Systems regulating the tra genes of derepressed F-like plasmids

A study was made of the ability of reference plasmids of the 6 known Fin-groups to inhibit the functions of transfer genes (tra-genes) of the 4 derepressed F-like plasmids (pAP22-2, pAP38, pAP43, pAP53). It was shown that unlike the derepressed Flac plasmid, the conjugation transfer of pAP38 and pAP53 plasmids was inhibited only by, the FinV plasmid, whereas pAP22-2 plasmids by Fin V and Fin V plasmids. The formation of donor-specific pili in case of pAP38 plasmid was inhibited by Fin Q, Fin U and Fin V plasmids, in case of pAP43 plasmid by Fin U Fin V and Fin W plasmids.     

Five control systems preventing transfer of Escherichia coli K-12 sex factor F.

The transfer inhibition systems of 28 Fin+ plasmids have been characterized, using Flac mutants insensitive to inhibition by R100 or R62. All F-like plasmids (except R455) and one N group plasmid determined systems analogous to that of R100; this is designated the FinOP system. None of these plasmids could supply a FinP component of the transfer inhibitor able to replace that of F itself. In addition to the FinOP and R62 transfer inhibition systems described previously, new systems were encoded by the F-like plasmid R455, the I-like plasmid JR66a, and the group X plasmid R485. Besides inhibiting F transfer, JR66a also inhibited F pilus formation and surface exclusion, whereas R485 inhibited only pilus formation and R455 inhibited neither. All three R factors inhibited transfer of J-independent Flac elements, indicating that they act directly on one or more genes (or products) of the transfer operon, rather than directly via traJ. The tral products and transfer origin sequences of two Fin+ F-like plasmids, ColB2 and R124, appear to have similar specificities to those of F itself.     

Characterization of the ColE1 mobilization region and its protein products

Summary A third of the 6.6 kb genome of ColE1 is devoted to mobilization (mob) genes necessary to promote its specific transfer in the presence of conjugative plasmids. Themob region is genetically complex: twomob genes are entirely overlapped by a third. Oligonucleotide-directed mutagenesis was used to insert an amber codon into one of the overlapped genes and make possible a full complementation analysis ofmob. Fourmob genes essential for mobilization by R64drd11 were thus identified. Fragments ofmob were subcloned under control of the P_tac promoter in a suitable vector, overexpressed in minicells and the mobilization proteins visualized. A comprehensive alignment of themob region of ColE1 with those of its close relatives ColK and ColA demonstrating that the four essentialmob genes are conserved is also presented.     

Mutations of the genetic region Fin of the F-like plasmid pAP18-1]

With help of nitrosoguanidine 60 mutants of F-like plasmids pAP18-1 drd::Tn 5 and pAP18-1::Tn 9 were induced which determined resistance of E. coli cells of specific phage MS2. Mutational changes in fin-locus of those plasmids were accompanied by phenotypic reversion Fin(-)-Fin+.     

The role of lipopolysaccharide structure in the recipient cell during plasmid-mediated bacterial conjugation

The role of the lipopolysaccharide (LPS) structure in the recipient cell during bacterial conjugation was studied using a series of well-defined LPS mutations in Salmonella minnesota. The plasmids Flac (IncFI) and R1drd19 (IncFII) transferred at a high frequency to the smooth S218 parent strain and the rough LPS mutants. However, R64drd1 1 (IncI alpha) transferred poorly to the LPS mutants compared with transfer to the smooth LPS parent strain. The decrease in R64drd1 1 transfer frequency correlated with the extent of the defect in LPS structure, suggesting that intact LPS on the recipient is a major requirement for R64drd1 1 mating. Transfer of the P-group plasmid, RK2, was not affected by changes in LPS structure. These results show that plasmids use different cell surface structures during conjugation, and that LPS is particularly important for R64drd1 1 transfer.     

Plasmid Specificity of The Origin of Transfer of Sex Factor F

The ability of F-like plasmids to promote transfer from the F origin of transfer was determined. Chromosome transfer was measured from plasmid derivatives of RecA(-) Hfr deletion strains which had lost all the F transfer genes but which in some cases retained, and in others had also lost, the origin sequence. ColV2 and ColVBtrp could initiate transfer from the F origin, but R100-1, R1-19, and R538-1 drd could not. These results can be correlated with the plasmid specificity of the traI components of the different plasmid transfer systems, supporting the hypothesis that the origin of transfer is the site of action of the traI product. Most F-like plasmids, including R1-19 and R538-1 drd, could transfer ColE1, consistent with previous findings that the (plasmid-specific) traI product is not necessary for ColE1 transfer by Flac; ColE1 transfer may be initiated by a ColE1-or host-determined product. R100-1 and R136fin(-) could not transfer ColE1 efficiently, apparently because of differences residing in their pilus-forming genes.     

Genetic and physical analyses of a cluster of genes essential for xanthan gum biosynthesis in Xanthomonas campestris.

Xanthomonas campestris produces copious amounts of a complex exopolysaccharide, xanthan gum. Nonmucoid mutants, defective in synthesis of xanthan polysaccharide, were isolated after nitrosoguanidine mutagenesis. To isolate genes essential for xanthan polysaccharide synthesis (xps), a genomic library of X. campestris DNA, partially digested with SalI and ligated into the broad-host-range cloning vector pRK293, was constructed in Escherichia coli. The pooled clone bank was conjugated en masse from E. coli into three nonmucoid mutants by using pRK2013, which provides plasmid transfer functions. Kanamycin-resistant exconjugants were then screened for the ability to form mucoid colonies. Analysis of plasmids from several mucoid exconjugants indicated that overlapping segments of DNA had been cloned. These plasmids were tested for complementation of eight additional nonmucoid mutants. A 22-kilobase (kb) region of DNA was defined physically by restriction enzyme analysis and genetically by ability to restore mucoid phenotype to 10 of the 11 nonmucoid mutants tested. This region was further defined by subcloning and by transposon mutagenesis with mini-Mu(Tetr), with subsequent analysis of genetic complementation of nonmucoid mutants. A region of 13.5 kb of DNA was determined to contain at least five complementation groups. The effect of plasmids containing cloned xps genes on xanthan gum synthesis was evaluated. One plasmid, pCHC3, containing a 12.4-kb insert and at least four linked xanthan biosynthetic genes, increased the production of xanthan gum by 10% and increased the extent of pyruvylation of the xanthan side chains by about 45%. This indicates that a gene affecting pyruvylation of xanthan gum is linked to this cluster of xps genes.     

Functional and mutational analysis of conjugative transfer region 1 (Tra1) from the IncHI1 plasmid R27

The conjugative transfer region 1 (Tra1) of the IncHI1 plasmid R27 was subjected to DNA sequence analysis, mutagenesis, genetic complementation, and an H-pilus-specific phage assay. Analysis of the nucleotide sequence indicated that the Tra1 region contains genes coding for mating pair formation (Mpf) and DNA transfer replication (Dtr) and a coupling protein. Insertional disruptions of 9 of the 14 open reading frames (ORFs) in the Tra1 region resulted in a transfer-deficient phenotype. Conjugative transfer was restored for each transfer mutant by genetic complementation. An intergenic region between traH and trhR was cloned and mobilized by R27, indicating the presence of an origin of transfer (oriT). The five ORFs immediately downstream of the oriT region are involved in H-pilus production, as determined by an H-pilus-specific phage assay. Three of these ORFs encode proteins homologous to Mpf proteins from IncF plasmids. Upstream of the oriT region are four ORFs required for plasmid transfer but not H-pilus production. TraI contains sequence motifs that are characteristic of relaxases from the IncP lineage but share no overall homology to known relaxases. TraJ contains both an Arc repressor motif and a leucine zipper motif. A putative coupling protein, TraG, shares a low level of homology to the TraG family of coupling proteins and contains motifs that are important for DNA transfer. This analysis indicates that the Mpf components of R27 share a common lineage with those of the IncF transfer system, whereas the relaxase of R27 is ancestrally related to that of the IncP transfer system.     

Cloning of the neurodegeneration gene drop-dead and characterization of additional phenotypes of its mutation

Mutations in the Drosophila gene drop-dead (drd) result in early adult lethality and neurodegeneration, but the molecular identity of the drd gene and its mechanism of action are not known. This paper describes the characterization of a new X-linked recessive adult-lethal mutation, originally called lot's wife (lwf(1)) but subsequently identified as an allele of drd (drd(lwf)); drd(lwf) mutants die within two weeks of eclosion. Through mapping and complementation, the drd gene has been identified as CG33968, which encodes a putative integral membrane protein of unknown function. The drd(lwf) allele is associated with a nonsense mutation that eliminates nearly 80% of the CG33968 gene product; mutations in the same gene were also found in two previously described drd alleles. Characterization of drd (lwf) flies revealed additional phenotypes of drd, most notably, defects in food processing by the digestive system and in oogenesis. Mutant flies store significantly more food in their crops and defecate less than wild-type flies, suggesting that normal transfer of ingested food from the crop into the midgut is dependent upon the DRD gene product. The defect in oogenesis results in the sterility of homozygous mutant females and is associated with a reduction in the number of vitellogenic egg chambers. The disruption in vitellogenesis is far more severe than that seen in starved flies and so is unlikely to be a secondary consequence of the digestive phenotype. This study demonstrates that mutation of the drd gene CG33968 results in a complex phenotype affecting multiple physiological systems within the fly.     

Identification of functional regions of the colicinogenic plasmid ColA

Summary ColA is a colicinogenic plasmid of 6.72 kb. It is compatible with ColE1 but not with ColK. Transposon insertion mutagenesis as well as complementation studies have been carried out to investigate the location of the various functional regions of this plasmid. Four independent ColA::Tn1 and one ColA::Tn3 plasmids were isolated and the locations of insertions were determined. From these plasmids, six different deletion mutants were constructed. In addition, various restriction fragments of ColA have been cloned into pUC8 to carry out complementation studies. We have thus confirmed the location of the DNA regions involved in colicin production, colicin release and immunity function. The DNA region involved in conjugal mobility promoted by R64 drd11 has been identified and we have demonstrated that the ColE1 mobility proteins can act in trans on the bom (basis of mobility) site of ColA. The location of this site, as well as the region involved in stable maintenance of ColA, have also been determined. These results are discussed with regard to the homology in nucleotide sequence between ColA and ColE1.     

Genetic organization of transfer region of F-like plasmid pAP18-1

The results of complementation analysis of nitrosoguanidine-induced mutants of F-like drd-plasmid pAP18-1 (Tc, ColV) testified to the existence of at least 3 tra regions (tra1, tra2, tra3) and regulating locus fin V in the genome of this plasmid. By means of molecular cloning of tra2 region and locus fin V of plasmid pAP18-1drd were located in Sall-fragment f5 (3.9 MD).     

Partial suppression of the phenotype of Escherichia coli K-12 dnaG mutants by some I-like conjugative plasmids.

Three I-like conjugative plasmids, ColIdrd1, R144drd3, and R64drd11, which are derepressed for functions involved in conjugation, were found to suppress at least partially the phenotype of temperature-sensitive dnaG mutants of Escherichia coli K-12, as judged from the kinetics of deoxyribonucleic acid synthesis at elevated temperature in newly formed and established plasmid-containing strains. In contrast, the corresponding wild-type plasmids and three F-like derepressed conjugative plasmids, F101, R100drd1, and R1drd16, all failed to suppress. Suppression is presumably caused by a different plasmid-determined function from that which promotes survival of ultraviolet-irradiated bacteria, because both the wild-type I-like plasmids and their drd mutants protected irradiated bacteria. One possible interpretation of these results is that the product of a gene carried by certain I-like plasmids can substitute for the bacterial dnaG gene product during ongoing deoxyribonucleic acid replication.     

Genetic and physical characteristics of an enterotoxin plasmid.

We are engaged in the genetic and physical characterization of an enterotoxin (Ent) plasmid, Ent P307, which contains genes for the production of a hear-labile and a heat-stable enterotoxin. We are using an Escherichia coli K-12 strain, 711 (P307), constructed by S. Falkow, which contains no other plasmids besides Ent P307. Our genetic studies have shown that the plasmid is incompatible with the sex factor F, both in the integrated (Hfr) and the autonomous (F-prime) state. Ent P307 can thus be assigned to incompatibility group FI. An R factor, R386, which belongs to the same incompatibility group, was also found to be incompatibile with Ent P307, whereas five other R factors belonging to different incompatibility groups were compatible with Ent P307. In the presence of Ent P307, conjugal transfer and sensitivity to a male-specific phage of a derepressed F-like R factor, R1drd19, were repressed. Ent P307 is, thus, finO+. Presumably, it also causes repression of its own transfer genes since conjugal transfer of Ent P307 could not be demonstrated. Unlike F, it does not restrict the growth of female-specific phage phiII. From physical studies on extracted deoxyribonucleic acid, the molecular weight of Ent P307 was determined to be 54 X 10(6). By electron microscope heteroduplex analysis, the plasmid was found to be homologous with F in four regions, encompassing about half of its length. One long region and two short ones contain genes for conjugal transfer; the other short region carries genes for replication and incompatibility.     

Characterization of the Tra2 Region of the IncHI1 Plasmid R27

In this study, the DNA sequence of one of the transfer regions of the IncHI1 plasmid R27 was determined. This region, which corresponds to coordinates 0-40 on the R27 map has been called the Tra2 region, and is believed to be involved in mating pair formation. DNA sequence analysis of the transfer region identified 11 open reading frames which showed similarities to the transfer genes from other conjugative systems. The R27 transfer genes appear to most closely resemble the genes from the F plasmid and Sphingomonas aromaticivorans plasmid pNL1, both within the individual genes and in the overall gene order. The Tra2 region is also distinct in that replication, partitioning, and stability genes are found in the middle of the transfer region. The R27 Tra2 region also contains a gene, trhF, which appears to be related to the TraF genes of Agrobacterium and Rhizobium species. This, along with the temperature-sensitive transfer system found in both H plasmids and Agrobacterium, leads to the speculation that the R27 transfer region evolved from both ancestral F-like and P-like plasmids.     

The R1 conjugative plasmid increases Escherichia coli biofilm formation through an envelope stress response

Differential gene expression in biofilm cells suggests that adding the derepressed conjugative plasmid R1drd19 increases biofilm formation by affecting genes related to envelope stress (rseA and cpxAR), biofilm formation (bssR and cstA), energy production (glpDFK), acid resistance (gadABCEX and hdeABD), and cell motility (csgBEFG, yehCD, yadC, and yfcV); genes encoding outer membrane proteins (ompACF), phage shock proteins (pspABCDE), and cold shock proteins (cspACDEG); and phage-related genes. To investigate the link between the identified genes and biofilm formation upon the addition of R1drd19, 40 isogenic mutants were classified according to their different biofilm formation phenotypes. Cells with class I mutations (those in rseA, bssR, cpxA, and ompA) exhibited no difference from the wild-type strain in biofilm formation and no increase in biofilm formation upon the addition of R1drd19. Cells with class II mutations (those in gatC, yagI, ompC, cspA, pspD, pspB, ymgB, gadC, pspC, ymgA, slp, cpxP, cpxR, cstA, rseC, ompF, and yqjD) displayed increased biofilm formation compared to the wild-type strain but decreased biofilm formation upon the addition of R1drd19. Class III mutants showed increased biofilm formation compared to the wild-type strain and increased biofilm formation upon the addition of R1drd19. Cells with class IV mutations displayed increased biofilm formation compared to the wild-type strain but little difference upon the addition of R1drd19, and class V mutants exhibited no difference from the wild-type strain but increased biofilm formation upon the addition of R1drd19. Therefore, proteins encoded by the genes corresponding to the class I mutant phenotype are involved in R1drd19-promoted biofilm formation, primarily through their impact on cell motility. We hypothesize that the pili formed upon the addition of the conjugative plasmid disrupt the membrane (induce ompA) and activate the two-component system CpxAR as well as the other envelope stress response system, RseA-sigma(E), both of which, along with BssR, play a key role in bacterial biofilm formation.     

Genetic analysis of bacterial plasmid promiscuity

The genetic basis of the promiscuous behaviour of bacterial plasmids has been investigated by study of the incompatibility P-1 group of conjugative plasmids of gram-negative bacteria. Both transposon mutagenesis and the construction of minireplicons linking varying combinations of the plasmid genome have shown that specific genomic regions control the conjugational transfer and vegetative replication of the plasmid in specific bacterial hosts. These include the plasmid DNA primase gene, the origin of plasmid transfer, a region near the origin of transfer, the origin of plasmid vegetative replication, thetrans- acting gene essential for the initiation of plasmid replication and a region involved in its regulation. DNA sequence analysis has identified the requirement of specific direct repeats within the origin of replication for plasmid replication in some but not in other hosts. The cloning of some of the trans-acting genes onto multicopy cloning vectors and complementation tests have shown that the requirements of these gene products vary in different hosts and that the plasmid has evolved genetic strategies for their optimal expression.     

Cloning of the neurodegeneration gene drop-dead and characterization of additional phenotypes of its mutation

Mutations in the Drosophila gene drop-dead (drd) result in early adult lethality and neurodegeneration, but the molecular identity of the drd gene and its mechanism of action are not known. This paper describes the characterization of a new X-linked recessive adult-lethal mutation, originally called lot's wife (lwf1) but subsequently identified as an allele of drd (drdlwf); drdlwf mutants die within two weeks of eclosion. Through mapping and complementation, the drd gene has been identified as CG33968, which encodes a putative integral membrane protein of unknown function. The drdlwf allele is associated with a nonsense mutation that eliminates nearly 80% of the CG33968 gene product; mutations in the same gene were also found in two previously described drd alleles. Characterization of drdlwf flies revealed additional phenotypes of drd, most notably, defects in food processing by the digestive system and in oogenesis. Mutant flies store significantly more food in their crops and defecate less than wild-type flies, suggesting that normal transfer of ingested food from the crop into the midgut is dependent upon the DRD gene product. The defect in oogenesis results in the sterility of homozygous mutant females and is associated with a reduction in the number of vitellogenic egg chambers. The disruption in vitellogenesis is far more severe than that seen in starved flies and so is unlikely to be a secondary consequence of the digestive phenotype. This study demonstrates that mutation of the drd gene CG33968 results in a complex phenotype affecting multiple physiological systems within the fly.     

Mobilization of closely related plasmids pUB110 and pBC16 by Bacillus plasmid pXO503 requires trans-acting open reading frame beta.

Genetic analysis of the closely related nonconjugative plasmids pUB110 and pBC16 has demonstrated that the open reading frame beta (ORF-beta) region in pUB110 and the corresponding homologous region in pBC16 are essential for mobilization of these plasmids by pLS20 or its derivatives. Deletions in this region or insertions that interrupted ORF-beta severely impaired or eliminated the mobilization of pUB110::pUC18 and pBC16::pUC18 hybrids. In contrast, a hybrid in which pUC18 was inserted into pBC16 at a point outside ORF-beta transferred at a frequency comparable to that of intact pUB110 or pBC16 (10(-4) transcipients per donor cell). The defect of most transfer-deficient (Mob-) hybrid plasmids could be complemented by an intact sister plasmid (i.e., pBC16 for pUB110::pUC18 Mob- hybrids). The inability to complement certain constructs suggested that the origin of transfer might be located in an area 5' to ORF-beta. Furthermore, cloning the region 5' to ORF-beta onto a nonmobilizable pC194::pUC18 construct resulted in a hybrid plasmid, pUCCoriTBC16, that could be mobilized with complementation. These results indicate that mobilization of pUB110 and pBC16 by conjugative helper plasmids requires ORF-beta in trans and at least one other region, including the RSA sequence, which presumably functions as an origin of transfer, in cis.     

Synthesis of a trans-Acting Inhibitor of DNA Maturation by Prohead Mutants of Phage λ

Bacteriophage lambda with mutations in genes that control prohead assembly and other head precursors cannot mature their DNA. In this paper we present evidence that the failure of these phage mutants to mature DNA is a reflection of a mechanism that modulates terminase nicking activity during normal phage development. We have constructed plasmids that contain the lambda-cohesive end site (cos) and the genes that code for DNA terminase, the enzyme that matures DNA by cutting at cos. The DNA terminase genes are under control of a thermosensitive cI repressor. These plasmids lack most of the genes involved in prohead morphogenesis and other head precursors. However, when repression is lifted by destruction of the thermosensitive repressor, the terminase synthesized is able to cut almost 100% of the plasmids. Therefore, these plasmids can mature in the absence of proheads and other head gene products. The plasmids are also able to complement mutants of lambda deficient in terminase and DNA maturation. However, in these complementation experiments, if the phage carry mutations in prohead genes E or B, not only is phage DNA maturation blocked, but the plasmid also fails to mature. These experiments show that, in the absence of proheads, phage lambda produces a trans-acting inhibitor of maturation. The genetic determinant of this inhibitor maps in a region extending from the middle of gene B to the end of gene C. A model is proposed in which the nicking activity of DNA-bound terminase is inhibited by the trans-acting inhibitor. Prohead (and other factors) binding to this complex would release the block to allow DNA cleavage and packaging.     

Assembly of a functional phage PRD1 receptor depends on 11 genes of the IncP plasmid mating pair formation complex.

PRD1, a lipid-containing double-stranded DNA bacteriophage, uses the mating pair formation (Mpf) complex encoded by conjugative IncP plasmids as a receptor. Functions responsible for conjugative transfer of IncP plasmids are encoded by two distinct regions, Tra1 and Tra2. Ten Tra2 region gene products (TrbB to TrbL) and one from the Tra1 region (TraF) form the Mpf complex. We carried out a mutational analysis of the PRD1 receptor complex proteins by isolating spontaneous PRD1-resistant mutants. The mutations were distributed among the trb genes in the Tra2 region and accumulated predominantly in three genes, trbC, trbE, and trbL. Three of 307 phage-resistant mutants were weakly transfer proficient. Mutations causing a phage adsorption-deficient, transfer-positive phenotype were analyzed by sequencing.