Identification of the pifC gene and its role in negative control of F factor pif gene expression

The pif region of the F factor includes two genes, pifA and pifB, that lead to abortive T7 infection. We have identified a new gene in this region, pifC, by constructing an in vitro fusion of pif DNA at 41.6 kilobases on the F factor physical map to the lacZ gene. A PifC-LacZ fusion protein of 149,000 daltons has been identified by immunoprecipitation and polyacrylamide gel electrophoresis. This allows us to assign the N terminus of pifC to 42.5 kilobases on the F map. Using fusions of pifC, pifA, and pifB to lacZ, we have studied the regulation of pif gene expression and have shown that the product of pifC negatively controls its own expression and that of pifA and pifB.     

Isolation and characterization of Tn5 insertion mutants of Pseudomonas syringae pv. syringae altered in the production of the peptide phytotoxin syringotoxin.

A syringotoxin-producing strain of Pseudomonas syringae pv. syringae (B457) was subjected to Tn5 mutagenesis by the transposon vector pSUP1011. Analyses of auxotrophs obtained suggested simple random insertions of Tn5. Syringotoxin-negative mutants arose at a frequency of about 0.28%. In a Southern blot analysis, the loss of toxin production was associated with Tn5 insertions into chromosomal EcoRI fragments of about 10.5, 17.8, and 19.3 kilobases. Data from a Southern blot analysis of SstI-digested DNA from these mutants suggest that the 10.5- and 17.8-kilobase EcoRI fragments may be adjacent to or near each other. Mutants that produced only 3 to 4% wild-type toxin levels also were identified.     

Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region.

Rhodobacter capsulatus mutants unable to fix nitrogen were isolated by random transposon Tn5 mutagenesis. The Tn5 insertion sites of 30 Nif- mutants were mapped within three unlinked chromosomal regions designated A, B, and C. The majority of Tn5 insertions (21 mutants) map within nif region A, characterized by two ClaI fragments of 2.5 and 25 kilobases (kb). The 17-kb ClaI fragment of nif region B contains six nif::Tn5 insertions, and the three remaining mutations are located on a 32-kb ClaI fragment of nif region C. Hybridization experiments using all 17 Klebsiella pneumoniae nif genes individually as probes revealed homology to nifE, nifS, nifA, and nifB in nif region A. The nifHDK genes were localized in nif region B. About 2 kb away from this operon, a second copy of the DNA fragments homologous to nifA and nifB, originally found in nif region A, was identified.     

Synthesis of human insulin gene. VIII. Construction of expression vectors for fused proinsulin production in Escherichia coli

Analysis of Tn1725 insertions in the Pif+ plasmid pRS2496 showed the maximum limits of the F pif region to be between 43.7 and 47.15 on the 100-kb map of the F plasmid. The effect of these insertions on the expression of pif polypeptides indicated that two of the pif genes, pifA and pifC, lie within a polycistronic operon.     

Regulation of the F-factor pif operon: pifO, a site required in cis for autoregulation, titrates the pifC product in trans.

F factor pifC, pifA, and pifB gene expression is subject to negative regulation by the product of the pifC locus (J.F. Miller and M. H. Malamy, J. Bacteriol. 156:338-347, 1983). In this paper, we describe the properties of a new regulatory site in the pif region, pifO, which is required in cis for autoregulation of pif gene expression. Spontaneous pifO mutations were isolated that allow expression of a pifC-lacZ protein fusion in the presence of pifC product in trans. Recombination of these pifO mutations onto F'lac results in increased pifA and pifB activity. Thus, a single regulatory element, pifO, regulates pifC, pifA, and pifB expression in cis. The presence of multiple copies of a fragment from the pif region carrying wild-type pifO sequences (F coordinates 42.9 to 42.43 kilobases) in trans to F'lac results in an increase in pifA and pifB activity as measured by inhibition of T7 plating. When the pifO mutations are recombined onto a plasmid carrying pifO, the resulting recombinants are greatly decreased in the ability to increase F'lac pif expression. These results suggest that increased F'lac pifA and pifB expression caused by pifO sequences in trans is a consequence of titration of pifC product and derepression of the pif operon.     

Gene cluster of Pseudomonas syringae pv. "phaseolicola" controls pathogenicity of bean plants and hypersensitivity of nonhost plants

Loss of the ability of Pseudomonas syringae pv. "phaseolicola" NPS3121 to elicit a hypersensitive response on tobacco and other nonhost plants was associated with loss of pathogenicity on the susceptible host bean. Eight independent, prototrophic transposon Tn5 insertion mutants which had lost the ability to elicit a hypersensitive response on tobacco plants were identified. Six of these mutants no longer produced disease lesions on primary leaves of the susceptible bean cultivar Red Kidney and failed to elicit a hypersensitive response on the resistant bean cultivar Red Mexican and on the nonhost plants tomato, cowpea, and soybean. The two remaining mutants had reduced pathogenicity on Red Kidney bean and elicited variable hypersensitive responses on the other plants tested. Southern blot analysis indicated that each mutant carried a single independent Tn5 insertion in one of three EcoRI fragments of about 17, 7, and 5 kilobases. Marker exchange mutagenesis further supported the conclusion that the pleiotropic mutant phenotype was not associated with multiple Tn5 insertions. A genomic library of the wild-type strain was constructed in the cosmid vector pLAFR3. A recombinant plasmid, designated pPL6, that carried P. syringae pv. "phaseolicola" genomic sequences was identified by colony hybridization. This plasmid restored the wild-type phenotype to all but one mutant, suggesting that genes affected by the insertions were clustered. Structural analysis of pPL6 and the wild-type genome indicated that the 17- and 5-kilobase EcoRI fragments were contiguous in the strain NPS3121 genome.     

Isolation and characterization of pathogenicity genes of Pseudomonas syringae pv. tabaci.

Pseudomonas syringae pv. tabaci BR2 produces tabtoxin and causes wildfire disease on tobacco and bean plants. Approximately 2,700 Tn5 insertion mutants of a plasmid-free strain, PTBR 2.024, were generated by using suicide plasmid pGS9. Of these Tn5 mutants, 8 were no longer pathogenic on tobacco plants and 10 showed reduced symptoms. All of the eight nonpathogenic mutants caused typical wildfire disease symptoms on bean plants. Two of the nonpathogenic mutants failed to produce tabtoxin. The eight nonpathogenic mutants have Tn5 insertions into different EcoRI and SalI restriction fragments. The EcoRI fragments containing Tn5 from the eight nonpathogenic mutants were cloned into vector pTZ18R or pLAFR3. A genomic library of the parent strain was constructed in the broad-host-range cosmid pLAFR3. Three different cosmid clones that hybridized to the cloned Tn5-containing fragment from one of the nonpathogenic mutants, PTBR 4.000, were isolated from the genomic library. These clones contained six contiguous EcoRI fragments (a total of 57 kilobases [kb]). A 7.2-kb EcoRI fragment common to all three restored pathogenicity to mutant PTBR 4.000. None of the six EcoRI fragments hybridized to Tn5-containing fragments from the other seven mutants. The 7.2-kb fragment was conserved in P. syringae pv. tabaci and P. syringae pv. angulata, but not in other pathovars or strains. Because the mutants retained pathogenicity on bean plants and because of the conservation of the 7.2-kb EcoRI fragment only in pathovars of tobacco, we suggest that genes on the fragment might be related to host specificity.     

Restriction nuclease and enzymatic analysis of transposon-induced mutations of the Rac prophage which affect expression and function of recE in Escherichia coli K-12.

Fourteen Tn5-generated mutations of the Rac prophage, called sbc because they suppress recB21 recC22, were found to fall into two distinct types: type I mutations, which were insertions of Tn5, and type II mutations, which were insertions of IS50. Both orientations of Tn5 and IS50 were represented among the mutants and were arbitrarily labeled A and B. All 14 of the Tn5 and IS50 insertions occurred in the same location (+/- 100 base pairs) approximately 5.6 kilobases from one of the hybrid attachment sites. Eleven of the mutants contained essentially the same amount of exonuclease VIII, the product of recE. The possibility that a promoter for recE was created by the insertion of Tn5 and IS50 was considered. Two IS50 mutants in which such a promoter could not have been created showed three to four times as much exonuclease VIII, and another showed one-half as much as the majority. The possibility was considered that a promoter internal to IS50 is responsible for this heterogeneity. Restriction alleviation was measured in all 14 mutants. An insertion of the transposon Tn10 which reduces expression of exonuclease VIII (recE101::Tn10) was located within the Rac prophage at a position 2.35 kilobases from the left hybrid attachment site. Location and orientation of the Rac prophage on the Escherichia coli genetic map are discussed in light of these results.     

Transfer functions of the Streptococcus faecalis plasmid pAD1: organization of plasmid DNA encoding response to sex pheromone.

The conjugative plasmid pAD1 (59.6 kilobases) of Streptococcus faecalis shows a 10,000-fold increase in transfer frequency following induction by the sex pheromone cAD1. Mutagenesis of the plasmid with transposon Tn917 was undertaken to determine the region(s) of pAD1 required for the mating response. The relevant genetic material was found to be distributed over a 31.2-kilobase contiguous region of the plasmid. Although insertions in two previously identified regions (traA and traB) exhibited increased transfer frequencies, insertions in five new regions (D, E, F, G, and H) decreased the ability of pAD1 to transfer. Insertions in region H allowed the cells to form visible mating aggregates, but the plasmid transfer frequency was decreased to levels below detection during a 1-h broth mating. Mutants with mutations in region G were able to form aggregates; however, insertions in regions D, E, and F prevented aggregate formation. Insertions in region C decreased the sensitivity of the cell to exogenous cAD1 and exhibited increased activity of the pheromone inhibitor iAD1. Surface protein profiles produced by a number of these mutants were examined, and in some cases were found to be different from those of the wild type. A map showing the various regions is presented, and related aspects of the regulation of the pAD1 mating response are discussed.     

Identification and cloning of genes involved in phaseolotoxin production by Pseudomonas syringae pv. "phaseolicola"

Genes involved in the production of phaseolotoxin by Pseudomonas syringae pv. "phaseolicola" NPS3121 were identified by Tn5 mutagenesis and cosmid cloning. A total of 5,180 kanamycin-resistant colonies were screened for the loss of phaseolotoxin production by a microbiological assay. Six independent, prototrophic, Tox- mutants were isolated that had Tn5 insertions in five different EcoRI fragments. All six mutants had Tn5 inserted in the same KpnI fragment, which had a length of ca. 28 kilobases including Tn5. The mutants produced residual toxin in vitro. An EcoRI fragment containing Tn5 and flanking sequences from mutant NPS4336 was cloned and used to probe a wild-type genomic library by colony hybridization. Seven recombinant plasmids showing homology to this probe were identified. Each Tox- mutant was restored in OCTase-specific toxin production by two or more of the recombinant plasmids. The data suggest that at least some of the genes involved in phaseolotoxin production were clustered in a large KpnI fragment. No homology was detected between the Tn5 target fragment cloned from mutant NPS4336 and the total genomic DNA from closely or distantly related bacteria that do not produce phaseolotoxin.     

Isolation of Zoogloea ramigera I-16-M exopolysaccharide biosynthetic genes and evidence for instability within this region.

The genetics of the biosynthesis of an exocellular polysaccharide (EPS) from Zoogloea ramigera I-16-M is being investigated. Tn5 insertion mutants deficient in EPS production were isolated by screening for the absence of fluorescence on plates containing the dye Cellufluor (Polysciences Chemicals). Complementation of these mutations was achieved with a Z. ramigera I-16-M gene library constructed in a broad-host-range cosmid vector and introduced into the I-16-M mutants by conjugation. Four recombinant plasmids able to restore EPS production to all of these mutants were found to contain at least 14 kilobases of common insert DNA. Subcloning of the common region and restriction mapping the locations of Tn5 insertions have identified two complementation groups contained within a chromosomal segment of DNA that is between 4.6 and 6.5 kilobases in size. We have clearly demonstrated genetic instability in this region which leads to spontaneous deletions and possibly rearrangements resulting in the loss of EPS production.     

Cloning and expression of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli.

The transport of cyanocobalamin (vitamin B12) in cells of Escherichia coli is dependent on a receptor protein (BtuB protein) located in the outer membrane. A 9.1-kilobase pair BamHI fragment carrying the btuB gene was cloned from a specialized transducing phage into multicopy plasmids. Insertions of transposon Tn1000 which prevented production of the receptor localized btuB to a 2-kilobase pair region. Further subcloning allowed isolation of this region as a 2.3-kilobase pair Sau3A fragment. The BtuB+ plasmids were shown by maxicell analysis to encode a polypeptide with a molecular weight of 66,000 in the outer membrane. This polypeptide was missing in cells with Tn1000 insertions in btuB and was reduced in amount upon growth of plasmid-bearing cells in repressing concentrations of vitamin B12. Several Tn1000 insertions outside the 5' end of the coding region exhibited reduced production of receptor. A deletion at the 3' end of btuB resulted in formation of an altered receptor. Amplified production of this polypeptide was associated with increased levels of binding of the receptor's ligands (vitamin B12 and phage BF23), increased rates of vitamin B12 uptake, and altered susceptibility to the group E colicins. Deficiency in various major outer membrane proteins did not affect production of the btuB product, and the amplified levels of this protein partially reversed the tolerance to E colicins seen in these mutants.     

Physical mapping of transposon Tn5 insertions defines a gene cluster functional in nitrous oxide respiration by Pseudomonas stutzeri.

By transposon Tn5 mutagenesis, 19 strains of Pseudomonas stutzeri were acquired that had defects in nitrous oxide respiration (Nos- phenotype). A physical map of the mutants showed nearly random Tn5 insertions into genomic DNA within a single region ca. 8 kilobases long. Mutants were characterized immunochemically, enzymatically, and chemically. Several functions related to the synthesis and regulation of nitrous oxide reductase were associated with this DNA region, indicating that in P. stutzeri part of the genetic information necessary to respire nitrous oxide is clustered.     

Genetic analysis of the pAD1 pheromone response in Streptococcus faecalis, using transposon Tn917 as an insertional mutagen

The conjugative plasmid pAD1 (56.7 kilobases) in Streptococcus faecalis has been shown to confer a mating response to the sex pheromone cAD1 excreted by recipient strains. The response is characterized by the synthesis of a proteinaceous adhesin which coats the surface of the pAD1 -containing donor cell and facilitates the formation of mating aggregates. Donors exposed to cAD1 -containing filtrates of recipients undergo self-aggregation (clumping), an event believed to be associated with an interaction between the adhesin and a binding substance always present on the surface of both recipients and donors. To analyze the molecular processes involved in the mating response, mutants were generated by the erythromycin resistance transposon Tn917 . Transpositions to pAD1 in S. faecalis DS16 gave rise to a number of derivatives that exhibited "constitutive clumping" and the ability to transfer at high frequencies in short (10-min) matings. These mutants fell into two subclasses, which exhibited colony morphologies that were "dry" or "normal". The Tn917 insertions were mapped by restriction enzyme analysis to two separate clusters, designated traA and traB. The dry colony subclass corresponded to traA and represented a span of 1.5 kilobases, whereas the normal subclass corresponded to traB and spanned 1.3 kilobases. The two clusters were separated by 1.7 kilobases in which insertions of Tn917 did not affect the ability to respond normally to cAD1 . Neither type of constitutive clumper produced cAD1 . Another series of insertions exhibited reduced donor potential. In two cases, the reduction in transfer was three to four orders of magnitude; these mapped in traA . In two other cases, the reduction was one to two orders of magnitude. These mapped outside of traA and traB, and one was associated with an increase in plasmid copy number.     

chlC (nar) operon of Escherichia coli includes structural genes for alpha and beta subunits of nitrate reductase.

The synthesis of the alpha and beta subunits of nitrate reductase by 20 chlC::Tn5 insertion mutants of Escherichia coli was determined by immune precipitation of the subunits from fractions of cell extracts. Only two of the mutants produced either subunit in detectable amounts; these two accumulated the alpha subunit, but no beta subunit. In both cases the alpha subunit was present in the cytosolic fraction, in contrast to wild-type cells, in which both subunits are present mainly in the membrane fraction. EcoRI restriction fragments containing the Tn5 inserts from five of the mutants were cloned into pBR322. The insertions were localized on two contiguous EcoRI fragments spanning a 5.6-kilobase region that overlapped the contiguous ends of the two fragments. An insertion that permitted alpha subunit formation defined one end of the 5.6-kilobase region. The results indicated that the genes encoding the alpha and beta subunits of nitrate reductase were part of a chlC (nar) operon that is transcribed in the direction alpha leads to beta.