons in Haemophilus influenzae.J Bacteriol18664096421
  48. 48. Warming S, Costantino N, Court DL, Jenkins NA, Copeland NG (2005) Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res 33: e36.S. WarmingN. CostantinoDL CourtNA JenkinsNG Copeland2005Simple and highly efficient BAC recombineering using galK selection.Nucleic Acids Res33e36
  49. 49. Sauvonnet N, Gounon P, Pugsley AP (2000) PpdD type IV pilin of Escherichia coli K-12 can Be assembled into pili in Pseudomonas aeruginosa. J Bacteriol 182: 848–854.N. SauvonnetP. GounonAP Pugsley2000PpdD type IV pilin of Escherichia coli K-12 can Be assembled into pili in Pseudomonas aeruginosa.J Bacteriol182848854
  50. 50. Sauvonnet N, Vignon G, Pugsley AP, Gounon P (2000) Pilus formation and protein secretion by the same machinery in Escherichia coli. Embo J 19: 2221–2228.N. SauvonnetG. VignonAP PugsleyP. Gounon2000Pilus formation and protein secretion by the same machinery in Escherichia coli.Embo J1922212228
  51. 51. Tarry M, Jaaskelainen M, Paino A, Tuominen H, Ihalin R, et al. (2011) The extra-membranous domains of the competence protein HofQ show DNA binding, flexibility and a shared fold with type I KH domains. J Mol Biol 409: 642–653.M. TarryM. JaaskelainenA. PainoH. TuominenR. Ihalin2011The extra-membranous domains of the competence protein HofQ show DNA binding, flexibility and a shared fold with type I KH domains.J Mol Biol409642653
  52. 52. Kooistra J, Venema G (1976) Effect of adenosine 5′-triphosphate-dependent deoxyribonuclease deficiency on properties and transformation of Haemophilus influenzae strains. J Bacteriol 128: 549–556.J. KooistraG. Venema1976Effect of adenosine 5′-triphosphate-dependent deoxyribonuclease deficiency on properties and transformation of Haemophilus influenzae strains.J Bacteriol128549556
  53. 53. Wilcox KW, Smith HO (1975) Isolation and characterization of mutants of Haemophilus influenzae deficient in an adenosine 5′-triphosphate-dependent deoxyribonuclease activity. J Bacteriol 122: 443–453.KW WilcoxHO Smith1975Isolation and characterization of mutants of Haemophilus influenzae deficient in an adenosine 5′-triphosphate-dependent deoxyribonuclease activity.J Bacteriol122443453
  54. 54. Cameron AD, Volar M, Bannister LA, Redfield RJ (2008) RNA secondary structure regulates the translation of sxy and competence development in Haemophilus influenzae. Nucleic Acids Res 36: 10–20.AD CameronM. VolarLA BannisterRJ Redfield2008RNA secondary structure regulates the translation of sxy and competence development in Haemophilus influenzae.Nucleic Acids Res361020
  55. 55. Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T, Inamoto E, et al. (2005) Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res 12: 291–299.M. KitagawaT. AraM. ArifuzzamanT. Ioka-NakamichiE. Inamoto2005Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research.DNA Res12291299
  56. 56. Bartolome B, Jubete Y, Martinez E, de la Cruz F (1991) Construction and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivatives. Gene 102: 75–78.B. BartolomeY. JubeteE. MartinezF. de la Cruz1991Construction and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivatives.Gene1027578
  57. 57. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, et al. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 2006 0008.T. BabaT. AraM. HasegawaY. TakaiY. Okumura2006Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.Mol Syst Biol22006 0008
  58. 58. Tracy E, Ye F, Baker BD, Munson RS Jr (2008) Construction of non-polar mutants in Haemophilus influenzae using FLP recombinase technology. BMC Mol Biol 9: 101.E. TracyF. YeBD BakerRS Munson Jr2008Construction of non-polar mutants in Haemophilus influenzae using FLP recombinase technology.BMC Mol Biol9101
  59. 59. Appleyard RK (1954) Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K12. Genetics 39: 440–452.RK Appleyard1954Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K12.Genetics39440452
  60. 60. Bachmann BJ (1972) Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev 36: 525–557.BJ Bachmann1972Pedigrees of some mutant strains of Escherichia coli K-12.Bacteriol Rev36525557