Transforming DNA Uptake Gene Orthologs Do Not Mediate Spontaneous Plasmid Transformation in Escherichia coli

Spontaneous plasmid transformation of Escherichia coli occurs on nutrient-containing agar plates. E. coli has also been reported to use double-stranded DNA (dsDNA) as a carbon source. The mechanism(s) of entry of exogenous dsDNA that allows plasmid establishment or the use of DNA as a nutrient remain(s) unknown. To further characterize plasmid transformation, we first documented the stimulation of transformation by agar and agarose. We provide evidence that stimulation is not due to agar contributing a supplement of Ca²⁺, Fe²⁺, Mg²⁺, Mn²⁺, or Zn²⁺. Second, we undertook to inactivate the E. coli orthologues of Haemophilus influenzae components of the transformation machine that allows the uptake of single-stranded DNA (ssDNA) from exogenous dsDNA. The putative outer membrane channel protein (HofQ), transformation pseudopilus component (PpdD), and transmembrane pore (YcaI) are not required for plasmid transformation. We conclude that plasmid DNA does not enter E. coli cells as ssDNA. The finding that purified plasmid monomers transform E. coli with single-hit kinetics supports this conclusion; it establishes that a unique monomer molecule is sufficient to give rise to a transformant, which is not consistent with the reconstitution of an intact replicon through annealing of partially overlapping complementary ssDNA, taken up from two independent monomers. We therefore propose that plasmid transformation involves internalization of intact dsDNA molecules. Our data together, with previous reports that HofQ is required for the use of dsDNA as a carbon source, suggest the existence of two routes for DNA entry, at least across the outer membrane of E. coli.The spontaneous transformation of Escherichia coli with plasmid DNA on nutrient-containing agar plates was described in at least three independent articles (14, 23, 24). However, no attempt to characterize the mechanism of plasmid DNA uptake has been reported. Genomic analysis revealed the presence in E. coli of a set of genes homologous to those required for DNA uptake in naturally transformable species, including the gram-positive Bacillus subtilis and Streptococcus pneumoniae and the gram-negative Haemophilus influenzae and Neisseria gonorrhoeae (9). The machine they potentially encode would allow the uptake of single-stranded DNA (ssDNA) from an exogenous double-stranded DNA (dsDNA) substrate in E. coli (Fig. ​(Fig.1).1). HofQ (called ComE in reference 7) is the ortholog of the PilQ secretin of N. gonorrhoeae, which constitutes a transmembrane channel required for exogenous dsDNA to traverse the outer membrane (OM) and reach the so-called transformation pseudopilus (8). According to the Bacillus subtilis paradigm (8), assembly of the pseudopilus requires a prepilin peptidase (PppA; called PilD in reference 7), a traffic NTPase (HofB; called PilB in reference 7), and a polytopic membrane protein (HofC; called PilC in reference 7). The pseudopilus, which would include PpdD (called PilA in reference 7), provides access for dsDNA to its receptor, YbaV (called ComE1 in reference 7), through the peptidoglycan. Degradation of one strand by an unidentified nuclease (N) would allow uptake of ssDNA through YcaI (called Rec2 in reference 7), a channel in the inner membrane. Finally, DprA (also named Smf) would be required to protect internalized ssDNA from endogenous nucleases, as shown in S. pneumoniae (4), and to assist the processing of ssDNA into transformants (16).Open in a separate windowFIG. 1.Diagrammatic representation of the putative E. coli DNA uptake machine. The E. coli orthologues of proteins required involved in the uptake of transforming DNA in naturally transformable species, including B. subtilis, S. pneumoniae, H. influenzae, and N. gonorrhoeae, were identified by genomic analysis (9). GspD is a PilQ paralogue (25% identity over 278 residues), which was considered in the present study as a possible alternative route for dsDNA across the OM. A prepilin peptidase (PppA; called PilD in reference 7) required for maturation and export of proteins constituting the transformation pseudopilus (see Table S1 in the supplemental material) is not drawn on this diagram. (Additional information regarding the relationship between E. coli and H. influenzae transformation genes, and a table listing the various alternative names used in the literature are available in the supplemental material.). Red crosses indicate components of the putative DNA uptake machine inactivated during this work. IM, inner membrane.In H. influenzae, transformation genes are preceded by unusual CRP (for cyclic AMP receptor protein) binding sites, now called CRP-S (7), that absolutely require a second protein, Sxy (also called TfoX), in addition to CRP for induction (19). Interestingly, bioinformatics analysis revealed the conservation of CRP-S sites in front of the corresponding E. coli genes (7), including all of the genes encoding the proteins shown in Fig. ​Fig.11 (except GspD). Furthermore, some of these genes were experimentally demonstrated to require CRP, cAMP (CRP''s allosteric effector), and Sxy for induction in E. coli, providing support to the view that CRP-S sites control a bona fide transformation regulon in this bacterium (7). However, the involvement of E. coli transformation genes in DNA uptake has not been documented, except for hofQ, which was reported to be required for the use of dsDNA as a nutrient (11, 18). Although the functionality of the E. coli transformation genes has not been confirmed experimentally, it is of note that the bioinformatics identification of a complete set of transformation genes in two other species not previously known to be naturally transformable, Streptococcus thermophilus and Bacillus cereus, opened the way to the demonstration of genetic transformation in these species (6, 15a).To characterize further spontaneous plasmid transformation in E. coli, we first identified parameters affecting plasmid transformation frequencies on plates. We then undertook to inactivate genes encoding the putative transformation-related DNA uptake machinery of E. coli (Fig. ​(Fig.1)1) and to compare the rate of spontaneous plasmid transformation in the corresponding mutants and in their wild-type parent. In addition, to get an insight into the process of plasmid DNA entry, we characterized the kinetics of plasmid monomer transformation because it was shown in S. pneumoniae that regeneration of an intact plasmid replicon requires the independent uptake (via the transformation machine) of complementary ssDNA from two monomers (21). Finally, we discuss the possible significance of our data regarding the entry of exogenous dsDNA in E. coli in the light of previous findings on the use of dsDNA as a carbon source in this species (11, 18).     

The global nutritional regulator CodY is an essential protein in the human pathogen Streptococcus pneumoniae

CodY is a global regulator highly conserved in low-G+C Gram-positive bacteria. It plays a key role in the adaptation of Bacillus subtilis to nutritional limitation through repression of a large gene set during exponential growth and relief of repression upon starvation. In several pathogenic bacteria, CodY regulates major virulence genes. Our interest in Streptococcus pneumoniae CodY originates from our observations that the oligopeptide permease Ami was involved in repression of competence for genetic transformation. We hypothesized that peptide uptake through Ami feeds amino acid pools, which are sensed by CodY to repress competence. As our initial attempts at inactivating codY failed, we launched an in-depth analysis into the question of the essentiality of codY. We report that codY cannot be inactivated unless a complementing ectopic copy is present. We obtained genetic evidence that a recently published D39 codY knock-out contains additional mutations allowing survival of codY mutant cells. Whole genome sequencing revealed mutations in fatC, which encodes a ferric iron permease, and amiC. This combination of mutations was confirmed to allow tolerance of codY inactivation. The amiC mutation is in itself sufficient to account for the strong derepression of competence development observed in D39 codY cells.     

Semi-synthesis, topoisomerase I and kinases inhibitory properties, and antiproliferative activities of new rebeccamycin derivatives

In the course of structure-activity relationship studies, new rebeccamycin derivatives substituted in 3,9-positions on the indolocarbazole framework, and a 2',3'-anhydro derivative were prepared by semi-synthesis from rebeccamycin. The antiproliferative activities against nine tumor cell lines were determined and the effect on the cell cycle of murine leukemia L1210 cells was examined. Their DNA binding properties and inhibitory properties toward topoisomerase I and three kinases PKCzeta, CDK1/cyclin B, CDK5/p25 and a phosphatase cdc25A were evaluated. The 3,9-dihydroxy derivative is the most efficient compound of this series toward CDK1/cyclin B and CDK5/p25. It is also characterized as a DNA binding topoisomerase I poison. Its broad spectrum of molecular activities likely accounts for its cytotoxic potential. This compound which displays a tumor cell line-selectivity may represent a new lead for subsequent drug design in this series of glycosylated indolocarbazoles.     

Rebeccamycin analogues bearing amine substituents or other groups on the sugar moiety

In the course of structure-activity relationship studies on rebeccamycin analogues, a series of compounds bearing an amino function on the sugar moiety were synthesized with the aim of improving the solubility and interaction with the macromolecular target(s). The syntheses of amino derivatives and the corresponding chloro, iodo and azido intermediates are described. Their interaction with DNA and effects on human DNA topoisomerases I and II were investigated. Their antimicrobial activities against two Gram-positive bacteria, Bacillus cereus and Streptomyces chartreusis, a Gram-negative bacterium Escherichia coli and a yeast Candida albicans were also determined. 6'-Amino compound 7 and 6'-N-methylamino 14 very efficiently inhibit the growth of E. coli. The introduction of an amino group at the 6'-position strongly enhances the capacity of the drugs to interact with DNA but almost abolishes their poisoning effect on topoisomerase I. Unlike the vast majority of rebeccamycin analogues previously studied, the newly designed compounds do not stimulate DNA cleavage by topoisomerase I. The enhanced capacity of the 6'-amino glycosyl rebeccamycin derivatives to bind to DNA likely account for the improved biological profiles. DNA and topoisomerase I represent two independent targets which can both be used for the development of antitumor rebeccamycin derivatives.     

Different Regioselectivity of Two Commercial Acetylesterases for Hydrolysis of Peracetylated Maltose

Two commercial acetylesterases from Aspergillus niger with an associated amyloglucosidase degraded 6% and 25% of peracetylated maltose. This difference is explained by the regioselectivity of deacetylation : the acetylesterase from Sigma attacked the C1 and C2 atoms of the glucosidic ring whereas acetylesterase from Viscozyme attacked the C1 and C6 atoms and cleaved the bond 1 4 of the substrate.     

Regional difference in the distribution of vasoactive intestinal polypeptide-immunoreactive nerve fibres along the uterus and between myometrial muscle layers in the rat

To investigate a possible regional variation of the vasoactive intestinal polypeptide innervation in the uterus of the cyclic rat, the distribution of vasoactive intestinal polypeptide-containing nerve fibres from the cervix to the oviduct end of the uterine horns was studied using immunohistochemistry. Immunoreactive nerve fibres were most concentrated in the cervix, where they formed a dense plexus in association with the musculature and surrounding blood vessels. In the uterus, a clear regional distribution of the vasoactive intestinal polypeptide innervation was observed. Numerous vascular and non-vascular immunoreactive nerve fibres were present in the lower part of the uterine horns, whereas they were sparse in the median region and absent at the oviduct end. Moreover, non-vascular peptide innervation was mostly concentrated in the circular layer of the myometrium and also occurred in the endometrium. Only a very few immunoreactive nerve fibres were presen t in the longitudinal muscle layer. No change in the peptide innervation pattern was observed during the different stages of the sexual cycle. The marked regional distribution of the peptide innervation in the rat uterus suggests that the regulatory effects of the peptide occur mainly in the lower part of the organ and principally affect the circular muscle layer in the myometrium.     

Insect muscle actins differ distinctly from invertebrate and vertebrate cytoplasmic actins

Summary Invertebrate actins resemble vertebrate cytoplasmic actins, and the distinction between muscle and cytoplasmic actins in invertebrates is not well established as for vertebrate actins. However, Bombyx and Drosophila have actin genes specifically expressed in muscles. To investigate if the distinction between muscle and cytoplasmic actins evidenced by gene expression analysis is related to the sequence of corresponding genes, we compare the sequences of actin genes of these two insect species and of other Metazoa. We find that insect muscle actins form a family of related proteins characterized by about 10 muscle-specific amino acids. Insect muscle actins have clearly diverged from cytoplasmic actins and form a monophyletic group emerging from a cluster of closely related proteins including insect and vertebrate cytoplasmic actins and actins of mollusc, cestode, and nematode. We propose that muscle-specific actin genes have appeared independently at least twice during the evolution of animals: insect muscle actin genes have emerged from an ancestral cytoplasmic actin gene within the arthropod phylum, whereas vertebrate muscle actin genes evolved within the chordate lineage as previously described.Offprint requests to.: N. Mounier