The Hydrophobic TraM Protein of pKM101 Is Required for Conjugal Transfer and Sensitivity to Donor-Specific Bacteriophage

pKM101 is a self-transmissible plasmid of the IncN incompatibility group. Analysis of the DNA sequences of the genes required for conjugal transfer suggested the existence of a previously uncharacterized open reading frame, designatedtraM,that might be required for conjugation. Merodiploid strains containing transposon insertion mutations either intraMor in neighboringtragenes were used to demonstrate thattraMconstitutes a new complementation group essential for conjugation and donor phage sensitivity. The hydrophobicity profile of TraM suggests that it contains a signal sequence. The remainder of TraM is also composed predominantly of hydrophobic amino acids but contains one possible surface exposed loop. TraM–alkaline phosphatase and TraM–β-galactosidase fusion proteins supported the hypothesis that TraM has a small cytoplasmic loop. We were unable to detect heterologous complementation between anytramutation and its homolog from thevirBoperon ofAgrobacterium tumefaciens.     

Transfer of the plJ101 plasmid in Streptomyces lividans requires a cis-acting function dispensable for chromosomal gene transfer

The tra gene of Streptomyces lividans plasmid plJ101 is required for both plasmid DNA transfer and plJ101-induced mobilization of chromosomal genes during mating. We show that a chromosomally inserted copy of tra mediates transfer of chromosomal DNA at high frequency but promotes efficient transfer of plasmids only when they contain a previously unknown locus, here named clt. Insertional mutation or deletion of clt from plJ101 reduced plasmid transfer mediated by either plasmid-borne or chromosomally located tra by at least three orders of magnitude, abolished the transfer-associated pocking phenomenon, and interfered with the ability of tra⁺ plasmids to promote transfer of chromosomal DNA. Our results indicate that plasmid transfer in S. lividans involves a cis-acting function dispensable for chromosomal gene transfer and imply that either the S. lividans chromosome encodes its own clt-like function or, alternatively, that transfer of plasmid and chromosomal DNA occurs by different mechanisms.     

Some group N plasmids makeEscherichia coli K-12 strain AB1157 dependent on exogenous purine

Escherichia coli K-12 strain AB1157 given the conjugative group N plasmid R46 (or its derivative, pKM101, or plasmid R384N) grew only very slowly on defined medium containing the known growth-factor requirements of AB1157, which do not include any purine. Addition of adenine or hypoxanthine (or their nucleosides) restored normal growth; guanine and xanthine (and their nucleosides) were ineffective, because of thegpt defect caused by deletionproA2. Variants of AB1157(R46) able to grow rapidly on defined medium without purine were tetracycline-sensitive and/or transfer-defective; an, R46 gene,slo, causing purine auxotrophy, is inferred to be betweentet andtra.     

The TraK accessory factor activates substrate transfer through the pKM101 type IV secretion system independently of its role in relaxosome assembly

A large subfamily of the type IV secretion systems (T4SSs), termed the conjugation systems, transmit mobile genetic elements (MGEs) among many bacterial species. In the initiating steps of conjugative transfer, DNA transfer and replication (Dtr) proteins assemble at the origin-of-transfer (oriT) sequence as the relaxosome, which nicks the DNA strand destined for transfer and couples the nicked substrate with the VirD4-like substrate receptor. Here, we defined contributions of the Dtr protein TraK, a predicted member of the Ribbon-Helix-Helix (RHH) family of DNA-binding proteins, to transfer of DNA and protein substrates through the pKM101-encoded T4SS. Using a combination of cross-linking/affinity pull-downs and two-hybrid assays, we determined that TraK self-associates as a probable tetramer and also forms heteromeric contacts with pKM101-encoded TraI relaxase, VirD4-like TraJ receptor, and VirB11-like and VirB4-like ATPases, TraG and TraB, respectively. TraK also promotes stable TraJ–TraB complex formation and stimulates binding of TraI with TraB. Finally, TraK is required for or strongly stimulates the transfer of cognate (pKM101, TraI relaxase) and noncognate (RSF1010, MobA relaxase) substrates. We propose that TraK functions not only to nucleate pKM101 relaxosome assembly, but also to activate the Tra_pKM101 T4SS via interactions with the ATPase energy center positioned at the channel entrance.     

Localization of the plasmid (pKM101) gene(s) involved in recA+lexA+-dependent mutagenesis

Summary Twenty Tn5 insertion mutants of the drug resistance plasmid pKM101 have been isolated that are unable to enhance mutagenesis with ultraviolet (UV) irradiation or methyl methanesulfonate. By restriction mapping, the Tn5 insertion in each of these pKM101 mutants was shown to be within a 1.9 kb region of the plasmid genome. We have termed this segment of the pKM101 map the muc (mutagenesis: UV and chemical) gene(s). Characterization of these mutants indicated that any Tn5 insertion within the muc gene(s) abolished the ability of pKM101 to: (a) enhance spontaneous, UV and chemical mutagenesis, (b) increase host survival following UV-irradiation, (c) increase the survival of UV-irradiated phage plated on irradiated or unirradiated cells, and (d) suppress the repair and mutagenesis deficiencies of a umuC ^– mutant. Possible models to explain the role of the pKM101 muc gene(s) in mutagenesis and repair are discussed.     

Plasmid pKM101-dependent repair and mutagenesis in Escherichia coli cells with mutations lexB30 tif and zab-53 in the recA gene

Bacterial survival after UV irradiation was increased in E. coli K12 lexB30 and tif zab-53 mutants harboring plasmid pKM101. Mutagenesis in response to UV was observed in these bacteria which, in absence of pKM101, are not UV-mutable. The mutator effect observed in unirradiated wild-type cells containing pKM101 was higher after incubation at 30°C with adenine than at 37°C. This effect was still enhanced by tif mutation, even in the tif zab-53 strain, but it was abolished by lexB30 mutation. In the tif zab-53 (pKM101) strain, repair and mutagenesis of UV-irradiated phage λ was observed, but not in the lexB30 mutant carrying pKM101. The pKM101 mutant, pGW1, was unable to protect tif zab-53 bacteria against killing by UV, whereas the protection of lexB30 was intermediate; moreover, it did not promote the mutator effect at 30°C or enhance phage repair and mutagenesis in tif zab-53 cells. All UV-induced bacterial mutations in lexB30 (pKM101) strain were suppressors; in contrast, true revertants were found after UV irradiation of the tif zab-53 (pKM101) cells.We suggest that the constitutive activity of RecA protein is enough for the production of UV-promoted suppressor mutations, whereas true reversions require a more active form of this protein which could exert its effects directly or by acting at a regulatory level on other cellular functions.     

R46-derived recombinant plasmids affecting DNA repair and mutation in E. coli

Summary Recombinant plasmids which render their host less mutable and more sensitive to some DNA-damaging agents have been isolated from the N-group plasmid R46. These plasmids have been physically mapped and found to originate from the region of R46 that has been deleted in pKM101. This deleted region is well removed from the muc region of R46 and pKM101 which is responsible for the mutator effects of these plasmids.The effect of these anti-mutagenic plasmids on the ability of pKM101 to complement umuC mutations has been examined, and they have been found to inhibit the complementation of such strains. We propose that these plasmids may code for a negative control function acting on the muc gene.     

The novel gene(s) ARD of plasmid pKM101: alleviation of EcoK restriction

Summary The host-controlled K restriction of unmodified phage was 10-100-fold alleviated in the wild-type strain E. coli K12, carrying plasmid pKM101 of incompability group N. pKM101-mediated release of K restriction was also observed in lexA ^-, recA ^-, and recB ^- strains of E. coli K12. By restriction mapping Tn5 insertions in pKM101, which reduced pKM101-mediated alleviation of restriction, were shown to be located within the BglIIB fragment approximately 11 kb anticlockwise from the RI site of pKM101. We have termed the gene(s) promoting the alleviation of K restriction of phage ard (alleviation of restriction of DNA). It was shown (1) that ard function affected only the EcoK restriction system and not the EcoB, EcoRI, EcoRIII, or EcoPI system, (2) ard gene(s) did not mediate EcoK type modification of DNA and did not increase the modification activity of the EcoK system in a way similar to that observed with gene ral of bacteriophage .     

The large plasmids found in enterohemorrhagic and enteropathogenic Escherichia coli constitute a related series of transfer-defective Inc F-IIA replicons

Forty-six of 52 (88.5%) enterohemorrhagic Escherichia coli (EHEC) strains screened carried a “common” plasmid of about 90 kb which encoded sequences homologous to the Inc F-IIA replicon. A similarly high incidence of Inc F-IIA plasmid-containing strains was observed in other groups of diarrheagenic E. coli, but not in random environmental coliform isolates. Enteropathogenic E. coli (EPEC) contain plasmids of similar properties and share a 23-kb DNA fragment with plasmids from EHEC. The common region encodes the F-IIA replication region and sequences homologous to the transfer operon of the Inc F-II plasmid R1. Sequence homology varied between plasmids isolated from different EHEC/EPEC strains with >80% showing homology to the regions encoding the rep and par genes. Only 5% of plasmids from EHEC strains had intact sequences homologous to the DNA between these two regions, including the oriT site. Some plasmids with an apparently intact tra operon still failed to plaque F-pilus-specific phages. This is consistent with observations that the large plasmids of EHEC and EPEC are phenotypically nonconjugative. These results suggest that the large plasmids of EHEC/EPEC constitute a family of transfer-deficient Inc F-IIA plasmids with varying degrees of deletion in tra function. The evolutionary ramifications of this finding are considered.     

Unraveling the essential role in conjugation of the Tra protein of Streptomyces lividans plasmid pIJ101

Plasmid pIJ101 from Streptomyces lividans encodes a single gene, tra, that is essential for both plasmid transfer and mobilization of chromosomes during mating. The tra gene product (Tra) is a membrane protein, a portion of which shows similarity to transfer proteins of other streptomycete plasmids as well as additional bacterial chromosome partitioning proteins. This paper reviews past and present work that has focused on elucidating the precise role of the Tra protein of pIJ101 in conjugation in Streptomyces.     

Weakening of bacteriophage lambda EcoK DNA restriction in the presence of plasmid pKM101 ard+. I. General characteristics and genetic localization

The host-controlled K-restriction of unmodified phage lambda is ten to hundred-fold alleviated in the E. coli K12 strain, carring plasmid pKM101 of N-incompatibility group. By restriction mapping Tn5 insertion in pKM101, which reduced pKM101-mediated alleviation of K-restriction, was shown to by located within BglII-B-fragment approximately 9 kb anticlockwise from the EcoRI-site of pKM101. We have termed the gene(s) promoting the alleviation of K-restriction ARD (Alleviation of Restriction of DNA). It was shown that (i) plasmid pKM101-mediated alleviation of K-restriction did not depend on bacterial genes LexA, RecBC, umuC and plasmid gene muc; (ii) ard gene did not mediate EcoK type modification of DNA and did not enhance the modification activity of EcoK system in a way similar to that observed with RAL gene of phage lambda. Action of Ard gene of plasmid pKM101 is highly specific: alleviation of restriction of DNA lambda takes place only in K-strains of E. coli and is practically absent in B-strains and also in E. coli strains which have restricting enzymes of 11 type, EcoRI and EcoRIII.     

Inhibition of Homologous Recombination by the Plasmid MucA′B Complex

By its functional interaction with a RecA polymer, the mutagenic UmuD′C complex possesses an antirecombination activity. We show here that MucA′B, a functional homolog of the UmuD′C complex, inhibits homologous recombination as well. In F⁻ recipients expressing MucA′B from a P_tac promoter, Hfr × F⁻ recombination decreased with increasing MucA′B concentrations down to 50-fold. In damage-induced pKM101-containing cells expressing MucA′B from the native promoter, recombination between a UV-damaged F lac plasmid and homologous chromosomal DNA decreased 10-fold. Overexpression of MucA′B together with UmuD′C resulted in a synergistic inhibition of recombination. RecA[UmuR] proteins, which are resistant to UmuD′C inhibition of recombination, are inhibited by MucA′B while promoting MucA′B-promoted mutagenesis efficiently. The data suggest that MucA′B and UmuD′C contact a RecA polymer at distinct sites. The MucA′B complex was more active than UmuD′C in promoting UV mutagenesis, yet it did not inhibit recombination more than UmuD′C does. The enhanced mutagenic potential of MucA′B may result from its inherent superior capacity to assist DNA polymerase in trans-lesion synthesis. In the course of this work, we found that the natural plasmid pKM101 expresses around 45,000 MucA and 13,000 MucB molecules per lexA(Def) cell devoid of LexA. These molecular Muc concentrations are far above those of the chromosomally encoded Umu counterparts. Plasmid pKM101 belongs to a family of broad-host-range conjugative plasmids. The elevated levels of the Muc proteins might be required for successful installation of pKM101-like plasmids into a variety of host cells.     

Genetic and biochemical analysis of an endonuclease encoded by the IncN plasmid pKM101.

The IncN plasmid pKM101 nuc gene encodes a periplasmically localized endonuclease. DNA sequence analysis indicates that this gene encodes a hydrophilic protein of about 19.5 kDa containing a hydrophobic signal sequence. nuc is homologous to a partially sequenced open reading frame adjacent to the sog gene of the plasmid CollB-P9, a plasmid known to encode an endonuclease similar to that of pKM101. A partially sequenced tra gene directly upstream of nuc is homologous to the virB11 gene of Agrobacterium tumefaciens. We have partially purified the pKM101 nuclease by osmotic shock and cation exchange chromatography, and used this enzyme preparation to sequence the protein's amino terminus. The first 13 amino acids of the mature protein match amino acids 23 to 35 of the predicted sequence, indicating that the protein is proteolytically processed to a molecular mass of approximately 17 kDa, probably during export to the periplasmic space. The enzyme was able to attack many sites along an end labelled duplex DNA substrate, but showed clearly preferred cleavage sites, and may cleave preferentially at purine-rich regions.     

Plasmid (pKM101)-mediated enhancement of repair and mutagenesis: Dependence on chromosomal genes in Escherichia coli K-12

Summary The drug resistance plasmid pKM101 plays a major role in the Ames Salmonella/microsome carcinogen detecting system by enhancing chemical mutagenesis. It is shown that in Escherichia coli K-12 the plasmid pKM101 enhances both spontaneous and methyl methanesulfonate-caused reversion of an ochre mutation, bacterial survival after ultraviolet irradiation, and reactivation of ultraviolet-irradiated in unirradiated cells. All these effects are shown to be dependent on the recA ⁺ lexA⁺ genotype but not on the recB ⁺ recC⁺ or recF ⁺ genotypes. The recA lexA-dependence of the plasmid-mediated repair and mutagenesis suggests an interaction with the cell's inducible error-prone repair system. The presence of pKM101 is shown to cause an additional increase in methyl methanesulfonate mutagenesis in a tif mutant beyond that caused by growth at 42°. The presence of the plasmid raises the level of the Weigle-reactivation curve for the reactivation of ultraviolet-irradiated in E. coli and causes a shift of the maximum to a higher UV fluence. These observations suggest that pKM101 does not exert its effects by altering the regulation of the cell's error-prone repair system but rather by supplying a mechanistic component or components.     

Effect of the plasmid ColIb-P9 on cellular processes related to DNA repair

Summary The plasmid ColIb-P9 introduced into Escherichia coli K12 umuC mutant cells suppresses the deficiencies in mutagenesis and repair of mutants after UV-irradiation. These data suggest that ColIb-P9 encodes a product with a function similar to that of the chromosomal gene umuC. Tn5 insertion mutants of ColIb-P9 were isolated with an altered ability to restore UV-mutagenesis in the umuC mutant. The same plasmid mutations were shown to eliminate the effects of ColIb-P9 on UV-mutagenesis, survival after UV and mitomycin C treatment, reactivation of UV-irradiated in unirradiated cells, Weigle-reactivation, induction of colicin E1 synthesis. The ColIb-P9 genes responsible for the enhancement of UV-mutagenesis were cloned within a 14 Md SalI fragment. Their location was established by restriction analysis of the mutant plasmid ColIb 6-13::Tn5.While the action of the plasmids ColIb-P9 and pKM101 is similar, these plasmids were shown to have opposite effects on cell survival and colicin E1 synthesis after mitomycin C treatment. A study of the mutant plasmids ColIb::Tn5 and pGW12 (muc ^- mutant of pKM101) has shown the difference in the effects of ColIb-P9 and pKM101 to be associated with the plasmid genes responsible for the protective and mutagenesis-enhancing effects of these plasmids in UV-irradiated cells.Abbreviations MC mitomycin C - ICS induction of colicin synthesis     

Use of chimeric type IV secretion systems to define contributions of outer membrane subassemblies for contact‐dependent translocation

Recent studies have shown that conjugation systems of Gram‐negative bacteria are composed of distinct inner and outer membrane core complexes (IMCs and OMCCs, respectively). Here, we characterized the OMCC by focusing first on a cap domain that forms a channel across the outer membrane. Strikingly, the OMCC caps of the Escherichia coli pKM101 Tra and Agrobacterium tumefaciens VirB/VirD4 systems are completely dispensable for substrate transfer, but required for formation of conjugative pili. The pKM101 OMCC cap and extended pilus also are dispensable for activation of a Pseudomonas aeruginosa type VI secretion system (T6SS). Chimeric conjugation systems composed of the IMC_pKM101 joined to OMCCs from the A. tumefaciens VirB/VirD4, E. coli R388 Trw, and Bordetella pertussis Ptl systems support conjugative DNA transfer in E. coli and trigger P. aeruginosa T6SS killing, but not pilus production. The A. tumefaciens VirB/VirD4 OMCC, solved by transmission electron microscopy, adopts a cage structure similar to the pKM101 OMCC. The findings establish that OMCCs are highly structurally and functionally conserved – but also intrinsically conformationally flexible – scaffolds for translocation channels. Furthermore, the OMCC cap and a pilus tip protein coregulate pilus extension but are not required for channel assembly or function.     

Identification and mapping of regions of the plasmid pKM101 which influence the growth rate and resistance to phleomycin E of Escherichia coli WP2.

The effects of deletion of various regions of the pKM101 genome on several phenotypes conferred by pKM101 in Escherichia coli WP2 cells were investigated. Differences in the response of cells carrying pKM101 or various pKM101 deletion derivatives to the mutagenic effects of phleomycin E can be attributed to differences in sensitivity to the lethal effects of phleomycin E. Resistance to phleomycin E is conferred by the pKM101 mucAB genes (or an adjacent gene) but observed only with pKM101 derivatives which have lost a 2.2-kilobase (BalI-KpnI-2) segment which completely includes the pKM101 endonuclease gene nuc. A pKM101 slow-growth determinant, distinct from the slo gene, has also been identified and localized in the 2.4-kilobase (BalI-KpnI-3) segment which is adjacent to the nuc gene. Loss of this region does not appear to substantially influence the toxic or mutagenic effects of phleomycin E.     

The role of the transformer genes in the development of genitalia and analia of Drosophila melanogaster

The autosomal mutations transformer (tra) and transformer-2 (tra-2) of Drosophila convert chromosomal females (X/X) into phenotypical males. Our analysis aims at an understanding of the role which the transformer genes play in the development of the sexually dimorphic genital disc. In each Drosophila embryo, this disc starts development with a male and a female genital primordium, and an anal primordium. Our experiments involved the production of cell clones that were made homozygous for tra or tra-2 at different times of development. Homozygous clones were obtained by inducing mitotic recombination in three types of females heterozygous for tra or tra-2. The cells of the homozygous tra/tra or tra-2/tra-2 clones responded by changing from the female into the male pathway. Male genital structures developed if the clones were induced not later than 81 hr into development. In the analia, male clones appeared up to 120 hr. Our results show that the action of the wild-type alleles of tra⁺ and tra-2⁺ is required until late in larval development to repress the male genital primordium and to support development of the female primordium, as well as to maintain the anal primordium in the female pathway. Our data also suggest that the embryonic genital disc consists of two compartments, one containing the precursors for penis and analia, the other those of the male and female genitalia.     

Mutational Specificity of Ultraviolet Light in ESCHERICHIA COLI with and without the R Plasmid Pkm101

Plasmid pKM101 provides UV protection and increases the frequency of spontaneous and UV-induced mutations in Escherichia coli. By analyzing reversion patterns of defined trpA alleles, we showed that pKM101 altered the mutational specificity of UV-induced mutations. Certain UV-induced base-pair substitutions were strongly enhanced, while others were decreased in frequency in the presence of pKM101. This result suggests an interaction between cellular misrepair and an error-prone repair function(s) provided by pKM101. We have also examined UV mutational specificity in the absence of pKM101 and found the following: (1) UV preferentially enhances missense, as well as nonsense, intergenic suppressor mutations; (2) UV causes all possible base-pair substitutions as well as frameshift mutations; (3) G·C base pairs are more susceptible to UV mutagenesis than A·T base pairs at the same nucleotide positions; and (4) UV-induced mutations can occur at nucleotide positions that are not part of pyrimidine-pyrimidine sequences.     

Chromosomal locations of two DNA segments that flank ribosomal insertion-like sequences in Drosophila: Flanking sequences are mobile elements

We report the chromosomal locations of two repetitive DNA sequences that flank ribosomal insertion-like sequences in Drosophila melanogaster. The chromocentric region of D. melanogaster contains many copies of sequences that are homologous to type 1 ribosomal insertions. These insertion-like elements are interspersed with other DNA segments that we call flanking sequences. Two distinct flanking sequences derived from the same cloned DNA molecule pDmI 101, the HindIII fragments 101E and 101F, were studied. Whole genome Southern blots with DNA from the D. melanogaster stocks Oregon R (P2), gt-1, and gt-X11 showed complex restriction patterns that differed substantially between the three stocks. This and other data show that flanking sequences are members of diverged repetitive sequence families. In situ hybridization to salivary gland chromosomes of gt-1 and gt-X11 showed that both sequences are homologous to the chromocenter and to about 5 to 8 (101E) or 25 to 30 (101F) euchromatic sites in each stock. Most, if not all, of these sites differed in gt-1 and gt-X11. Both 101E and 101F are homologous to the chromocenter and very few euchromatic bands in D. simulans, but 101F is homologous to numerous bands in D. mauritiana. We conclude that the flanking sequences represented by 101E and 101F are mobile elements within the genome of Drosophila. These two sequences differ in several structural features from mobile DNA elements previously described in this organism.We dedicate this paper to Professor W. Beermann at the occasion of his 60th birthday