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.     

Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes.

A genetic determinant essential for hemolysin production by Listeria monocytogenes has been inactivated by insertion of transposon Tn916 into L. monocytogenes DNA. The transposon was transferred by means of conjugation of a streptomycin-resistant L. monocytogenes recipient strain with Streptococcus faecalis CG110 on membrane filters. Among the tetracycline-resistant transconjugants, mutants were detected which had lost hemolytic activity. When tested in a mouse model, these mutants appeared to have lost the virulence that characterizes the parental strain. An extracellular protein of 58,000 apparent molecular weight was eliminated in the nonhemolytic mutants. In some of the mutants, the decrease in the production of the 58,000-dalton protein was accompanied by the production of a new protein of 49,000 apparent molecular weight. Hemolytic revertants regained the hemolytic phenotype and virulence and produced the extracellular protein that characterizes the recipient strain. Hybridization studies with Tn916 DNA indicated that the transposon is present in EcoRI and HindIII fragments of the nonhemolytic mutants. Single copies of Tn916 were detected in the chromosomal DNA of two of the three nonhemolytic mutants that were studied in detail. In hemolytic, tetracycline-sensitive revertants Tn916 appeared to be completely excised from the chromosome.     

Rhizobium leguminosarum exopolysaccharide mutants: biochemical and genetic analyses and symbiotic behavior on three hosts

Ten independently generated mutants of Rhizobium leguminosarum biovar phaseoli CFN42 isolated after Tn5 mutagenesis formed nonmucoid colonies on all agar media tested and lacked detectable production of the normal acidic exopolysaccharide in liquid culture. The mutants were classified into three groups. Three mutants harbored Tn5 insertions on a 3.6-kilobase-pair EcoRI fragment and were complemented to have normal exopolysaccharide production by cosmids that shared an EcoRI fragment of this size from the CFN42 genome. The Tn5 inserts of five other mutants appeared to be located on a second, slightly smaller EcoRI fragment. Attempts to complement mutants of this second group with cloned DNA were unsuccessful. The mutations of the other two mutants were located in apparently adjacent EcoRI fragments carried on two cosmids that complemented those two mutants. The latter two mutants also lacked O-antigen-containing lipopolysaccharides and induced underdeveloped nodules that lacked nitrogenase activity on bean plants. The other eight mutants had normal lipopolysaccharides and wild-type symbiotic proficiencies on bean plants. Mutants in each of these groups were mated with R. leguminosarum strains that nodulated peas (R. leguminosarum biovar viciae) or clovers (R. leguminosarum biovar trifolii). Transfer of the Tn5 mutations resulted in exopolysaccharide-deficient R. leguminosarum biovar viciae or R. leguminosarum biovar trifolii transconjugants that were symbiotically deficient in all cases. These results support earlier suggestions that successful symbiosis with peas or clovers requires that rhizobia be capable of acidic exopolysaccharide production, whereas symbiosis with beans does not have this requirement.     

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.     

Cloning and characterization of hydrogen uptake genes from Rhizobium leguminosarum.

A gene library of genomic DNA from the hydrogen uptake (Hup)-positive strain 128C53 of Rhizobium leguminosarum was constructed by using the broad-host-range mobilizable cosmid vector pLAFR1. The resulting recombinant cosmids contained insert DNA averaging 21 kilobase pairs (kb) in length. Two clones from the above gene library were identified by colony hybridization with DNA sequences from plasmid pHU1 containing hup genes of Bradyhizobium japonicum. The corresponding recombinant cosmids, pAL618 and pAL704, were isolated, and a region of about 28 kb containing the sequences homologous to B. japonicum hup-specific DNA was physically mapped. Further hybridization analysis with three fragments from pHU1 (5.9-kb HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) showed that the overall arrangement of the R. leguminosarum hup-specific region closely parallels that of B. japonicum. The presence of functional hup genes within the isolated cosmid DNA was demonstrated by site-directed Tn5 mutagenesis of the 128C53 genome and analysis of the Hup phenotype of the Tn5 insertion strains in symbiosis with peas. Transposon Tn5 insertions at six different sites spanning 11 kb of pAL618 completely suppressed the hydrogenase activity of the pea bacteroids.     

Biochemical and genetic analyses of acetoin catabolism in Alcaligenes eutrophus

In genetic studies on the catabolism of acetoin in Alcaligenes eutrophus, we used Tn5::mob-induced mutants which were impaired in the utilization of acetoin as the sole carbon source for growth. The transposon-harboring EcoRI restriction fragments from 17 acetoin-negative and slow-growing mutants (class 2a) and from six pleiotropic mutants of A. eutorphus, which were acetoin-negative and did not grow chemolithoautotrophically (class 2b), were cloned from pHC79 gene banks. The insertions of Tn5 were mapped on four different chromosomal EcoRI restriction fragments (A, C, D, and E) in class 2a mutants. The native DNA fragments were cloned from a lambda L47 or from a cosmid gene bank. Evidence is provided that fragments A (21 kilobase pairs [kb]) and C (7.7 kb) are closely linked in the genome; the insertions of Tn5 covered a region of approximately 5 kb. Physiological experiments revealed that this region encodes for acetoin:dichlorophenol-indophenol oxidoreductase, a fast-migrating protein, and probably for one additional protein that is as yet unknown. In mutants which were not completely impaired in growth on acetoin but which grew much slower and after a prolonged lag phase, fragments D (7.2 kb) and E (8.1 kb) were inactivated by insertion of Tn5::mob. No structural gene could be assigned to the D or E fragments. In class 2b mutants, insertions of Tn5 were mapped on fragment B (11.3 kb). This fragment complemented pleiotropic hno mutants in trans; these mutants were impaired in the formation of a rpoN-like protein. The expression of the gene cluster on fragments A and C seemed to be rpoN dependent.     

Insertional inactivation by Tn3701 of pIP964 hemolysin expression in Enterococcus faecalis

Abstract Transposition of the conjugative mobile element (Tn 3701 ) of Etreptococcus pyogenes strain A454 onto the Enterococcus feacalis hemolysin plasmid pIP964 modified the expression of the genes involved in hemolysin production. By hemolysis complementation tests the genes coding for two components required for the hemolytic activity expression were found to be located on the Eco RI E and G fragments of pIP964.     

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.     

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.     

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.     

Physical and genetic organization of the plasmid R15

The conjugative IncN plasmid R15 (SmrSurHgr, 62.3 kb) is cleaved by the hexanucleotide-specific endonucleases BglII, HindIII, EcoRI, BamHI, SmaI, SalI, PstI and XhoI into 9, 9, 6, 5, 4, 4, 4 and 2 fragments, respectively. The restriction sites were located on the physical map of the R15 genome. Distribution of the cleavage sites is strongly asymmetric. 28 of 32 sites for BamHI, EcoRI, HindIII, SalI, SmaI and PstI were located close to or within the sequences of transposable elements Tn2353 and Tn2354. According to the results of analysis of R15::Tn1756 deletion derivatives and recombinant plasmids harboring fragments of R15, the genetic determinants for resistance to Sm, Su and Hg were mapped, as well as the regions necessary for EcoRII restriction--modification and for plasmid replication and conjugation. The features of physical and genetic structures of R15 and other IncN plasmids are discussed.     

Regeneration of insertionally inactivated streptococcal DNA fragments after excision of transposon Tn916 in Escherichia coli: strategy for targeting and cloning of genes from gram-positive bacteria

The conjugative transposon Tn916 (15 kilobases), originally identified in Streptococcus faecalis DS16, has been cloned as an intact element on the pBR322-derived vector pGL101 in Escherichia coli. The EcoRI F' (EcoRI F::Tn916) fragment of pAM211 (pAD1::Tn916) was cloned into the single EcoRI site of pGL101 to form the chimera, pAM120, by selecting for the expression of Tn916-encoded tetracycline resistance (Tcr). Interestingly, in the absence of continued selection for Tcr, Tn916 excised from pAM120 at high frequency. This excision event resulted in a plasmid species consisting of the pGL101 vector and a 2.7-kilobase restriction fragment comigrating with the EcoRI F fragment of pAD1 during agarose gel electrophoresis. Filter blot hybridization experiments showed the 2.7-kilobase fragment generated as a result of Tn916 excision to be homologous with the EcoRI F fragment of pAD1. Analogous results were obtained with another chimera, pAM170, generated by ligating the EcoRI D' (EcoRI D::Tn916) fragment of pAM210 (pAD1::Tn916) to EcoRI-digested pGL101. Comparison of the AluI and RsaI cleavage patterns of the EcoRI F fragment isolated after Tn916 excision with those from an EcoRI F fragment derived from pAD1 failed to detect any difference in the two fragments: data in support of a precise Tn916 excision event in E. coli. Subcloning experiments showed that an intact transposon was required for Tn916 excision and located the Tcr determinant near the single HindIII site on Tn916. Although excision occurred with high frequency in E. coli, Tn916 insertion into the E. coli chromosome was a much rarer event. Tcr transformants were not obtained when pAM120 DNA was used to transform a polA1 strain, E. coli C2368.     

Molecular cloning of genes that specify virulence in Pseudomonas solanacearum.

The suicide plasmid pSUP2021 was used to introduce Tn5 into the Pseudomonas solanacearum wild-type strain K60. We isolated eight avirulent mutants after screening 6,000 kanamycin-resistant transconjugants by inoculating eggplant (Solanum melongena L. cv. Black Beauty) and tobacco (Nicotiana tabacum L. cv. Bottom Special) seedlings. The Tn5-containing EcoRI fragments from the eight mutants were unique, suggesting that numerous genes specify virulence in this species. These EcoRI fragments were cloned into pBR322 or pUC12, and one of the clones, pKD810, was transformed into K60. All of the kanamycin-resistant, ampicillin-sensitive transformants were avirulent. Three randomly selected avirulent transformants were shown to carry the Tn5-containing fragment in place of the wild-type fragment and to exhibit the same hybridization pattern as the original KD810 mutant did. With pKD810 as a probe, we identified cosmids carrying the wild-type virulence genes by using a genomic library of K60 prepared in pLAFR3. Two of the homologous cosmids, pL810A and pL810C, when introduced into KD810 by transformation, restored virulence and normal growth of this mutant in tobacco. Altogether, these data indicate that the gene(s) interrupted by Tn5 insertion in KD810 is essential for the virulence of P. solanacearum. Further characterization of this gene is now being completed by subcloning, transposon mutagenesis, and complementation analysis.     

Cloning of the Alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli.

Eight mutants of Alcaligenes eutrophus defective in the intracellular accumulation of poly-beta-hydroxybutyric acid (PHB) were isolated after transposon Tn5 mutagenesis with the suicide vector pSUP5011. EcoRI fragments which harbor Tn5-mob were isolated from pHC79 cosmid gene banks. One of them, PPT1, was used as a probe to detect the intact 12.5-kilobase-pair EcoRI fragment PP1 in a lambda L47 gene bank of A. eutrophus genomic DNA. In six of these mutants (PSI, API, GPI, GPIV, GPV, and GPVI) the insertion of Tn5-mob was physically mapped within a region of approximately 1.2 kilobase pairs in PP1; in mutant API, cointegration of vector DNA has occurred. In two other mutants (GPII and GPIII), most probably only the insertion element had inserted into PP1. All PHB-negative mutants were completely impaired in the formation of active PHB synthase, which was measured by a radiometric assay. In addition, activities of beta-ketothiolase and of NADPH-dependent acetoacetyl coenzyme A (acetoacetyl-CoA) reductase were diminished, whereas the activity of NADPH-dependent acetoacetyl-CoA reductase was unaffected. In all PHB-negative mutants the ability to accumulate PHB was restored upon complementation in trans with PP1. The PHB-synthetic pathway of A. eutrophus was heterologously expressed in Escherichia coli. Recombinant strains of E. coli JM83 and K-12, which harbor pUC9-1::PP1, pSUP202::PP1, or pVK101::PP1, accumulated PHB up to 30% of the cellular dry weight. Crude extracts of these cells had significant activities of the enzymes PHB synthase, beta-ketothiolase, and NADPH-dependent acetoacetyl-CoA reductase. Therefore, PP1 most probably encodes all three genes of the PHB-synthetic pathway in A. eutrophus. In addition to PHB-negative mutants, we isolated mutants which accumulate PHB at a much lower rate than the wild type does. These PHB-leaky mutants exhibited activities of all three PHB-synthetic enzymes; Tn5-mob had not inserted into PP1, and the phenotype of the wild type could not be restored with fragment PP1. The rationale for this mutant type remains unknown.     

The capsule polysaccharide synthesis locus of streptococcus pneumoniae serotype 14: Identification of the glycosyl transferase gene cps14E.

To identify a chromosomal region of Streptococcus pneumoniae serotype 14 involved in capsule polysaccharide synthesis, two strategies were used: (i) Tn916 mutagenesis, followed by the characterization of four unencapsulated mutants, and (ii) cross-hybridization with a capsule polysaccharide synthesis gene (cps) probe from S. agalactiae, which has a structurally similar capsule. The two approaches detected the same chromosomal region consisting of two adjacent EcoRI fragments. One of these EcoRI fragments was cloned and hybridized with a cosmid library. This resulted in clone cMKO2. A similar cosmid clone was obtained from an unencapsulated Tn916 mutant, Spnl4.H. Sequence analysis of the two cosmid clones revealed that in the Tn916 mutant, a gene, cps14E, which is homologous to other bacterial genes encoding glycosyl transferases, had been inactivated. An open reading frame immediately downstream of cps14E, designated cps14F, shows no significant homology with any known genes or proteins. A functional assay showed that cps14E encodes a glycosyl transferase and that a gene-specific knockout mutant lacks this enzyme activity, whereas inactivation of cps14F does not have this effect.     

Isolation and characterization of Rhizobium (IC3342) genes that determine leaf curl induction in pigeon pea.

Nodulation by the Rhizobium strain IC3342 causes a leaf curl syndrome in certain tropical legumes such as pigeon pea (Cajanus cajan) (N.M. Upadhyaya, J.V.D.K. Kumar Rao, D.S. Letham, and P.J. Dart, Physiological and Molecular Plant Pathology 39:357-373, 1991). Transposon (Tn5) mutagenesis of this leaf curl-inducing (Curl+) Rhizobium strain yielded two Curl- Fix- and three Curl- Fix+ mutants. Plasmid visualization and subsequent Southern blot hybridization analyses with Tn5, nif and nod gene probes showed that the Tn5 element had inserted into the symbiotic (Sym) plasmid in three of the mutants. Restriction endonuclease analyses indicated that none of the Tn5 insertions were closely linked. Tn5-containing EcoRI fragments were cloned from each mutant and used as probes to isolate the corresponding wild-type DNA fragments from a cosmid (pLAFR3) genomic library. Fix+ and/or Curl+ phenotypes were restored in each mutant by the introduction of cosmids containing the corresponding wild-type DNA. A closely related but Curl- Rhizobium strain ANU240 was shown, by Southern hybridization, to contain conserved DNA sequences of all but one of the identified genetic regions of the Curl+ Rhizobium strain IC3342. Cosmids containing the genetic region unique to the strain IC3342, designated lcr1, conferred a Curl+ phenotype on the strain ANU240. DNA sequence analysis of the cloned lcr1 region revealed five open reading frames (ORFs). The ORF2 showed homology with the Escherichia coli regulatory gene ompR, and ORF4 showed homology with E. coli and Rhizobium meliloti regulatory genes fnr and fixK, respectively.     

Transposon-induced non-motile mutants of Vibrio cholerae

Non-motile mutants of Vibrio cholerae were isolated after transposon insertion mutagenesis with either Tn5 on a plasmid or Tn10ptac mini-kan in bacteriophage lambda. The physical location and number of transposon insertions was determined. Eighteen Tn5 insertion mutants and 11 Tn10ptac mini-kan insertion mutants had single unique insertion sites. The 18 Tn5 insertions were contained within six different EcoRI fragments and the 11 Tn10ptac mini-kan insertions were contained within eight different fragments of V. cholerae chromosomal DNA. These data suggest that multiple genes are involved in motility. Immunoblot analysis of non-motile mutants with antibody to wild-type flagellar core protein indicated that two of the non-motile mutants made flagellar core protein. Three additional mutants reacted weakly with the antibodies. However, these mutants with immunopositive reactions did not produce any structures which resembled flagella by transmission electron microscopy. In addition, none of the other non-motile mutants produced wild-type flagella. However, five mutants which did not react in the immunoblot produced a structure which resembled a flagellar sheath without the internal flagellar core. In addition to having no filamentous core, the sheaths often extended from the sides of the bacteria, rather than from the poles where the flagellum is normally located. The data suggest that sheath formation is independent of flagellar filament formation, but that proper positioning of the sheath may require the flagellar filament.     

Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within HupRhizobium leguminosarum Strains

Thirteen Rhizobium leguminosarum strains previously reported as H(2)-uptake hydrogenase positive (Hup) or negative (Hup) were analyzed for the presence and conservation of DNA sequences homologous to cloned Bradyrhizobium japonicum hup-specific DNA from cosmid pHU1 (M. A. Cantrell, R. A. Haugland, and H. J. Evans, Proc. Natl. Acad. Sci. USA 80:181-185, 1983). The Hup phenotype of these strains was reexamined by determining hydrogenase activity induced in bacteroids from pea nodules. Five strains, including H(2) oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H(2)-uptake hydrogenase activity and contained DNA sequences homologous to three probe DNA fragments (5.9-kilobase [kb] HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) from pHU1. The pattern of genomic DNA HindIII and EcoRI fragments with significant homology to each of the three probes was identical in all five strains regardless of the H(2)-dependent ATP generation trait. The restriction fragments containing the homology totalled about 22 kb of DNA common to the five strains. In all instances the putative hup sequences were located on a plasmid that also contained nif genes. The molecular sizes of the identified hup-sym plasmids ranged between 184 and 212 megadaltons. No common DNA sequences homologous to B. japonicum hup DNA were found in genomic DNA from any of the eight remaining strains showing no significant hydrogenase activity in pea bacteroids. These results suggest that the identified DNA region contains genes essential for hydrogenase activity in R. leguminosarum and that its organization is highly conserved within Hup strains in this symbiotic species.