Construction of Tn3-Containing Plasmids from Plant-Pathogenic Pseudomonads and an Examination of Their Biological Properties

Indigenous plasmids isolated from Pseudomonas tabaci ATCC 11528(pJP1), Pseudomonas angulata 45(pJP30), and P. tabaci BR2(pBPW1) (M. Obukowicz and P. D. Shaw, J. Bacteriol. 155:438-442, 1983) were labeled with Tn3, and the strains were subsequently cured of their respective plasmids. Plasmid-containing and cured isolates caused plant symptoms that were nearly indistinguishable, and the same amount of tabtoxin was produced by P. tabaci strains ATCC 11528 and BR2.     

Isolation and Characterization of Plasmid DNA in Streptococcus cremoris

Nine industrially important strains of Streptococcus cremoris (HP, AM₂, ML₁, WC, C₃, R₁, E₈, KH, and Wg₂) were shown to possess a diversity of plasmid molecules. Molecular weights of plasmids were determined from their relative mobilities after agarose gel electrophoresis and via electron microscopy. To illustrate the varied plasmid sizes, strain HP contained plasmids of 26, 18, 8.5, 3.3, and 2 megadaltons (Mdal); strain ML₁ contained plasmids of 29, 18, 9, 4, 2.2, and 1.8 Mdal; and strain AM₂ had plasmids of 42, 27, 16, and 8.4 Mdal. The numbers of plasmids observed in the other strains were 6, 5, 5, 7, 5, and 4 for C₃, E₈, KH, R₁, WC, and Wg₂, respectively. A spontaneous proteinase-negative (Prt⁻) mutant of HP was missing the 8.5-Mdal plasmid, which suggests that in this strain proteinase activity could be linked to this particular plasmid. A lactose-negative (Lac⁻) Prt⁻ mutant of ML₁ lacked the 2.2-Mdal plasmid. Under the conditions employed, antibiotic sensitivity and heavy-metal susceptibility did not correlate with the missing plasmid in Prt⁻ HP or in the Lac⁻ Prt⁻ ML₁. Curing experiments with AM₂, using acridine dyes and elevated temperatures, did not yield Lac⁻ variants. AM₂ was also cultured at high dilution rates in a chemostat for 168 h by using a buffered milk or lactic broth at 18 or 32°C with no selection of Lac⁻ derivatives. The inability to obtain Lac⁻ variants under conditions known to facilitate plasmid elimination suggests that lactose metabolism is not plasmid-mediated in AM₂.     

Characterization of Plasmid Deoxyribonucleic Acid in Streptococcus lactis subsp. diacetylactis: Evidence for Plasmid-Linked Citrate Utilization

The use of Streptococcus diacetylactis as a flavor producer in dairy fermentations is dependent upon its ability to produce diacetyl from citrate. Treatment of S. diacetylactis strains 18-16 and DRC1 with acridine orange resulted in the conversion of approximately 2% of the DRC1 population and 20% of the 18-16 population to citrate negative, which is indicative of the involvement of plasmid deoxyribonucleic acid (DNA). Growth in the presence of acridine orange also resulted in the appearance of 2% lactose-negative derivatives in S. diacetylactis 18-16 and 99% lactose-defective, proteinase-negative derivatives in S. diacetylactis DRC1. Cesium chloride-ethidium bromide equilibrium density gradients of cleared lysate material from each strain revealed the presence of covalently closed circular DNA. Samples of this covalently closed circular DNA were subjected to agarose gel electrophoresis to determine the plasmid composition of each strain. S. diacetylactis 18-16 was found to possess six plasmids, of approximately 41, 28, 6.4, 5.5, 3.4, and 3.0 megadaltons (Mdal). S. diacetylactis DRC1 contained six plasmids, of approximately 41, 31, 18, 5.5, 4.5, and 3.7 Mdal. Variants of S. diacetylactis 18-16 which failed to produce acetoin plus diacetyl from citrate (citrate negative) were missing a 5.5-Mdal plasmid. Lactose-negative mutants of the same strain were devoid of a 41-Mdal plasmid. Lactose-defective, proteinase-negative mutants of S. diacetylactis DRC1 were missing a 31-Mdal plasmid. The citrate-negative mutants of S. diacetylactis DRC1 isolated in this study did not possess a 5.5-Mdal plasmid. Thus, we have evidence that there is a correlation between the ability to utilize citrate and the presence of a 5.5-Mdal plasmid. A relationship was also noted between lactose fermentation and proteinase activity and plasmid DNA in S. diacetylactis.     

Molecular relationships among plasmids of Bacillus thuringiensis: conserved sequences through 11 crystalliferous strains

Summary Screening for the plasmid content of 11 strains belonging to nine different serotypes ofB. thuringiensis was carried out by electron microscope examination and electrophoresis in agarose gels. All the strains contained at least two covalently closed, circular (CCC) DNA species. In one strain (berliner 1715), 17 extrachromosomal elements could be distinguished with regard to their size, ranging from 3.9 to 180 Mdal. Southern hybridisation experiments showed that most of these plasmids fell into two categories (inferior to 15 Mdal and superior to 15 Mdal) which have no homology between them. Within these two size groups there is partial conservation of DNA sequences through various serotypes. Further relationships among the plasmids were investigated by a two dimensional version of the Southern's blotting technique.Possible homology between plasmids and the chromosomal DNA was studied. It was shown that the smaller plasmids from the berliner 1715 and kurstaki HD₁ strains contained no sequence related to chromosomal DNA, whereas among the larger plasmids a few showed homologous sequences.Abbreviations cry^- tacrystalliferous mutant - GCC covalently closed circular DNA - OC open circular DNA - Mdal megadalton - kb 1,000 base pairs     

Evidence for plasmid-associated crystal toxin production in Bacillus thuringiensis subsp. israelensis

Three crystalliferous (Cry⁺) strains of Bacillus thuringiensis subsp. israelensis (serotype 14) that produce parasporal protein crystals toxic to dipteran larvae and several acrystalliferous (Cry^?) mutants, either induced or spontaneously derived from a single Cry⁺ parent, were examined for the presence of covalently closed circular (CCC) DNA in attempts to correlate toxin production with the presence of a specific plasmid. The plasmid profiles of both Cry⁺ and Cry^? variants were analyzed by both a cleared lysate- and a modified Eckhardt lysateelectrophoresis technique. All of the Cry^? mutants derived from the Cry⁺ parental strain had lost a 4.0- to 4.4-megadalton (Mdal) plasmid. Bioassay data confirmed loss of toxin production by the Cry^? variants. All three Cry⁺ strains, including the parent of the Cry^? strains, contained CCC plasmids DNAs of the following approximate molecular weights: 4.0 to 4.4, 5.2 to 6.0, and 11.4 to 13.0 Mdal. One Cry⁺ strain contained an additional CCC plasmid of 6.7 to 7.2 Mdal. The plasmid patterns for several Cry^? derivatives differed in other respects from the pattern for their parent strain. The various Cry⁺ and Cry^? strains could be distinguished either by phenotypical differences in antibiotic sensitivity, crystal production, and toxicity, or by differences in their plasmid profiles.     

Structure of Acidic Polysaccharide Produced by Strain No. 626 of Aeromonas hydrophila

Lactobacillus acidophilus strain TK8912 was found to carry six plasmids having molecular masses of 40, 17, 10.5, 5, 2, and 1.8 megadaltons (Mdal). An acriflavin-induced, Lac^- mutant had lost a 17-Mdal plasmid (pLA102) and phospho-β-galactosidase (P-β-gal) activity. Since strain TK1-2TL (Lac⁺ transformant) restored P-β-gal activity, pLA102 is likely to encode a P-β-gal gene required for lactose metabolism in strain TK8912. It is also suggested that the gene for the tagatose 6-phosphate pathway, which is responsible for galactose metabolism, is encoded by the 40-Mdal plasmid pLA101.     

Characteristics of Ti plasmids from broad-host-range and ecologically specific biotype 2 and 3 strains of Agrobacterium tumefaciens.

Agrobacterium tumefaciens strains isolated from crown gall tumors on grapevines in California were consistently of the biotype 3 group. All 11 of these strains were limited in their host range and harbored Ti plasmids with molecular masses between 119 and 142 megadaltons (Mdal) as well as a larger cryptic plasmid of greater than 200 Mdal; occasionally a smaller cryptic plasmid of 65 Mdal was also present. Ti plasmids o these strains have DNA sequences in common with Ti plasmids of octopine and nopaline strains belonging to the biotype 1 group and exhibited sequence homologies with the conserved region of the T-DNA. Ten of the 11 strains utilized octopine as a sole source of carbon and nitrogen and 3 strains catabolized both octopine and nopaline, whereas 1 strain catabolized only nopaline. All of these strains were resistant to the bacteriocin agrocin-84, except one grapevine strain that belonged to the biotype 1 group and was agrocin sensitive; it is also differed in its plasmid and virulence characteristics. Isolations from Rubus ursinus ollalieberry galls yielded exclusively biotype 2 strains. These strans were insensitive to agrocin-84, utilized nopaline as a sole carbon and nitrogen source, and were highly virulent on all host plants tested. They contained Ti plasmids ranging between 100 and 130 Mdal and occasionally a cryptic plasmid of 69 Mdal. Their Ti plasmids have DNA sequences in common with Ti plasmids of biotype 1 strains and with the conserved region of the T-DNA.     

Comparison of the Deoxyribonucleic Acid Molecular Weights and Homologies of Plasmids Conferring Linked Resistance to Streptomycin and Sulfonamides

Bacterial strains showing linked resistance to streptomycin (Sm) and sulfonamides (Su) were chosen representing a wide taxonomic and geographical range. Their SmSu resistances were transferred to Escherichia coli K-12 and then plasmid deoxyribonucleic acid (DNA) was isolated by ethidium bromide CsCl centrifugation. The plasmid DNA was examined by electron microscopy and analyzed by sedimentation through 5 to 20% neutral sucrose gradients. Plasmid DNA from strains having transmissible SmSu resistance consisted of two or three molecular species, one of which had a molecular mass of about 5.7 Mdal (10(6) daltons), the others varying between 20 to 60 Mdal. By using transformation or F' mobilization, we isolated the SmSu-resistance determinant from any fellow resident plasmids in each strain and again isolated the plasmid DNA. Cosedimentation of each of these with a differently labeled reference plasmid DNA (R300B) showed 9 out of 12 of the plasmids to have a molecular mass not significantly different from the reference (5.7 Mdal); two others were 6.3 and 9.2 Mdal, but PB165 consisted of three plasmids of 7.4, 14.7, and 21.4 Mdal. Three separate isolations of the SmSu determinant from PB165 gave the same three plasmids, which we conclude may be monomer, dimer, and trimer, respectively. DNA-DNA hybridizations at 75 C demonstrated 80 to 93% homology between reference R300B DNA and each isolated SmSu plasmid DNA, except for the 9.2-Mdal plasmid which had 45% homology and PB165 which had 35%. All the SmSu plasmids were present as multiple copies (about 10) per chromosome. The conjugative plasmid of R300 (present as 1.3 copies per chromosome) has been shown to have negligible effect on the number of copies of its accompanying SmSu plasmid R300B. We conclude that the SmSu plasmids are closely related and probably have a common evolutionary origin.     

Transduction of Lactose Metabolism by Streptococcus cremoris C3 Temperate Phage

Temperate phage was induced from Streptococcus cremoris C3 and morphologically characterized by high-resolution electron micrographic techniques. Interspecies genetic transfer of lactose-fermenting ability by the temperate phage was demonstrated, using two lactose-negative (Lac⁻) S. lactis strains as recipients. Plasmid transfer was confirmed by agarose gel electrophoresis. Transductant plasmid profiles were of three types—those containing no visible plasmid deoxyribonucleic acid, those possessing a 23-megadalton (Mdal) plasmid, and those containing a 23-Mdal plasmid and a 30-Mdal plasmid. A Lac⁺ transductant could serve as a donor of the lac determinants during solid-surface matings. These results add to previously published reports of inter- and intraspecies genetic transfer in dairy starter cultures.     

Plasmid linkage of a bacteriocin-like substance in Streptococcus lactis subsp. diacetylactis strain WM4: transferability to Streptococcus lactis.

Streptococcus lactis subsp. diacetylactis strain WM4 transferred lactose-fermenting and bacteriocin-producing (Bac+) abilities to S. lactis LM2301, a lactose-negative, streptomycin-resistant (Lac- Strr), plasmid-cured derivative of S. lactis C2. Three types of transconjugants were obtained: Lac+ Bac+, Lac+ Bac-, and Lac-Bac+.S. diacetylactis WM4 possessed plasmids of 88, 33, 30, 5.5, 4.8, and 3.8 megadaltons (Mdal). In Lac+ Bac+ transconjugants, lactose-fermenting ability was linked to the 33-Mdal plasmid and bacteriocin-producing ability to the 88-Mdal plasmid. Curing the 33-Mdal plasmid from Lac+ Bac+ transconjugants resulted in loss of lactose-fermenting ability but not bacteriocin-producing ability (Lac- Bac+). These strains retained the 88-Mdal plasmid. Curing of both plasmids resulted in a Lac- Bac- phenotype. The Lac+ Bac- transconjugant phenotype was associated with a recombinant plasmid of 55 or 65 Mdal. When these transconjugants were used as donors in subsequent matings, the frequency of Lac transfer was about 2.0 X 10(-2) per recipient plated, whereas when Lac+ Bac+ transconjugants served as donors, the frequency of Lac transfer was about 2.0 X 10(-5) per recipient plated. Also, Lac- Bac+ transconjugants were found to contain the 88-Mdal plasmid. The data indicate that the ability of WM4 to produce bacteriocin is linked to an 88-Mdal conjugative plasmid and that lactose-fermenting ability resides on a 33-Mdal plasmid.     

Restriction endonuclease analysis of the lactose plasmid in Streptococcus lactis ML3 and two recombinant lactose plasmids.

We investigated the molecular relationship between the 60-megadalton (Mdal) recombinant lactose plasmids in ML 3 x LM2301 lactose-positive (Lac+) transconjugants and the genetic material of Streptococcus lactis ML3. Lactose metabolism is linked to the 33-Mdal plasmid pSK08 in ML3, and the recipient LM2301 is cured of plasmid DNA. The plasmids were analyzed with a series of restriction enzymes. We found that the 60-Mdal plasmids of Lac+ transconjugants contained pSK08 DNA, but were not simply dimers of pSK08. The 60-Mdal plasmids contained a segment of DNA not apparent in pSK08. The restriction patterns of the 60-Mdal plasmid in a Lac+ nonclumping transconjugant and that in a Lac+ clumping transconjugant were different. This suggested that there was a molecular differences between these two recombinant plasmids. We conclude that the segment of DNA in the 60-Mdal plasmids that was not present in pSK08 was the proposed transfer factor responsible for cell aggregation and high-frequency conjugation.     

The molecular relatedness among alpha-hemolytic plasmids from various incompatibility groups

Deoxyribonucleic acid (DNA) reassociation studies among α-hemolytic (Hly) plasmids from FVI and FIII–IV incompatibility groups showed a close similarity between the nucleotide sequences of plasmids from the same group. With respect to R plasmids from the F overgroup, they have 20–26 Mdal in common, an amount of DNA close to the amount involved in the traF operon. No more extensive sequence homology was found between pSU316 (IncFIII–IV) and the incompatible plasmids ColB-K98 (IncFIII) or R124 (IncFIV). The IncIα I2 plasmid pSU5 has only the α-hemolytic region (5 Mdal) in common with plasmid pSU316 but it is much more closely related to IncFVI plasmids where the DNA in common amounts to 22 Mdal. Finally, the genetically unrelated plasmid pSU233 shares 66% of its nucleotide sequences (40 Mdal) with the IncFVI plasmids and has 16–23 Mdal in common with various F-like plasmids.     

Conjugal transfer of beta-lactamase-producing plasmids of Neisseria gonorrhoeae to Neisseria meningitidis

Twenty clinical isolates of beta-lactamase-producing Neisseria gonorrhoeae from Japanese sources were studied to define their ability to serve as donors for their plasmids in conjugation with Neisseria meningitidis. These twenty strains of N. gonorrhoeae harbored the 4.5-megadalton (Mdal) beta-lactamase-producing plasmids and the 24.5-Mdal conjugative plasmids. We found that only three of twenty N. gonorrhoeae strains showed a detectable conjugation frequency (greater than 10(-5)) with N. meningitidis as the recipient although all strains were capable of mobilizing beta-lactamase-producing plasmids to N. gonorrhoeae and to Escherichia coli. The 4.5-Mdal beta-lactamase-producing plasmid was maintained in N. meningitidis, but the large 24.5-Mdal conjugative plasmid has not been found in N. meningitidis transconjugants.     

Genetic isolation and physical characterization of pAgK84, the plasmid responsible for agrocin 84 production

The plasmid responsible for agrocin 84 biosynthesis by Agrobacterium radiobacter strain K84 has been genetically isolated free from any opine-catabolic plasmids. This was accomplished by mobilizing the agrocin plasmid, pAgK84, into a Ti plasmid-free A. tumefaciens strain, A136. The mobilizing element, pAt84a, was then cured from such a transconjugant by cultivation at 37 °C. Derivatives of strain A136 harboring both plasmids or pAgK84 only produce agrocin 84. The agrocin plasmid isolated from these strains is indistinguishable by restriction endonuclease analysis from that in strain K84. A physical map of pAgK84 has been constructed with respect to six restriction endonucleases. The plasmid is cut only once by XbaI and twice by HpaI. Hybridization analysis shows that pAgK84 is closely related to pAtBo542a, a 25-Mdal plasmid from a virulent, agrocinogenic A. tumefaciens strain of European origin. Similar analyses indicate, however, that pAgK84 shows no detectable homology to octopine or nopaline-type Agrobacterium plasmids.     

Evidence for plasmid-mediated resistance ofPseudomonas putida to hexahydro-1,3,5-triethyl-s-triazine

Resistance to the industrial biocide hexahydro-1,3,5-triethyl-s-triazine (HHTT) by a strain ofPseudomonas putida was shown to be encoded by a 32.5-megadalton (Mdal) plasmid as evidenced: (a) by visualization of the plasmid DNA by agarose gel electrophoresis, (b) by the loss of HHTT resistance as well as the loss of the 32.5-Mdal plasmid upon curing with novobiocin, and (c) by conjugal concomitant transfer of HHTT resistance and the 32.5-Mdal plasmid by mating the novobiocin-cured HHTT-sensitive derivative with the HHTT-resistant strain. The 32.5 Mdal did not encode for heavy metal or antibiotic resistance, and it was shown not to be one of the degradative plasmids ofPseudomonas. The mechanism of HHTT resistance was not discerned from these studies.     

Restriction endonuclease analysis of the lactose plasmid in Streptococcus lactis ML3 and two recombinant lactose plasmids

We investigated the molecular relationship between the 60-megadalton (Mdal) recombinant lactose plasmids in ML 3 x LM2301 lactose-positive (Lac+) transconjugants and the genetic material of Streptococcus lactis ML3. Lactose metabolism is linked to the 33-Mdal plasmid pSK08 in ML3, and the recipient LM2301 is cured of plasmid DNA. The plasmids were analyzed with a series of restriction enzymes. We found that the 60-Mdal plasmids of Lac+ transconjugants contained pSK08 DNA, but were not simply dimers of pSK08. The 60-Mdal plasmids contained a segment of DNA not apparent in pSK08. The restriction patterns of the 60-Mdal plasmid in a Lac+ nonclumping transconjugant and that in a Lac+ clumping transconjugant were different. This suggested that there was a molecular differences between these two recombinant plasmids. We conclude that the segment of DNA in the 60-Mdal plasmids that was not present in pSK08 was the proposed transfer factor responsible for cell aggregation and high-frequency conjugation.     

Essential virulence determinants of different Yersinia species are carried on a common plasmid

Plasmids of 44.4–46 Mdal were identified in conditional virulent Yersinia species. All virulent strains studied are unable to grow on oxalate-containing plates at 37 °C (OX^? phenotype) which is a characteristic property of strains producing the essential virulence VW antigens. The phenotopic transition from OX^? to OX⁺ in these strains is concomitant with loss of virulence and loss of this plasmid. The similarity in size and in the DNA fragmentation patterns, generated by HindIII, of the plasmids isolated from either Y. pseudotuberculosis or two conditional virulent Y. pestis strains, suggests that a common plasmid—pSB2—is carried by these strains. A plasmid of a similar size, ~42 Mdal, and function was recently identified (P. Gemski, J. R. Lazere, and T. Casey, 1980, Infect. Immunity27, 682–685; D. L. Zink, J. C. Feeley, J. G. Wells, C. Vanderzant, J. C. Vickery, W. D. Roof, and G. A. O'Donovan, 1980, Nature (London)283, 224–225) in virulent Y. enterocolitica. We conclude that pSB2 in Y. pseudotuberculosis and Y. pestis and its counterpart in Y. enterocolitica carry genetic information essential for virulence common to the Yersinia species, probably related to VW antigen production. Several additional plasmids were identified in several strains of Y. pestis. One of these plasmids, designated pSB3 (12.5 Mdal), appears to be associated with pesticin production.     

Mobilization of nonconjugative antibiotic resistance plasmids in Haemophilus ducreyi.

A clinical isolate of Haemophilus ducreyi was found to harbor three plasmids: a 23.5-megadalton (Mdal) phenotypically cryptic plasmid, a 7.0-Mdal ampicillin resistance plasmid, and a 4.0-Mdal sulfonamide resistance plasmid. The two smaller plasmids were transferable by conjugation to Haemophilus recipients, but only if the donor cell harbored the 23.5-Mdal plasmid as well, indicating that this large plasmid had mobilizing capabilities. Transfer was also possible to Escherichia coli recipients. Haemophilus influenzae transconjugants which had acquired both the 23.5-Mdal plasmid and one of the R-plasmids could subsequently retransfer the R-plasmid to other Haemophilus recipients at higher frequencies. A derivative of the 23.5 Mdal plasmid was isolated which was shown by restriction endonuclease analysis to contain an ampicillin resistance transposon and to have retained its conjugative ability.     

Identification and mobilization by cointegrate formation of a nodulation plasmid in Rhizobium trifolii.

A nodulation plasmid, pRtr-514a, of molecular size 180 megadaltons (Mdal) was identified in Rhizobium trifolii strain NZP514. This plasmid was absent in both spontaneous and heat-cured Nod- derivatives of NZP514, and these strains were unable to induce root hair curling. The ability to nodulate clover was transferred from the wild-type strain to a Nod- derivatives, PN104, with the broad-host-range plasmid R68.45 (39 megadaltons) at a cotransfer frequency of about 4 X 10(-3). Most of the Nod+ transconjugants were resistant to kanamycin, tetracycline, and carbenicillin and had received a plasmid approximately 36 or 70 Mdal larger than pRtr514a but did not contain a plasmid of the size of R68.45, indicating that pRtr-514a was mobilized as a cointegrate plasmid containing either one or possibly two copies of R68.45. Use of these cointegrate-containing strains as donors in further crosses with the Nod- derivative strain PN118 resulted in high-frequency transfer of Nod+ (10(-3) to 10(-4), with cotransfer frequencies with kanamycin of up to 100%. Introduction of R68.45 into a derivative of NZP514 containing the broad-host-range plasmid pJP4 (52 Mdal) resulted in a high frequency of transconjugants carrying a cointegrate plasmid composed of pRtr-514a and pJP4. When used as donors to Nod- derivatives, such strains cotransferred Nod+ with kanamycin plus mercury at a frequency of 67%. The identification of stable cointegrates between pRtr-514a and the broad-host-range plasmids R68.45 and pJP4 should enable several genetic manipulations to be carried out with this nodulation plasmid, including the transfer of the plasmid to most gram-negative bacterial genera.     

Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMG5.

Large plasmids from Agrobacterium tumefaciens, Salmonella typhimurium, Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa were routinely and consistently isolated using a procedure which does not require ultracentrifugation but includes steps designed to separate large-plasmid DNA from the bacterial folded chromosome. It also selectively removes fragments of broken chromosome. A variety of large plasmids was readily visualized with agarose gel electorphoresis, including five between 70 and 85 megadaltons (Mdal) in size, six between 90 and 143 Mdal, one that was larger than 200 Mdal, and one that was larger than 300 Mdal. This isolation procedure allowed initial estimation of the molecular sizes of the two IncP2 plasmids, pMG1 and pMG5, which were 312 and 280 Mdal, respectively. A standard curve for size determination by gel electrophoresis including plasmids between 23 and 143 Mdal in size did not extrapolate linearly for plasmids of the 300-Mdal size range. Unique response of different plasmids to the isolation procedure included sensitivity of IncP1 plasmids to high pH and the co-isolation of a 20-Mdal "cryptic" plasmid in conjunction.