Transposon Library Research Articles

Flagellar basal body flg operon as a virulence determinant of Vibrio vulnificus

Vibrio vulnificus, a halophilic estuarine bacterium causing a rapidly progressing fatal septicemia, is highly cytotoxic to eukaryotic cells. To identify new virulence factors associated with cytotoxicity, we constructed a mariner-based transposon (Tn Himar1) library of the highly virulent clinical isolate MO6-24/O having a double mutation in the hemolysin and protease genes. The Himar1 mutant library was extensively screened for the mutants showing decreased cytotoxicity to HeLa cells. We selected a cytotoxicity defective mutant having a Himar1 insertion in an open reading frame showing 96% identity to Vibrio parahaemolyticus FlgC, a flagella basal body rod protein. The Tn Himar1 insertion mutation also resulted in a significant decrease in motility, adhesion, cytotoxicity, and lethality to mice. This is the first report showing that flg genes, which are components of the flagellum biogenesis gene cluster, might play an important role in the virulence of V. vulnificus.

Mutagenesis of Streptococcus equi and Streptococcus suis by transposon Tn 917

Genetic tools for studying streptococci are much less sophisticated than those that are available for many other bacterial genera. In this paper, we describe the development of a transposon mutagenesis system that we have used to mutate two important veterinary streptococci, Streptococcus equi and Streptococcus suis. The system uses a temperature-sensitive suicide vector to deliver Tn 917 via electroporation, transposing Tn 917 into the chromosomal DNA of the two streptococci. The transposon insertions can be rescued from the streptococcal chromosomes by plasmid rescue and selection in E. coli, with subsequent insertion site analysis by DNA sequencing. Transposition appeared to have occurred in an essentially random fashion when chromosomal DNA of S. suis and S. equi mutants was analysed by Southern blotting. However, when analysis of 60 S. equi mutants was carried out using the S. equi genome sequence database, 60% of transposon insertions had occurred within a 15 kb region of the genome whereas the other insertions appeared to have occurred essentially randomly. This finding suggests that Southern blot analysis for assessing the randomness of transposon libraries may need to be interpreted with caution. However, this observation notwithstanding, the Tn 917 based system described in this paper will facilitate the study of S. suis and S. equi.

Characterization of a Mycobacterium tuberculosis H37Rv transposon library reveals insertions in 351 ORFs and mutants with altered virulence.

A library of Mycobacterium tuberculosis insertional mutants was generated with the transposon Tn5370. The junction sequence between the transposon and the mycobacterial chromosome was determined, revealing the positions of 1329 unique insertions, 1189 of which were located in 351 different ORFs. Transposition was not completely random and examination of the most susceptible genome regions revealed a lower-than-average G+C content ranging from 54 to 62 mol%. Mutants were obtained in all of the recognized M. tuberculosis functional protein-coding gene classes. About 30% of the disrupted ORFs had matches elsewhere in the genome that suggested redundancy of function. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated in a severe combined immune deficiency (SCID) mouse model. A range of phenotypes was observed in these mutants, the most notable being the severe attenuation in virulence of a strain disrupted in the Rv1290c gene, which encodes a protein of unknown function. The library described in this study provides a resource of defined mutant strains for use in functional analyses aimed at investigating the role of particular M. tuberculosis genes in virulence and defining their potential as targets for new anti-mycobacterial drugs or as candidates for deletion in a rationally attenuated live vaccine.

An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones.

We describe an efficient high-throughput method for accurate DNA sequencing of entire cDNA clones. Developed as part of our involvement in the Mammalian Gene Collection full-length cDNA sequencing initiative, the method has been used and refined in our laboratory since September 2000. Amenable to large scale projects, we have used the method to generate >7 Mb of accurate sequence from 3695 candidate full-length cDNAs. Sequencing is accomplished through the insertion of Mu transposon into cDNAs, followed by sequencing reactions primed with Mu-specific sequencing primers. Transposon insertion reactions are not performed with individual cDNAs but rather on pools of up to 96 clones. This pooling strategy reduces the number of transposon insertion sequencing libraries that would otherwise be required, reducing the costs and enhancing the efficiency of the transposon library construction procedure. Sequences generated using transposon-specific sequencing primers are assembled to yield the full-length cDNA sequence, with sequence editing and other sequence finishing activities performed as required to resolve sequence ambiguities. Although analysis of the many thousands (22 785) of sequenced Mu transposon insertion events revealed a weak sequence preference for Mu insertion, we observed insertion of the Mu transposon into 1015 of the possible 1024 5mer candidate insertion sites.

Adaptive divergence in experimental populations of Pseudomonas fluorescens. I. Genetic and phenotypic bases of wrinkly spreader fitness.

A central feature of all adaptive radiations is morphological divergence, but the phenotypic innovations that are responsible are rarely known. When selected in a spatially structured environment, populations of the bacterium Pseudomonas fluorescens rapidly diverge. Among the divergent morphs is a mutant type termed "wrinkly spreader" (WS) that colonizes a new niche through the formation of self-supporting biofilms. Loci contributing to the primary phenotypic innovation were sought by screening a WS transposon library for niche-defective (WS(-)) mutants. Detailed analysis of one group of mutants revealed an operon of 10 genes encoding enzymes necessary to produce a cellulose-like polymer (CLP). WS genotypes overproduce CLP and overproduction of the polymer is necessary for the distinctive morphology of WS colonies; it is also required for biofilm formation and to maximize fitness in spatially structured microcosms, but overproduction of CLP alone is not sufficient to cause WS. A working model predicts that modification of cell cycle control of CLP production is an important determinant of the phenotypic innovation. Analysis of >30 kb of DNA encoding traits required for expression of the WS phenotype, including a regulatory locus, has not revealed the mutational causes, indicating a complex genotype-phenotype map.

The generation of a transposon-mutagenized Burkeholderia glumae library to isolate novel mutants

The gram-negative bacterium, Burkeholderia glumae, is a pathogen of rice. As a step toward identifying pathogenicity determinants, a transposon mutagenized B. glumae library was generated. A transposition frequency of 2.1×10 6 cfu/μg of DNA was achieved using a recently developed Tn 5-derived transposon system. Southern-blot analysis of several randomly selected colonies indicated that the transpositions were single, random events. Two genetic screens were conducted to assess the mutagenic frequency of the generated library. Screening of 1000 colonies for auxotrophs resulted in the identification of 46 mutants. The 46 mutants were subdivided into five classes based on their ability to grow on supplemented medias. Exposure of 1000 mutagenized colonies to UV light, as part of a second screen, identified two fluorescent mutants. The transposon library will be a useful tool in the investigation of metabolic pathways in B. glumae and allow a direct route to the affected gene. Current emphasis is directed toward identifying the factors required for plant infection.

Effect of selenite on growth and protein synthesis in the phototrophic bacterium Rhodobacter sphaeroides.

The effect of selenite on the growth rate and protein synthesis has been investigated in Rhodobacter sphaeroides. This photosynthetic bacterium efficiently reduces selenite with intracellular accumulation under both dark aerobic and anaerobic photosynthetic conditions. Addition of 1 mM selenite under these two growth conditions does not affect the final cell density, although a marked slowdown in growth rate is observed under aerobic growth. The proteome analysis of selenite response by two-dimensional gel electrophoresis shows an enhanced synthesis of some chaperones, an elongation factor, and enzymes associated to oxidative stress. The induction of these antioxidant proteins confirms that the major toxic effect of selenite is the formation of reactive oxygen species during its metabolism. In addition, we show that one mutant unable to precipitate selenite, selected from a transposon library, is affected in the smoK gene. This encodes a constituent of a putative ABC transporter implicated in the uptake of polyols. This mutant is less sensitive to selenite and does not express stress proteins identified in the wild type in response to selenite. This suggests that the entry of selenite into the cytoplasm is mediated by a polyol transporter in R. sphaeroides.

Aux1p/Swa2p is required for cortical endoplasmic reticulum inheritance in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the endoplasmic reticulum (ER) is found at the periphery of the cell and around the nucleus. The segregation of ER through the mother-bud neck may occur by more than one mechanism because perinuclear, but not peripheral ER, requires microtubules for this event. To identify genes whose products are required for cortical ER inheritance, we have used a Tn3-based transposon library to mutagenize cells expressing a green fluorescent protein-tagged ER marker protein (Hmg1p). This approach has revealed that AUX1/SWA2 plays a role in ER inheritance. The COOH terminus of Aux1p/Swa2p contains a J-domain that is highly related to the J-domain of auxilin, which stimulates the uncoating of clathrin-coated vesicles. Deletion of the J-domain of Aux1p/Swa2p leads to vacuole fragmentation and membrane accumulation but does not affect the migration of peripheral ER into daughter cells. These findings suggest that Aux1p/Swa2p may be a bifunctional protein with roles in membrane traffic and cortical ER inheritance. In support of this hypothesis, we find that Aux1p/Swa2p localizes to ER membranes.

Tn 10 insertional mutations of Bacillus subtilis that block the biosynthesis of bacilysin

Transposon mutagenesis was employed to isolate the gene(s) related with the biosynthesis of dipeptide antibiotic in Bacillus subtilis PY79 (a prototrophic derivative of the standard 168 strain). The blocked mutants were phenotypically selected from the transposon library by bioassay and the complete loss of biosynthetic ability was verified through ESI-mass spectrometry analysis. Four different bacilysin nonproducer mutants (Bac −::Tn 10( ori-spc)) were isolated from the transposon library. The genes involved in bacilysin biosynthesis were identified as thyA (thymidilate synthetase), ybgG (unknown; similar to homocysteine methyl transferase) and oppA (oligopeptide permease), respectively. The other blocked gene was yvgW (unknown; similar to heavy metal-transporting ATPase); however, backcross studies did not verify its involvement in bacilysin biosynthesis. This gene, on the other hand, appeared to be necessary for efficient sporulation and transformation. Opp involvement was significant as it suggested that bacilysin biosynthesis is under or a component of the quorum sensing pathway which has been shown to be responsible for the establishment of sporulation, competence development and onset of surfactin biosynthesis. For verification, it was necessary to check the involvement of peptide pheromones (PhrA or PhrC) internalized by the Opp system and response regulator ComA as the essential components of this global control. phrA, phrC and comA deleted mutants of PY79 were thus constructed and the latter two genes were shown to be essential for bacilysin biosynthesis.

Pleiotropic pigmentation mutants of Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram-negative, black pigmented oral anaerobe associated with adult periodontitis. The adherence of the bacterium to junctional epithelial cells is the first step in infection and colonization. The molecular mechanisms and genetics of colonization are, as yet, not well understood, although it has been demonstrated thatP. gingivalis fimbriae are involved in adhesion. In addition, cell surface cysteine proteinases may play a role either directly as adhesins or indirectly through their involvement in the biogenesis of fimbriae. A link has been established between cysteine proteinase-hemagglutinating activity and colongy pigmentation on blood agar. In this study a P. gingivalis ATCC 33277 transposon library was screened for white mutants. Pleiotropic mutants were identified with altered pigmentation, proteinase, hemagglutinin and haemolytic activities. Although the mutants fell into two classes based on the above phenotypes, by electron microscopy both classes showed increased fimbriation and decreased vesicle formation. Sequencing of genomic DNA flanking the transposon insertions revealed that one class of mutants carried disruptions in the gene encoding Lys-gingipain (kgp) and the other in a gene homologous to a glycosyl transferase. Potential roles for these genes in pigmentation, fimbriation, vesicle formation and attachment to epithelial cells are discussed.

Antibiotic resistance as a stress response: complete sequencing of a large number of chromosomal loci in Staphylococcus aureus strain COL that impact on the expression of resistance to methicillin.

Tn551 inactivation has identified several determinants--fem or auxiliary genes--that, in addition to the mecA gene, are also critical for the expression of high-level and homogeneous resistance to methicillin. Genetic and/or biochemical analysis has shown that of the nearly dozen aux mutations described so far most are in genes involved in cell wall synthesis (murE, pbp2, glmM, glnR, femA/B, llm, etc.) or in complex regulatory functions (sigmaB), suggesting that optimal expression of resistance may involve the cooperative functioning of a number of genes in cell wall metabolism as well as stress response. The exact mechanism of these functions is not known. In an attempt to explore this unusual aspect of methicillin resistance more fully, a Tn551 transposon library, constructed in the background of the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL, was screened for all independent insertional mutants in which the level of methicillin resistance of the parental strain (MIC, 1,600 microg/ml) was reduced by at least 15-fold and up to 500-fold. We now describe the sequencing of 21 Tn551-inactivated genes and their vicinities in 23 new auxiliary mutants that have been studied before. Using the inverted polymerase chain reaction (IPCR), we amplified fragments corresponding to the right and left junction of the Tn551 insertions, which were then sequenced by primer walking. The two largest groups of these new auxiliary genes encoded either proteins of unknown functions (6 genes) or showed homology with genes encoding proteins involved with putative sensory/regulatory activities (7 genes: protein kinases, ABC transporters, and a catabolite control protein). Sequencing upstream and downstream allowed the identification of a number of additional open reading frames, some of which may also include functions relevant for the expression of antibiotic resistance.

Iron starvation of Bordetella avium stimulates expression of five outer membrane proteins and regulates a gene involved in acquiring iron from serum.

Iron starvation of Bordetella avium induced expression of five outer membrane proteins with apparent molecular masses of 95, 92, 91.5, 84, and 51 kDa. Iron-responsive outer membrane proteins (FeRPs) of similar sizes were detected in six of six strains of B. avium, suggesting that the five FeRPs are common constituents of the outer membrane of most, if not all, strains of B. avium. Iron-regulated genes of B. avium were targeted for mutagenesis with the transposon TnphoA. Two mutants with iron-responsive alkaline phosphatase activities were isolated from the transposon library. The transposon insertion did not alter the iron-regulated expression of the five FeRPs in mutant Pho-6. The mutant Pho-20 exhibited a loss in expression of the 95-kDa FeRP and the 84-kDa FeRP. Both Pho-6 and Pho-20 were able to use free iron as a nutrient source. However, Pho-20 was severely compromised in its ability to use iron present in turkey serum. The data indicated that the mutation in Pho-20 affected expression of one or more components of an uptake machinery that is involved in acquisition of iron from organic ferricomplexes.

Transposon mutagenesis with IS6100 in the avermectin-producer Streptomyces avermitilis.

The insertion sequence IS6100 was shown to undergo intermolecular transposition from a temperature-sensitive delivery plasmid to the genome of the avermectin-producer Streptomyces avermitilis, creating cointegrates. Evidence from both Southern hybridization and the range of auxotrophic mutations present in a transposon library was consistent with random transposition. It was not possible to increase transposase expression by readthrough transcription from a copy of the tipA promoter located adjacent to the insertion sequence. This was in part due to the absence of a homologue of the Streptomyces lividans transcriptional activator TipAL in S. avermitilis. However, recombinant S. avermitilis strains carrying the S. lividans tip operon were also deficient for induction of the promoter. The frequency of reversion of different auxotrophic mutations by precise excision, involving recombination across 8 bp direct repeats, was shown to vary by at least five orders of magnitude. This dependence of recombination frequency on chromosomal location may contribute to the stability of repetitive modular type I polyketide biosynthetic genes.

TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae.

The production of several virulence factors in Vibrio cholerae O1, including cholera toxin and the pilus colonization factor TCP (toxin-coregulated pilus), is strongly influenced by environmental conditions. To specifically identify membrane proteins involved in these signal transduction events, we examined a transposon library of V. cholerae generated by Tnbla mutagenesis for cells that produce TCP when grown under various nonpermissive conditions. To select for TCP-producing cells we used the recently described bacteriophage CTX phi-Kan, which uses TCP as its receptor and carries a gene encoding resistance to kanamycin. Among the isolated mutants was a transposon insertion in a gene homologous to nqrB from Vibrio alginolyticus, which encodes a subunit of a Na(+)-translocating NADH:ubiquinone oxidoreductase, and tcpI, encoding a chemo-receptor previously implicated in the negative regulation of TCP production. A third transposon mutant had an insertion in tcpP, which is in an operon with tcpH, a known positive regulator of TCP production. However, TcpP was shown to be essential for TCP production in V. cholerae, as a tcpP-deletion strain was deficient in pili production. The amino-terminal region of TcpP shows sequence homology to the DNA-binding domains of several regulatory proteins, including ToxR from V. cholerae and PsaE from Yersinia pestis. Like ToxR, TcpP activates transcription of the toxT gene, an essential activator of tcp operon transcription. Furthermore, TcpH, with its large periplasmic domain and inner membrane anchor, has a structure similar to that of ToxS and was shown to enhance the activity of TcpP. We propose that TcpP/TcpH constitute a pair of regulatory proteins functionally similar to ToxR/ToxS and PsaE/PsaF that are required for toxT transcription in V. cholerae.

Massive reduction in methicillin resistance by transposon inactivation of the normal PBP2 in a methicillin-resistant strain of Staphylococcus aureus.

Screening of a large transposon library constructed in the background of a highly and homogeneously methicillin-resistant Staphylococcus aureus (MRSA) strain (methicillin MIC 1,600 micrograms/ml) for Tn551 mutants with reduced resistance, identified mutant RUSA130 with a methicillin MIC of 12 micrograms/ml. Cloning in E. coli followed by sequencing located the Tn551 insert omega 703 near the C-terminal of the PBP2 gene. Penicillin-binding assays with mutant RUSA130 showed the presence of normal amounts of penicillin-binding protein 2A (PBP2A) but the absence of PBP2. These observations suggest that the mecA gene product PBP2A is not the sole catalyst of peptidoglycan synthesis in MRSA growing in the presence of beta-lactam antibiotics, since an intact PBP2 is also essential for the optimal expression of methicillin resistance in MRSA.

Identification of sigma S-regulated genes in Salmonella typhimurium: complementary regulatory interactions between sigma S and cyclic AMP receptor protein.

sigma S (RpoS)-regulated lacZ transcriptional fusions in Salmonella typhimurium were identified from a MudJ transposon library by placing the rpoS gene under the control of the araBAD promoter and detecting lacZ expression in the presence or absence of arabinose supplementation. Western blot (immunoblot) analysis of bacteria carrying PBAD::rpoS demonstrated arabinose-dependent rpoS expression during all phases of growth. sigma S-dependent gene expression of individual gene fusions was confirmed by P22-mediated transduction of the MudJ insertions into wild-type or rpoS backgrounds. Analysis of six insertions revealed the known sigma S-regulated gene otsA, as well as five novel loci. Each of these genes is maximally expressed in stationary phase, and all but one show evidence of cyclic AMP receptor protein-dependent repression during logarithmic growth which is relieved in stationary phase. For these genes, as well as for the sigma S-regulated spvB plasmid virulence gene, a combination of rpoS overexpression and crp inactivation can result in high-level expression during logarithmic growth. The approach used to identify sigma S-regulated genes in this study provides a general method for the identification of genes controlled by trans-acting regulatory factors.

A novel locus of Yersinia enterocolitica serotype O:3 involved in lipopolysaccharide outer core biosynthesis.

Yersinia enterocolitica serotype O:3 strain 6471/76-c (YeO3-c) was sensitive to bacteriophage phi R1-37 when grown at 37 degrees C but not when grown at 22 degrees C because of steric hindrance by abundant lipopolysaccharide (LPS) O-side chain (O-antigen) expressed at 22 degrees C. The transposon library of YeO3-c was grown at 37 degrees C and screened for phage phi R1-37-resistant transposon insertion mutants. Three types of mutant were isolated: (i) phage receptor mutants expressing O-antigen (LPS-smooth), (ii) phage receptor mutants not expressing O-antigen (LPS-rough), and (iii) LPS-smooth mutants with the phage receptor constitutively sterically blocked. Mutant type (i) was characterized in detail; the transposon insertion inactivates an operon, named the trs operon. The main findings based on this mutant are: (i) the frs operon is involved in the biosynthesis of the LPS outer core in YeO3-c; the nucleotide sequence of the trs operon revealed eight novel genes showing similarly to known polysaccharide biosynthetic genes of various Gram-negative bacteria as well as to capsule biosynthesis genes of Staphylococcus aureus; (ii) the biosynthesis of the core of YeO3-c involves at least two genetic loci; (iii) the trs operon is required for the biosynthesis of the bacteriophage phi R1-37 receptor structures; (iv) the homopolymeric O-antigen of YeO3-c is ligated to the inner core in Y. enterocolitica O:3; (v) the trs operon is located between the adk-hemH and galE-gsk gene pairs in the Y. enterocolitica chromosome; and (vi) the phage phi R1-37 receptor is present in many but not in all Y. enterocolitica serotypes. The results also allow us to speculate that the trs operon is a relic of the ancestral rfb region of Y. enterocolitica O:3 carrying genes indispensable for the completion of the core polysaccharide biosynthesis.

Reassessment of the number of auxiliary genes essential for expression of high-level methicillin resistance in Staphylococcus aureus.

A new transposon library constructed in the background of the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL yielded 70 independent insertional mutants with reduced levels of antibiotic resistance. Restriction analysis with HindIII, EcoRV, EcoRI, and PstI and then Southern hybridization with probes for the transposon and for the femA-femB gene demonstrated that 41 of the 70 Tn551 mutants carried distinct and novel, as yet undescribed insertion sites, all of which were outside of the mecA gene and were also outside the already-characterized auxiliary genes femA, femB, femC, and femD. All previously described Tn551 mutations of this type were in genes located either on SmaI fragment A or SmaI fragment I. In contrast, inserts of the new library were located in 7 of the 16 SmaI chromosomal fragments, fragments A, B, C, D, E, F, and I. In all of the mutants, expression of methicillin resistance became heterogeneous, and the MIC for the majority of cells was reduced (1.5 to 200 micrograms ml-1) from the homogeneous methicillin MIC (1,600 micrograms ml-1) of the parental cells. Although identification of the exact number of genes inactivated through the new set of transposon inserts will require cloning and sequencing, a rough estimate of this number from mapping data suggests a minimum of at least 10 to 12 new genetic determinants, all of which are needed together with femA, femB, femC, and femD for the optimal expression of methicillin resistance.

Characterization of a gene, pilU, required for twitching motility but not phage sensitivity in Pseudomonas aeruginosa

Type 4 fimbriae (or pilli) are associated with a form of bacterial surface translocation known as twitching motility. Fimbriae are also associated with sensitivity to certain bacteriophages such as PO4. Transposon mutagenesis was used to generate a library of Pseudomonas aeruginosa mutants which lack the spreading-colony morphology characteristic of twitching motility. In four of these mutants the transposon was found to be located in the vicinity of the previously described pilT locus, but in only one case was it found to have inserted within the pilT coding sequence. Two twitching-motility mutants originally isolated by Bradley, K2.2, and PAO2001.2, which have been widely used in studies of P. aeruginosa fimbrial structure and expression, were also shown to affect pilT and to comprise a small deletion and a frameshift mutation, respectively. The other three transposon mutations were found to have occurred within a new gene located directly downstream of pilT. This gene, termed pilU, encodes a 382-amino-acid protein closely related to PilT and to other members of a family of putative nucleotide-binding proteins which are involved in the assembly of cell surface-associated complexes. Furthermore, the pilT and pilU genes appear to be independently expressed. Like pilT mutants, the pilU mutants were hyperfimbriate, but in neither case was this associated with an increase in transcription of the fimbrial subunit gene pilA. However, in contrast to pilT mutants, the pilU mutants had not also acquired resistance to infection by bacteriophage PO4. A broader survey showed differential patterns of sensitivity to various fimbrial-specific phages among the pilU mutants and other twitching-motility mutants in the transposon library. The fact that twitching motility is not obligatorily associated with phage sensitivity suggests that the latter may not be directly dependent upon fimbrial function but rather may be a consequence of some common factor(s) involved in their assembly or export pathways.

Isolation of an R- M+ mutant of Yersinia enterocolitica serotype O:8 and its application in construction of rough mutants utilizing mini-Tn5 derivatives and lipopolysaccharide-specific phage.

A generally applicable procedure was used to isolate a spontaneous restriction-deficient mutant of Yersinia enterocolitica serotype O:8. Transposition frequency in the mutant strain 8081-res was approximately 6.7 x 10(-6) per recipient, while it was practically zero in the wild-type strain 8081-c. Mobilization frequency into 8081-res was 10(5) times higher than that into the wild-type strain. The mutant had lost the ability to express the YenI restriction endonuclease activity present in serotype O:8 strains. This allowed the construction of a transposon library in 8081-res. Insertion mutants with transposons in the genes of the rfa region were selected from this library.