Tn7 insertion mutations affecting the host range of the promiscuous IncP-1 plasmid R18

The genetic basis of plasmid host range has been investigated by Tn7 insertion mutagenesis of the promiscuous plasmid R18 in Pseudomonas aeruginosa. Six mutants have been isolated on the basis of greatly reduced transferability into Escherichia coli C while retaining normal transferability within P. aeruginosa. Their physical mapping shows that two of them map at coordinate 11.72 ± 0.14 kb, in the region of the origin of plasmid replication (oriV) and one at 18.0 ± 0.3 kb, in the trans-acting gene essential for initiation of replication at oriV (trfA). Three map at 48.4 ± 0.5 kb in the region of the origin of plasmid transfer (oriT) and the site at which a single-strand nick is introduced in the plasmid DNA-protein relaxation complex (rlx). Consistent with the postulated defective replication of the oriV and trfA mutants was their inability to transform E. coli C or K12 while being able to transform P. aeruginosa. As expected the oriT/rlx mutants transformed both hosts as effectively as R18. Furthermore the trfA mutant was readily curable by mitomycin C in a DNA polymerase I-proficient P. aeruginosa and spontaneously lost from a polymerase-deficient mutant of P. aeruginosa suggesting a role of this polymerase in the replication of R18. Extensive transfer tests from P. aeruginosa into a range of enteric bacteria, other Pseudomonas species and into other Gram-negative bacteria indicated a complex host range pattern for these mutants. It appears that both plasmid replication and conjugation genes are responsible for host range in addition to the involvement of host gene products.     

Cloning and molecular analysis of the finO region from the antibiotic-resistance plasmid R6-5

The cloning of the finO region from the antibiotic-resistance plasmid R6-5 is reported. On the basis of DNA deletion analysis and Tn5 transposon insertional mutagenesis of finO+ chimeric plasmids, finO has been located within the coordinates 94.0-94.85 on the R6-5 map. A 32,000-Da polypeptide (32K), which is encoded within 92.75-94.25R6-5, has been identified and shown not to be associated with the FinO phenotype.     

Mutation leading to gene fusion in the histidine operon of Salmonella typhimurium

A spontaneous polar mutation located in the region of an intercistronic border in the hiatidine operon of Salmonella was isolated in our laboratory. The mutant, R81, tests as a frameshift in reversion experiments but is prototrophic, capable of growth without histidine supplements despite lowered levels of certain histidine enzymes. The mutation affects the operator distal end of the D gene, causing production of an active histidinol dehydrogenase enzyme with an altered C-terminus. The mutation severely affects expression of the immediately succeeding gene in the translation sequence, hisC, suggesting either that the D–C border and possibly hisC are physically altered or that their normal function in translation is seriously impaired. We have previously described the fortuitous production from R81 of a non-polar derivative with fused D and C genes. This strain produces a bifunctional enzyme with normally separate dehydrogenase and aminotransferase activities present on dimers or multimers of a single fused polypeptide chain. We have now investigated in greater detail the R81 mutation by amino acid sequencing of the C-terminus of altered histidinol dehydrogenase. We find that the R81 mutation causes the addition of a “tail” of four amino acid residues to an otherwise normal dehydrogenase polypeptide chain. The results support our previous suggestion that the R81 mutation profoundly effects the D–C gene border and that this effect is prerequisite to gene fusion.     

A study of the evolution of repeated DNA sequences in primates and the existence of a new class of repetitive sequences in primates.

A new class of lowly repetitive DNA sequences has been detected in the primate genome. The renaturation rate of this sequence class is practically indistinguishable from the renaturation rate of single-copy sequences. Consequently, this lowly repetitive sequence class has not been previously observed in DNA renaturation rate studies. This new sequence class is significant in that it might occupy a major fraction of the primate genome.Based on a study of the thermal stabilities of DNA heteroduplexes constructed from human DNA and either bonnet monkey or galago DNAs, we are able to compare the relative mutation rates of repetitive and single-copy sequences in the primate genome. We find that the mutation rate of short, interspersed repetitive sequences is either less than or approximately equal to the mutation rate of single-copy sequences. This implies that the base sequence of these repetitive sequences is important to their biological function.We also find that numerous mutations have accumulated in interspersed repeated sequences since the divergence of galago and human. These mutations are only recognizable because they occur at specific sites in the repeated sequence rather than at random sites in the sequence. Although interspersed repetitive sequences from human and galago can readily cross-hybridize, these site-specific mutations identify them as being two distinct classes. In contrast, far fewer site-specific mutations have occurred since the divergence of human and monkey.     

Study of the incompatibility and replication of the 70-kb virulence plasmid of Yersinia

The 70-kb virulence plasmid, vir, from four Yersinia enterocolitica and one Y. pseudotuberculosis strains are incompatible with IncFI plasmids FLac and R386 while they are compatible with plasmids representing nine other incompatibility groups. Hybridization experiments carried out on one of these virulence plasmids showed that it contains the F incompatibility determinant D, incD. This determinant was cloned onto pACYC184 and the recombinant clone expressed incompatibility with FLac. We conclude that the incompatibility observed between F or R386 and the 70-kb virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis is mediated by incD. Replication genes (rep) from the same plasmid were cloned independently in Escherichia coli. Rep and incD map on two different BamHI fragments. Surprisingly, the replicon isolated is not sensitive to inc D incompatibility. Apart from incD, vir and F share extremely little homology. In particular, there is no evidence for the presence of an F-like transfer operon on vir.     

Conditional lethal mutants of the kilB determinant of broad host range plasmid RK2

We have examined the relationship of kilB to the other known determinants which map in the 14'-22' region of RK2. These are trfA, which encodes a diffusible replication function, and tra3, which specifies a function required for plasmid transmissibility. We found that, in addition to kilB, both tra3 and trfA functions are expressed by the cloned 14'-22' region of RK2. Four temperature-sensitive mutants of kilB were isolated by in vitro mutagenesis of the cloned segment. At 42 degrees C these mutant plasmids can be maintained in Escherichia coli cells which lack a korB+ helper plasmid. At 30 degrees C the helper plasmid is required. Our analysis of these mutants revealed that kilB function is distinct from those of trfA and tra3. One mutant plasmid was temperature-sensitive for maintenance of an RK2 ori plasmid, but this phenotype was shown to be independent of the KilB(ts) phenotype. Thus, kilB appears to be a separate new locus in this portion of the RK2 genome. In addition, these mutants allowed us to test for the existence of an essential replication determinant (trfB) in the 50.4'-56.4' region of RK2. Our results demonstrate that this region is non-essential for replication from the RK2 ori in E. coli. We propose an alternative hypothesis to explain the role of the RK2 trfB region for plasmid maintenance in E. coli.     

Complementation analysis in Pseudomonas aeruginosa of the transfer genes of the wide host range R plasmid R18

A method of transductional complementation was developed in Pseudomonas aeruginosa to identify the cistrons involved in the conjugal transfer of the wide host range R plasmid R18. This used the P. aeruginosa bacteriophage E79tv-2 and has led to the identification of eight tra cistrons encoded by this plasmid. Plasmids mutant in six cistrons, traA, traB, traC, traD, traE, and traG were resistant to donor-specific phage (Dps^?) while traF and traH mutant plasmids retained phage sensitivity. Some traB mutants were unable to inhibit the replication of phage G101 (Phi(G101)^?) while some were also deficient in entry exclusion (Eex^?). Two traB mutants which were also Eex^? were suppressible by an amber suppressor. Three tra mutants selected directly as being Phi(G101)^? were found to be also Dps^?Eex^? and mutant in traB. These data suggest a relationship between traB, Eex, and Phi(G101). In order to facilitate future genetic comparison of the tra genes of R18 and other wide host range plasmids and the role of the host in conjugation, R18 DNA was compared with that of RP4, by restriction enzyme fragment patterns and found to be identical.     

Molecular cloning and selection of genes regulated in Aspergillus development

Over 350 clones homologous to poly(A)+ RNAs that are significantly more prevalent in conidiating cultures of Aspergillus nidulans than in somatic cells have been selected from a recombinant DNA library formed between nuclear DNA and lambda Charon 4A. The procedure used for this selection involved in situ hybridization to a cDNA probe which had been selectively depleted of sequences represented in somatic cells by complement hybridization. Five of these clones have been characterized further. All but one encoded poly(A)+ RNAs that were at least ten times more prevalent in conidiating cultures than in somatic cells. One clone hybridized to a single, developmentally regulated RNA. The three others were complementary to several RNAs having different molecular weights, each of which was more prevalent in condiating cultures than in vegetative cells. These results and quantitative aspects of the selection procedure suggest that developmentally controlled poly(A)+ RNA coding regions may not be distributed randomly in the Aspergillus genome.     

A host-vector system for gene cloning in the cyanobacterium Anacystis nidulans R2

We describe the construction of a series of vectors suitable for gene cloning in the Cyanobacterium Anacystis nidulans R2. From the indigenous plasmid pUH24, derivatives were constructed with streptomycin as the selective marker; one of these plasmids was used to construct pUC303, a shuttle vector capable of replication in A. nidulans R2 as well as in Escherichia coli K12. It has two markers, streptomycin and chloramphenicol resistance, and three unique restriction sites. Instability of recombinant plasmids was overcome by using a derivative of A. nidulans R2 cured of the indigenous plasmid pUH24. This strain, R2-SPc, can be transformed stably and at high frequency by the plasmids described in this paper. The combination of the cured strain R2-SPc and the new plasmid pUC303 serves as a suitable host-vector system for gene cloning in cyanobacteria.     

pFJ265, a new cloning vehicle for Streptomyces

A 9.3-kb plasmid, pNM100, was isolated from Streptomyces virginiae (NRRL 15156) and characterized. Streptomyces genes for thiostrepton and neomycin resistance were cloned into pNM100 to yield a small plasmid derivative, pFJ265, that is suitable for Streptomyces gene cloning. pFJ265 is a 9.2-kb nonconjugative plasmid and has a copy number of several hundred per chromosome.     

Sulfhydryl groups of the F1 adenosine triphosphatase of Escherichia coli and the stoichiometry of the subunits

The distribution and total number of sulfhydryl groups present in the F1 adenosine triphosphatase of Escherichia coli were used to calculate the stoichiometry of the alpha-delta subunits. Titration with 5,5'-dithiobis (2-nitrobenzoate) gave 19.1 +/- 2.2 sulfhydryl groups/mol ATPase. Labeling with [14C]iodoacetamide and [14C]N-ethylmaleimide showed that 11.9, 3.1, 1.9, and 1.8 sulfhydryl groups per molecule of ATPase were associated with the alpha, beta, gamma, and delta subunits, respectively. The epsilon subunit was not labeled. Application of the method of Creighton [Nature (London) (1980) 284, 487-489] showed that 4, 1, and 2 sulfhydryl groups were present in the alpha, beta, and gamma subunits, respectively. This, together with published data for the delta subunit, allowed a subunit stoichiometry of alpha 3 beta 3 gamma delta to be calculated. The presence of four cysteinyl residues in the alpha subunit, as shown by several different methods, does not agree with the results of DNA sequencing of the ATPase genes [H. Kanazawa, T. Kayano, K. Mabuchi, and M. Futai (1981) Biochem. Biophys. Res. Commun. 103, 604-612; N. J. Gay and J. E. Walker (1981) Nucl. Acids Res. 9, 2187-2194] where three cysteinyl residues/alpha subunit have been found. It is suggested that post-translational modification of the alpha subunit to add a fourth cysteinyl residue might occur.     

A host-dependent hybrid plasmid suitable as a suicidal carrier for transposable elements

Plasmid pAS8Tc^s rep-1::Tn7 (abbreviated pAS8Rep-1), a derivative of the RP4-ColE1 hybrid plasmid pAS8 displaying ColE1-dependent replication/maintenance, was found capable of the introduction of transposon Tn7 into the genome of phytopathogenic Pseudomonas. The plasmid is potentially useful as a general purpose suicidal Tn carrier for bacteria that do not support stable replication/maintenance of ColE1 but are within the conjugational host range of RP4.     

Specific pattern of instability of Escherichia coli HisG gene cloned in Bacillus subtilis via the Staphylococcus aureus plasmid pCS194

The plasmid pCS194, generated in vivo by recombination of two Staphylococcus aureus plasmids, pC194 and pS194, coding, respectively, for chloramphenicol (Cm) and streptomycin (Sm) resistance, can be replicated also in Bacillus subtilis in the presence of either of the two antibiotics. In their absence, no segregation of the individual components is observed, but the whole plasmid is lost at a rate of about 10% per generation. The unique EcoRI site of pCS194 is located in the Sm^R determinant. EcoRI-cleaved pCS194 has been joined to an EcoRI-linearized Escherichia coli replicon, the in vitro recombinant pHisG plasmid, composed of the vector pBR313 plus a BglII-segment of E. coli chromosomal DNA, containing a functional hisG gene. The ligation mixture has been used to transform either E. coli or B. subtilis. Following E. coli transformation and selection for Ap^R and Cm^R (the latter is expressed in E. coli by the pC194 determinant), two his⁺ clones were picked at random and the plasmids extracted. These appear identical and contain the original segments. Conversely, after transformation of B. subtilis and selection for Cm^R, only his^? clones have been obtained. From them, deleted plasmids have been extracted. They have lost part or, more frequently, all of the E. coli DNA insert. In the latter case also most of the bracketing pS194 sequence has been lost, and the resulting plasmids are almost identical to pC194, the Cm^R parent of pCS194. When the intact recombinant plasmids, isolated from his⁺ Ap^R Cm^RE. coli clones, have been used to transform B. subtilis cells for Cm^R, again deleted plasmids almost identical to pC194 have been obtained. The events causing these rearrangements occur after in vitro ligation, during either transformation or early propagation of the plasmids, and are probably caused by a translocatable element present in pCS194. A detailed physical map of pC194, carrying the restriction sites for HindIII, HaeIII, HpaII, MboII, AluI, HhaI, and BglI, is presented.     

Tn5 insertions in the agrocin 84 plasmid: the conjugal nature of pAgK84 and the locations of determinants for transfer and agrocin 84 production

The kanamycin-resistance transposon Tn5 was randomly introduced into pAgK84, a 47.7-kb plasmid coding for agrocin 84 production in Agrobacterium. Using such marked plasmids, pAgK84 was found to be conjugal. It could be transferred to several Agrobacterium strains including those harboring octopine- or nopaline-type Ti plasmids. Its presence has no effect on Ti plasmid functions such as opine utilization and tumorigenicity, but it does confer agrocin 84 immunity upon previously sensitive strains. The plasmid could also be conjugally transferred to a Nod+ Fix+ strain of Rhizobium meliloti. The production of agrocin 84 is expressed in all Agrobacterium and Rhizobium transconjugants tested. The agrocin plasmid could not be introduced into restrictionless Escherichia coli or Pseudomonas aeruginosa recipients by conjugation or transformation. The sites of 92 independent Tn5 insertions were mapped on pAgK84. These insertions are dispersed over the entire length of the plasmid. Analysis of the sites and effects of the Tn5 insertions has allowed us to construct a functional map of pAgK84. Forty-three of these insertions, spanning a 20-kb segment of the plasmid, abolished or greatly reduced the production of agrocin 84. The presence of two insertions within this segment having an effect on agrocin production suggests that at least three regions of the plasmid are involved in agrocin 84 biosynthesis. Fourteen of the Tn5 insertion derivatives are no longer conjugally transferable. These insertions all map to a single region of the plasmid and define about 3.5-kb as being associated with transfer functions.     

Human monocyte spreading induced by activated factor B of the complement alternative pathway: differential effects of Fab' and F(ab')2 antibody fragments directed to C5, C6, and C7

Human peripheral blood mononuclear phagocytes are induced by activated Factor B (Bb) of the complement alternative pathway to undergo morphological shape changes in vitro which have been described as spreading. The spreading reaction induced by Bb has previously been shown to depend upon the enzymatic activity of Bb and to be inhibited by Fab' antibody fragments directed to C5 (but not anti-C3 Fab'). The possibility that Bb may exert its effect on monocytes by initiating assembly of terminal complement complexes comprised of C5b, 6, 7, C5b-8, or C5b-9 was addressed in the present study. The effects were tested of Fab' and F(ab')2 antibody fragments directed to C5, C6, C7, and C8 and to neoantigens expressed in the assembling terminal complement complexes on the monocyte spreading reaction induced by Bb. Differential effects of monovalent Fab' and divalent F(ab')2 antibody fragments were observed. Anti-C5, C6, and C7 Fab' were found to inhibit the spreading reaction induced by Bb in an immunologically specific manner. Divalent F(ab')2 fragments directed to these same proteins (but not to C3, C4, C8, or C9) induced monocyte spreading in the complete absence of Bb or other recognized inducing agents. Monocyte spreading induced by hybridoma immunoglobulin (Ig) directed to C5 and C7 was found to be correlated with the binding of 10(6) molecules Ig per cell. These findings support the notion that C5, C6, and C7 (or an analogous system of cellular proteins) are associated with the surface of human peripheral blood monocytes and that these proteins may play a role in certain reactions by which mononuclear phagocytes are induced to altered states of cellular physiology.     

An expandable gene that encodes a Drosophila glue protein is not expressed in variants lacking remote upstream sequences

The Drosophila melanogaster gene Sgs4 encodes one of the glue polypeptides, sgs-4, synthesized in the larval salivary gland. We have examined the structure and expression of Sgs4 in five strains that produce abundant amounts of sgs-4 and its mRNA and in four that do not. The nonproducers include three Japanese strains that accumulate trace amounts of mRNA and one strain, BER-1, that contains no detectable Sgs4 RNA. Sgs4 carries a tandem array of repeated 21 bp elements within its coding sequence. The number of elements per array varies, causing considerable differences in the lengths of Sgs4 and its mRNA among the strains. These differences in length are not correlated with differences in mRNA abundance; rather, the low or zero abundance in nonproducers correlates with the loss of DNA upstream from the gene. The Japanese nonproducers carry a 52 bp deletion 305 bp upstream from the 5′ end of Sgs4, and BER-1 carries a 95 bp deletion 392 bp upstream. Curiously, each deletion encompasses one or more of the salivary-gland-specific DNAase I-hypersensitive sites which are known to flank the Sgs4 gene.     

A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants

Transposon-insertion mutants with vir^? Ti plasmids were characterized and then used in complementation experiments. One of the mutants (LBA 1517) had a mutation in a newly discovered vir locus called virF. The virF mutation led to a strongly diminished virulence on tomato and tobacco, but not on certain other plant species. Also a mutant (LBA 1505) was isolated with a mutation somewhere in the bacterial genome but outside the octopine Ti plasmid that caused a restriction in host range for tumor induction. Introduction of a nopaline Ti plasmid or an Ri plasmid into LBA 1505 did not restore normal virulence, showing that the vir gene affected in LBA 1505 determines a factor which is essential for normal tumor induction both by different types of Ti plasmids and by the Ri plasmid. The introduction of R primes containing part or all of the octopine Ti plasmid virulence region led to a restoration of virulence in strains with a vir^? nopaline Ti plasmid. Also the transfer of an Ri plasmid to a large number of different vir^? octopine or nopaline Ti plasmid mutants rendered these strains virulent. These results indicate that the octopine Ti plasmid, the nopaline Ti plasmid, and the Ri plasmid each have a similar virulence system which can mediate the transfer of T-DNA to plant cells from different types of Ti or Ri plasmids. In complementation experiments between vir^? octopine Ti plasmid mutations and vir^? nopaline Ti plasmid mutations it was found that equivalent functions are determined by the areas of DNA homology in the virulence regions of these two types of Ti plasmids. The previously defined octopine Ti plasmid virC locus appeared to consist of two different loci. One of these loci was found to be in a region of the octopine Ti plasmid which does not share DNA homology with the nopaline Ti plasmid, and was therefore called virO (octopine Ti plasmid specific). For the other locus the name virC was retained. Whereas mutations in the virC locus were avirulent on all plant species tested, mutations in virO were avirulent on tomato and pea, but virulent on sunflower and Nicotiana rustica. VirO^? mutants produced rooty tumors on Kalanchoë tubiflora.