Single Transposon Insertion Research Articles

Genome‐Wide Transposon Mutagenesis in Yeast

This unit provides comprehensive protocols for the use of insertional libraries generated by shuttle mutagenesis. From the basic protocol, a small aliquot of insertional library DNA may be used to mutagenize yeast, producing strains containing a single transposon insertion within a transcribed and translated region of the genome. This transposon-mutagenized bank of yeast strains may be screened for any desired mutant phenotype. Alternatively, since the transposon contains a reporter gene lacking its start codon and promoter, transposon-tagged strains may also be screened for specific patterns of gene expression. Strains of interest may be characterized by vectorette PCR (protocol provided) in order to locate the precise genomic site of transposon insertion within each mutant. A method by which Cre/lox recombination may be used to reduce the transposon in yeast to a small insertion element encoding an epitope tag is described. This tag serves as a tool by which transposon-mutagenized gene products may be analyzed further (e.g., localized to a discrete subcellular site).

Identification of Pasteurella multocida virulence genes in a septicemic mouse model using signature-tagged mutagenesis

P. multocida is the causative agent of several economically significant veterinary diseases occurring in numerous species worldwide. Signature-tagged mutagenesis (STM) is a powerful genetic technique used to simultaneously screen multiple transposon mutants of a pathogen for their inability to survive in vivo. We have designed an STM system based on a mini-Tn10 transposon, chemiluminescent detection and semi-quantitative analysis and have identified transposon insertions into genes of Pasteurella multocida that attenuate virulence in a septicemic mouse model. A bank of 96 transposons containing strongly-hybridizing tags was used to create 19 pools of P. multocida transposon mutants containing approximately 70–90 mutants/pool. A total of 62 mutants were attenuated when checked individually, and 25 unique single transposon insertion mutations were identified from this group. The sequence of the disrupted ORF for each attenuated mutant was determined by either cloning or PCR-amplifying and sequencing the flanking regions. The attenuated mutants contained transposon insertions in genes encoding biosynthetic enzymes, virulence factors, regulatory components and unknown functions. This study should contribute to an understanding of the pathogenic mechanisms by which P. multocida and other pathogens in thePasteurellaceae family cause disease and identify novel live vaccine candidates and new potential antibiotic targets.

Inactivation of a novel gene produces a phenotypic variant cell and affects the symbiotic behavior of Xenorhabdus nematophilus.

Xenorhabdus nematophilus is an insect pathogen that lives in a symbiotic association with a specific entomopathogenic nematode. During prolonged culturing, variant cells arise that are deficient in numerous properties. To understand the genetic mechanism underlying variant cell formation, a transposon mutagenesis approach was taken. Three phenotypically similar variant strains of X. nematophilus, each of which contained a single transposon insertion, were isolated. The insertions occurred at different locations in the chromosome. The variant strain, ANV2, was further characterized. It was deficient in several properties, including the ability to produce antibiotics and the stationary-phase-induced outer membrane protein, OpnB. Unlike wild-type cells, ANV2 produced lecithinase. The emergence of ANV2 from the nematode host was delayed relative to the emergence of the parental strain. The transposon in ANV2 had inserted in a gene designated var1, which encodes a novel protein composed of 121 amino acid residues. Complementation analysis confirmed that the pleiotropic phenotype of the ANV2 strain was produced by inactivation of var1. Other variant strains were not complemented by var1. These results indicate that inactivation of a single gene was sufficient to promote variant cell formation in X. nematophilus and that disruption of genetic loci other than var1 can result in the same pleiotropic phenotype.

Active detachment of Streptococcus mutans cells adhered to epon–hydroxylapatite surfaces coated with salivary proteins in vitro

Although the formation of biofilms has been much studied, detachment of adherent cells from biofilms has been relatively neglected. Recent results have shown that adherent Streptococcus mutans cells can actively detach from epon–hydroxylapatite (EHA) rods conditioned with hog gastric mucin. The mechanisms for adherence and detachment of Strep. mutans cells in this system was uncertain. In the present study, resting Strep. mutans cells were used to form a simple monolayer on EHA rods coated with saliva and salivary agglutinin (SAG). Preliminary experiments defined the variables for conditioning EHA with saliva and SAG and establishing the adherence of Strep. mutans to the conditioned surfaces. The results showed that salivary proteins including SAG adsorbed rapidly to EHA and that a relatively stable Strep. mutans NG8 monolayer was formed within 60 min of incubation. The monolayers were subsequently used for detachment studies. The results showed that adherent Strep. mutans cells detached in a temperature-dependent manner and responded to the addition of a preparation of surface protein-releasing enzyme (SPRE) obtained from Strep. mutans in a dose-dependent fashion. The effect of the exogenous SPRE on detachment could be abrogated by pronase treatment. Two putative SPRE-defective mutants (A and E) were generated by Tn 917 mutagenesis. Both mutants possessed a single transposon insertion as demonstrated by Southern hybridization and appeared to be different from one another based on the hybridization patterns. Mutant A displayed an increased quantity of cell-surface antigen P1, an adhesin that interacts with SAG. At the same time mutant A was unable to release P1 and other high molecular-weight proteins from the cell surface. Mutant A detached at a significantly lower rate (21%) than the parent strain (37%) ( p=0.05). SPRE prepared from mutant A was unable to release Strep. mutans NG8 adherent cells as compared to SPRE obtained from the wild-type cells. Collectively, these results suggest that the detachment of Strep. mutans adherent cells formed on salivary protein-coated EHA was an active process mediated by the action of SPRE.

Importance of pfkA for rapid growth of Enterobacter cloacae during colonization of crop seeds.

Enterobacter cloacae A-11 is a prototrophic, glycolytic mutant of strain 501R3 with a single transposon insertion in pfkA. The populations of strain A-11 on cucumber and radish seeds were smaller than the populations of strain 501R3 in natural soil, but the populations of these two strains on pea, soybean, sunflower, and sweet corn seeds were similar (D. P. Roberts, P. D. Dery, I. Yucel, J. Buyer, M. A. Holtman, and D. Y. Kobayashi, Appl. Environ. Microbiol. 65:2513-2519, 1999). The net effect of the mutation in pfkA in vitro was a shift from rapid growth on certain carbohydrates detected in seed exudates to much slower growth on other carbohydrates, amino acids, and organic acids. The impact of the mutation in pfkA was greatest on the growth rate of E. cloacae on the seeds that released the smallest quantities of fructose, other carbohydrates, and amino acids. Corn, pea, soybean, and sunflower seeds released total amounts of carbohydrates and amino acids at rates that were approximately 10- to 100-fold greater than the rates observed with cucumber and radish seeds for the first 24 h after inhibition began. The growth rate of strain A-11 was significantly less (50% less) than the growth rate of strain 501R3 on radish seeds, and the growth rate of strain A-11 was too low to estimate on cucumber seeds in sterile sand for the first 24 h after inhibition began. The growth rate of strain A-11 was also significantly lower on soybean seeds, but it was only 17% lower than the growth rate of strain 501R3. The growth rates of strains 501R3 and A-11 were similar on pea, sunflower, and corn seeds in sterile sand for the first 30 h after imbibition began. Large reductions in the growth rates of strain A-11 on seeds were correlated with subsequent decreased levels of colonization of seeds compared to the levels of colonization of strain 501R3. The strain A-11 populations were significantly smaller than the strain 501R3 populations only on radish and cucumber seeds. The mutation in pfkA appears to decrease the level of colonization by E. cloacae for seeds that release small quantities of reduced carbon compounds by decreasing the size of the pool of compounds that support rapid growth by this bacterium.

Identification of a pathogenicity island, which contains genes for virulence and avirulence, on a large native plasmid in the bean pathogen Pseudomonas syringae pathovar phaseolicola.

The 154-kb plasmid was cured from race 7 strain 1449B of the phytopathogen Pseudomonas syringae pv. phaseolicola (Pph). Cured strains lost virulence toward bean, causing the hypersensitive reaction in previously susceptible cultivars. Restoration of virulence was achieved by complementation with cosmid clones spanning a 30-kb region of the plasmid that contained previously identified avirulence (avr) genes avrD, avrPphC, and avrPphF. Single transposon insertions at multiple sites (including one located in avrPphF) abolished restoration of virulence by genomic clones. Sequencing 11 kb of the complementing region identified three potential virulence (vir) genes that were predicted to encode hydrophilic proteins and shared the hrp-box promoter motif indicating regulation by HrpL. One gene achieved partial restoration of virulence when cloned on its own and therefore was designated virPphA as the first (A) gene from Pph to be identified for virulence function. In soybean, virPphA acted as an avr gene controlling expression of a rapid cultivar-specific hypersensitive reaction. Sequencing also revealed the presence of homologs of the insertion sequence IS100 from Yersinia and transposase Tn501 from P. aeruginosa. The proximity of several avr and vir genes together with mobile elements, as well as G+C content significantly lower than that expected for P. syringae, indicates that we have located a plasmid-borne pathogenicity island equivalent to those found in mammalian pathogens.

Targeting and retrofitting pre-existing libraries of transposon insertions with FRT and oriV elements for in-vivo generation of large quantities of any genomic fragment.

A procedure is described that converts the pre-existing transposon insertion libraries to a collection of 'pop-out' strains, each allowing generation of 20- to 100-kb genomic fragments directly from the genome. The procedure consists of two steps: (1) single transposon insertions are targeted and retrofitted with excision and amplification elements (FRT and oriV), by homologous recombination with an FRT-oriV-carrying plasmid; and (2) two retrofitted neighbouring transposons are brought together by P1 transduction. From each strain, a 20- to 100-kb genomic fragment, bound by a pair of retrofitted transposons, could be excised and amplified upon supplying in trans the excision (Flp) and replication (TrfA) functions. To enhance the efficiency of crossing-in the FRT-oriV cassette, we transiently increased the copy number of our retrofitting plasmids using a temperature-sensitive TrfA-supplying helper plasmid. Using FRT-oriV and helper plasmids, we retrofitted four Tn10KmR and three Tn10CmR insertions. Subsequently, the FRT-oriV retrofitted insertions were crossed with each other in pairs (KmRxCmR), using P1 phage transductions. The resulting CmRFRT-[28-65-kb]-KmRFRT strains were transformed with a plasmid expressing FLP and trfA genes from the tightly controlled Ptet promoter. Induction of this tightly repressed promoter by autoclaved chlortetracycline (cTc) resulted in the efficient excision and amplification of genomic fragments located between FRT sites, but only in productive strains, i.e. having two parallel FRTs. We have shown that genomic fragments of 28-, 40-, 50- and 65-kb were efficiently excised and amplified. Furthermore, we could convert non-productive strains (having FRTs in non-parallel orientation), to productive combination of parallel FRTs, because one of the FRT elements was flanked by two convergent loxP sites, and thus could be inverted by the Cre function delivered either by the P1 phage or by a specially constructed temperature-sensitive Plac-cre plasmid. Although several microbial genomes were recently sequenced, the described method will help in supplying large quantities of any genomic fragment (prepared without the conventional cloning and its artifacts) for refined sequence comparison among strains and species, and for further analysis of uncharacterized ORFs, various mutations, and regulatory elements or functions. The excised and circularized DNA fragments (plasmids) could be propagated like any other large plasmids but only in hosts that could supply the appropriate Rep function. Our original 'pop-out' method [Pósfai et al. (1994) Nucleic Acids Res. 22, 2392-2398] was already employed for sequencing of the E. coli genome [Blattner et al. (1997) Science 277, 1453-1462]. Moreover, the Flp-mediated recombination between two FRT elements resulted in bacterial strains with large deletions (for parallel FRT orientations) or with large inversions (for inverted FRT orientations).

Isolation and characterization of transposon-induced mutants ofPorphyromonas gingivalisdeficient in fimbriation

Fimbriae are considered to be an important virulence factor ofPorphyromonas gingivalis. In order to identify genes essential for fimbriation, other thanfimAwhich encodes the major subunit protein of fimbriae, transposon mutagenesis and immunological screening techniques were used to isolate fimbria-deficient mutants. R751::ast;Ω4, a suicide vector that carries Tn4351, was transferred fromEscherichia colitoP. gingivalisby conjugation. Twenty-two independent fimbria-deficient mutants were identified among the resulting transformants. Southern hybridization analysis with pBlue 4351, a transposon-specific probe, and R751 indicated that 45% of the mutants resulted from single transposon insertions and that the remaining 55% of the mutants resulted from cointegration of R751 sequences. Southern hybridization analysis with pUCBg12.1, a probe for thefimAregion, indicated that nine of the mutants contained insertions within the 2.5 kbSacI DNA fragment ofP. gingivalisthat containsfimA, ORF1 (which encodes a 15 kDa protein), and the C-terminal portion of ORF5 (which encodes a 63 kDa protein). Polymerase chain reaction (PCR) analysis and further Southern hybridization analysis indicated that the insertion site(s) for all nine of these mutants was within thefimAgene. Southern hybridization analysis also indicated that the remaining thirteen mutants contained insertions somewhere outside the 10 kbfimAregion. Analysis by pulsed field gel electrophoresis (PFGE) revealed that insertions for most of the thirteen mutants mapped to a 300 kbNotI fragment and are located at least approximately 200 kb away fromfimA. These results identify genetic loci other thanfimA, that are required for fimbriation ofP. gingivalis. Future cloning and characterization of these genetic loci should be straightforward since they are now marked by antibiotic resistance genes carried by the transposon.

Identification of a gene essential for O-acetylation of the Staphylococcus aureus type 5 capsular polysaccharide.

The Staphylococcus aureus serotype 5 capsular polysaccharide (CP5) has a trisaccharide repeating unit of (-->4)-3-O-Ac-beta-D-ManNAcAp-(1-->4)-alpha-L-FucNAcp-(1-->3 )-beta-D-FucNAcp-(1-->). Tn918 mutagenesis of strain Reynolds yielded a mutant that produced wild-type levels of O-deacetylated CP5. The site and orientation of the single transposon insertion in mutant JL232 were determined by analysis of Southern blots and amplification of DNA flanking the transposon. DNA sequencing revealed that Tn918 was inserted within an open reading frame of 627 bp. The predicted amino acid sequence encodes a protein of approximately 26 kDa with homology to members of the NodL-LacA-CysE family of bacterial acetyltransferases. Southern blot analysis showed that genes similar to cap5H were present only in strains of S. aureus belonging to capsular serotypes 2, 4 and 5. In an in vitro assay, the parental strain was more resistant to opsonophagocytic killing than the mutant strain. In a mouse model of staphylococcal infection, the parental strain was able to seed the bloodstream from the peritoneal cavity and colonize the kidneys more efficiently than the O-deacetylated mutant. When cap5H was provided to the mutant in trans, it fully restored CP5 O-acetylation. The virulence of the complemented mutant strain closely approximated that of the parental strain.

Characterisation of a Transposon-induced Pleiotropic Mutant ofClostridium acetobutylicumP262

Transposon-induced metronidazole resistance was used as a selection system for the isolation ofClostridium acetobutylicumP262 mutants with altered electron transport pathways. The metronidazole resistant transconjugant of interest, mutant 3R, displayed resistance to DNA damaging agents, UV and bleomycin, and harboured a single transposon insertion within a structural gene, designatedsum(susceptibility tometronidazole). Thesumgene encoded a 334 amino-acid protein, with 36% identity and 57–58% similarity at the amino acid level to two archaebacterial protein sequences which appear to represent a class of uncharacterised reductase enzymes. Physiological studies of mutant 3R revealed a number of pleiotropic characteristics which included enhanced autolysin activity, increased motility, impaired clostridial cell formation, and resistance to the toxic tripeptide analogue, bialaphos. The introduction of thesumgene in multiple copies on a plasmid vector into the related strainClostridium beijerinckiiNCIMB 8052, resulted in inhibition of cell division, motility and autolysin activity. Thesumgene appears to be a member of a new subgroup of activases with reducing activity, which may control a regulon affecting different stationary phase processes such as clostridial differentiation and sporulation inC. acetobutylicumP262. The metronidazole resistant phenotype of thesummutant can be attributed to an increased capacity for DNA repair.

New genetic techniques for group B streptococci: high-efficiency transformation, maintenance of temperature-sensitive pWV01 plasmids, and mutagenesis with Tn917.

Three techniques were developed to improve the genetic manipulation of group B streptococci (GBS). We first optimized a protocol for transformation of GBS by electroporation, which provided transformation efficiencies of 10(5) CFU/microgram. Variables that influenced the transformation efficiency were the glycine content of the competent cell growth media, the electric field strength during electroporation, the electroporation buffer composition, the host origin of the transforming plasmid, and the concentration of selective antibiotic at the final plating. Our transformation protocol provides an efficiency sufficient for cloning from ligation reactions directly into GBS, obviating an intermediate host such as Escherichia coli. Second, temperature-sensitive plasmids of the pWV01 lineage were shown to transform GBS, and their temperature-sensitive replication was confirmed. Lastly, the temperature-sensitive pWV01 plasmid pTV1OK, which contains Tn917, was used as a transposon delivery vector for the construction of genomic Tn917 mutant libraries. We have shown, for the first time, that Tn917 transposes to the GBS chromosome and at a frequency of 10(-3)/CFU. Furthermore, representative clones from a Tn917 library contained single transposon insertions that were randomly located throughout the chromosome. These techniques should provide useful methods for cloning, mutagenesis, and characterization of genes from GBS.

The Vibrio cholerae O139 serogroup antigen includes an O-antigen capsule and lipopolysaccharide virulence determinants.

Vibrio cholerae serogroup O139 emerged on the Indian subcontinent in October 1992 to become the first non-O1 V. cholerae serogroup documented to cause epidemic cholera. Although related to V. cholerae El Tor O1 strains, O139 strains have unique surface structures that include a capsular surface layer and lipopolysaccharide (LPS). Immunoblot analysis of either whole-cell lysates or LPS preparations revealed three electrophoretic forms of the O139 antigen: two slowly migrating forms and one rapidly migrating form that appeared identical to O139 LPS. All three forms of the antigen shared an epitope defined by an O139-specific monoclonal antibody. A serum-sensitive nonencapsulated mutant was isolated that lacks only the slow migrating forms. The slow migrating forms did not stain with silver whereas the rapidly migrating form did, suggesting that the former might constitute highly polymerized O-antigen side-chain molecules that were not covalently bound to core polysaccharide and lipid A (an "O-antigen capsule"). A single transposon insertion resulted in the loss of immunoreactivity of both the LPS and the O-antigen capsule, implying that there are genes common to the biosynthesis of both these macromolecules. The O139 LPS and O-antigen capsule were both important for colonization of the small intestine of the newborn mouse and for serum resistance, demonstrating that both of these forms of the O139 serogroup antigen are virulence factors.

Transposon-induced mutants ofListeria monocytogenesincapable of growth at low temperature (4°C)

Transposon mutagenesis was used to pursue the molecular basis of cold resistance in Listeria and its possible involvement in pathogenesis. We have generated transposon-induced mutants of Listeria monocytogenes which were unable to grow at 4 degrees C but grew at 8 degrees C, 22 degrees C and 37 degrees C. The transposon was localized in the same 1.8-kb EcoRI fragment in two independently derived mutants. Mutants which carried single transposon insertions had normal hemolytic activity, motility and invasion of cultured fibroblasts. These results indicate that in L. monocytogenes cold sensitivity can be single transposon insertions and that loss of cold resistance may not interfere with other phenotypes, including invasion in a cell culture model.

Cloning of a phenazine biosynthetic locus of Pseudomonas aureofaciens PGS12 and analysis of its expression in vitro with the ice nucleation reporter gene.

Pseudomonas aureofaciens PGS12 produces three phenazine antibiotics, in addition to siderophores, hydrogen cyanide, pyrrolnitrin, and indoleacetic acid. Tn5-259.7 transposon mutagenesis was carried out to identify and clone a chromosomal locus involved in phenazine biosynthesis. Three classes of mutants were obtained: mutants deficient in phenazine production (Phz-), mutants deficient in hydrogen cyanide production (HCN-), and mutants deficient in the production of both compounds. EcoRI DNA fragments that contained the transposon and flanking regions were cloned from three mutants with single-transposon insertions, one from each phenotypic class. Phenazine and hydrogen cyanide production was restored by complementation of Phz- or HCN- mutants with selected cosmids from a PGS12 genomic library. No cosmids that complemented the doubly deficient Phz-HCN- mutant were obtained. A promoterless ice nucleation reporter gene was inserted in a phenazine biosynthetic locus by Tn3-spice transposon mutagenesis of a cosmid which complemented a phenazine-minus mutant. Reporter gene fusions that expressed the ice nucleation phenotype and no longer complemented phenazine production were introduced into the PGS12 chromosome by marker exchange. The expression of this locus was then monitored under different culture conditions. Expression decreased at pH levels below 7, and it was not affected by iron. Shikimic acid and phenylalanine favored higher expression levels. Expression was reduced in media with low substrate concentrations, indicating the importance of nutrient availability.

Analysis of Tn916-induced mutants ofClostridium acetobutylicum altered in solventogenesis and sporulation

The conjugative transposon Tn916 was used for mutagenesis of Clostridium acetobutylicum ATCC 824. Tetracycline-resistant mutants were screened for loss of granulose synthesis and five classes of granulose mutants, that contained single transposon insertions, were identified on the basis of altered solvent production. Class 1 mutants did not make acetone or butanol, lacked activity of enzymes induced during solventogenesis, and did not sporulate, indicating that they are regulatory mutants. The class 2 mutant strains also did not produce acetone but did form small amounts of butanol and ethanol, while the class 3 mutants produced low amounts of all solvents. Class 4 and 5 mutants produced essentially the same or higher amounts of solvents than the parent strain. Transposon insertions in the class 1 mutants were used as markers for in vitro synthesis of flanking chromosomal DNA using Tn916-specific primers. The DNA fragments were labeled to produce specific probes. Transposon insertion sites in the chromosomes of 13 different class 1 regulatory mutants were compared by hybridization of the specific probes to Southern blots of restriction endonuclease-digested parental chromosomal DNA. Insertions in two mutants appeared to be in the same region of the chromosome. These results predict that multiple regulatory elements are required to induce solvent production and sporulation.

Effects on virulence of mutations in a locus essential for hyaluronic acid capsule expression in group A streptococci

Mucoid or highly encapsulated strains of group A streptococci have been associated both with unusually severe infections and with acute rheumatic fever. Previously, we described an acapsular mutant, TX4, derived from a mucoid M-type 18 strain of a group A streptococcus by transposon mutagenesis (M. R. Wessels, A. E. Moses, J. B. Goldberg, and T. J. DiCesare, Proc. Natl. Acad. Sci. USA 88:8317-8321, 1991). We now report studies further characterizing strain TX4 as well as an additional acapsular mutant, TX72. Strain TX4 was found to contain a 9.5-kb deletion of chromosomal DNA adjacent to the site of transposon Tn916 insertion. Cloned chromosomal DNA from TX4 flanking the transposon insertion site was used as a probe to demonstrate the presence of homologous regions in 11 of 11 wild-type group A streptococcal strains of various M protein types. A second acapsular mutant, TX72, had a single transposon insertion and had no apparent deletion of chromosomal DNA. The Tn916 insertion in TX72 was mapped to the hasA locus (encoding hyaluronate synthase), which lies within the chromosomal region deleted in TX4. Strain TX72 was avirulent in mice and sensitive to phagocytic killing in vitro. Transduction of either the insertion-deletion mutation from TX4 or the simple insertion mutation from TX72 to a type 24 group A streptococcus strain also resulted in loss of capsule expression, demonstrating that a homologous region of the chromosome controls capsule expression in another serotype of group A streptococci. We conclude that the hyaluronic acid capsule plays an important role in virulence and that a region of the chromosome essential for capsular polysaccharide expression is conserved among diverse group A streptococcal strains.

Phospholipid and fatty acid compositions of Rhizobium leguminosarum biovar trifolii ANU843 in relation to flavone-activated pSym nod gene expression.

The phospholipid and associated fatty acid compositions of the bacterial symbiont of clover, Rhizobium leguminosarum biovar trifolii wild-type ANU843, was analyzed by two-dimensional silica thin-layer chromatography, fast atom bombardment-mass spectrometry, flame-ionization detection gas-liquid chromatography and combined gas-liquid chromatography/mass spectrometry. The phospholipid composition included phosphatidylethanolamine (15%), N-methylphosphatidylethanolamine (47%), N,N-dimethylphosphatidylethanolamine (9%), phosphatidylglycerol (19%), cardiolipin (5%) and phosphatidylcholine (2%). Fatty acid composition included predominantly cis-11-octadecenoic acid, lower levels of cis-9-hexadecenoic acid, hexadecanoic acid, 11-methyl-11-octadecenoic acid, octadecanoic acid, 11,12-methyleneoctadecanoic acid, eicosanoic acid and traces of branched, and di- and triunsaturated fatty acids. The influence of expression of the "nodulation" genes encoding symbiotic functions on the composition of these membrane lipids was examined in wild-type cells grown with or without the flavone inducer, 4',7-dihydroxyflavone and in mutated cells lacking the entire symbiotic plasmid where these genes reside, or containing single transposon insertions in selected nodulation genes. No significant changes in phospholipid or associated fatty acid compositions were detected by the above methods of analysis.

In-vitro and in-vivo characteristics of TnphoA mutant strains of Salmonella serotype Gallinarum not invasive for tissue culture cells.

Insertion mutants of a strain of Salmonella serotype Gallinarum, the cause of fowl typhoid, were produced with transposon TnphoA. Eight mutants were identified as being less invasive in cell culture than the parent strain. Neither the parent strain nor the mutants showed mannose-sensitive haemagglutination of various red blood cell species. Although two mutants gave mannose-resistant (MR) haemagglutination of red cells of different animal species these MR activities could not be correlated with other characteristics in vitro or in vivo. The mutant strains were divisible into three classes by their patterns of invasiveness and adhesiveness in vitro and by changes in membrane proteins. Strains of classes 1 and 2 had single transposon insertions, detected by Southern hybridisation, of 8.9 and 2.4 kb EcoRV-digested chromosomal-fragments, respectively, and were slightly less invasive than the parent strain. They were no less virulent for chickens by the oral route than a mutant strain shown to be as invasive in vitro as the parent strain. The class-2 mutant was also less adhesive than the other strains. The single mutant strain of class 3 which contained insertions in several chromosomal fragments, was non-invasive in Vero cells (less than log10 1.0 cfu/ml recovered compared with log10 2.88 for the parent strain) and showed reduced virulence by both oral and intramuscular routes. The mutant strains of all classes were taken up equally rapidly from the blood by the spleen. Intramuscular immunisation of chickens with the class-3 mutant strain gave complete protection against oral challenge 3 weeks later with 1000 oral LD50 doses of the virulent parent strain.

Targeted gene mutations in Drosophila.

A cloned gene can be of interest because of its expression in a particular tissue or at a certain developmental stage, or because of homology to an interesting gene from another organism. In Drosophila its location in the genome is readily determined by in situ hybridization to the banded larval salivary gland polytene chromosomes, but it is more difficult to isolate mutations that may reveal its function. This paper describes a general method for detecting transposable element insertions into the gene in question. This "reverse genetics" then offers the possibility of observing a consequent mutant phenotype, providing a key to the normal function of the gene. The sensitivity of the polymerase chain reaction makes it possible to detect the occurrence of a single appropriate P-element transposon insertion among a population of mutagenized flies. This is accomplished by the use of oligonucleotide primers--one a sequence from within the cloned gene and the other homologous to the terminal sequence of the P-element DNA--to prime synthesis into the DNA flanking an insertion site. A segment of DNA, bounded by the two primers, will be a target for amplification only in a fly in which a P-element has inserted within about 2 kilobases of the gene primer. This technique has been used to detect P-element insertions near a gene expressed in the Drosophila compound eye. Potential problems with the technique and possible refinements in the screen are discussed. In principle, it could be utilized to detect insertion of a foreign element into any gene for which at least a partial sequence is known and could be extended to other organisms.