Identification of a siderophore receptor required for ferric ornibactin uptake in Burkholderia cepacia

Ornibactins are linear hydroxamate siderophores produced by Burkholderia cepacia with peptide structures similar to that of pyoverdines produced by the fluorescent pseudomonads. The gene encoding the outer membrane receptor (orbA) was identified, sequenced, and demonstrated to have significant homology with hydroxamate receptors produced by other organisms. The orbA precursor was predicted to be a protein with a molecular mass of 81 kDa. An orbA mutant was constructed and demonstrated to be unable to take up (59)Fe-ornibactins or to grow in medium supplemented with ornibactins. Outer membrane protein profiles from the parent strain, K56-2, revealed an iron-regulated outer membrane protein of 78 kDa that was not detectable in the K56orbA::tp mutant. When this mutant harbored a plasmid containing the orbA gene, the 78-kDa protein was present in the outer membrane protein profiles and the mutant was able to utilize ornibactin to acquire iron. The orbA mutant was less virulent in a chronic respiratory infection model than the parent strain, indicating that ornibactin uptake and utilization are important in the pathogenesis of B. cepacia respiratory infections.     

Siderophore Production by Cystic Fibrosis Isolates of Burkholderia cepacia

Sixty-one Burkholderia cepacia isolates from patients with cystic fibrosis (CF) and four plant isolates were screened for production of the siderophores salicylic acid (SA), pyochelin, cepabactin, and ornibactins and fingerprinted by a PCR-based randomly amplified polymorphic DNA (RAPD) method. Of the 24 RAPD types determined, 22 (92%) were associated with isolates that produced SA, 21 (87%) were associated with isolates that produced ornibactins, 15 (60%) were associated with isolates that produced pyochelin, and 3 (12%) were associated with isolates that produced cepabactin. Of the 24 RAPD types plus 2 phenotypic variants of types 1 and 9, 3 were associated with isolates that produced all four siderophores, 8 were associated with isolates that produced three siderophores, 12 were associated with isolates that produced two siderophores, and 3 were associated with isolates that produced only one siderophore. These results suggest that the numbers and types of siderophores produced by CF isolates of B. cepacia correlate with RAPD type and that SA and ornibactins are the most prevalent siderophores produced.     

Comparative analysis of plant and animal models for characterization of Burkholderia cepacia virulence

A simple alfalfa model was developed as an alternative infection model for virulence studies of the Burkholderia cepacia complex. Symptoms of disease were observed in wounded alfalfa seedlings within 7 days following inoculation of 10(1) to 10(5) CFU of most strains of the B. cepacia complex. Strains from seven genomovars of the B. cepacia complex were tested for virulence in the alfalfa model, and the degree of virulence was generally similar in strains belonging to the same genomovar. Strains of Burkholderia multivorans and some strains of Burkholderia stabilis did not cause symptoms of disease in alfalfa seedlings. Representative strains were also tested for virulence using the rat agar bead model. Most of the strains tested were able to establish chronic lung infections; B. stabilis strains were the exception. Most of the strains that were virulent in the alfalfa infection model were also virulent in the lung infection model. The B. cepacia genomovar III mutants K56pvdA::tp and K56-H15 were significantly less virulent in the alfalfa infection model than their parent strain. Therefore, this alfalfa infection model may be a useful tool for assessing virulence of strains of the B. cepacia complex and identifying new virulence-associated genes.     

Identification of gene products involved in biofilm production by Moraxella catarrhalis ETSU-9 in vitro

Moraxella catarrhalis ETSU-9 was subjected to random transposon insertion mutagenesis to identify genes encoding products involved in the ability of the organism to form biofilms in vitro. Screening of approximately 3,000 transposon insertion mutants in the crystal violet-based biofilm assay system yielded six mutants that exhibited greatly reduced abilities to form biofilms. Three of these mutants had transposon insertions in the uspA2H gene, which encodes a surface protein previously shown to be involved in the ability of M. catarrhalis to both attach to human cell lines in vitro and resist killing by normal human serum. Random insertion mutagenesis of the uspA2H gene, involving the introduction of a 15-nucleotide fragment encoding 5 amino acids, was used to attempt to identify the domain(s) necessary for biofilm formation. Most of these insertions adversely affected biofilm formation, whereas the abilities of these same mutants to attach to Chang conjunctival epithelial cells in vitro were usually not reduced. Gain-of-function experiments showed that introduction of the M. catarrhalis ETSU-9 uspA2H gene into Escherichia coli conferred biofilm formation ability on this recombinant strain. Two of the other three M. catarrhalis ETSU-9 transposon insertion mutants that had greatly reduced abilities to form biofilms were shown to have insertions in genes encoding products predicted to be directly or indirectly involved in cell wall metabolism.     

Isolation of Citrobacter sp. mutants defective in decolorizing malachite green

To identify genes involved in the decolorization of malachite green, random mutants generated by transposon insertion in the malachite green-decolorizing bacterium, Citrobacter sp. were isolated. The resulting mutant bank yielded 24 mutants with complete defects in their abilities to decolorize malachite green. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 7 mutants, which appeared to have insertions at different sites of the chromosome. The Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. Based on a sequence database, the putative protein products encoded by the mg genes were identified as follows. mg3, an ABC transporter homolog; mg6, a LysR-type regulatory protein; m11, an oxidoreductase; mg17, a MalG protein in the maltose transport system; and mg21, a sugar kinase. The deduced sequences from two mg genes (mg7 and mg18) showed no significant similarity to any protein with a known function, suggesting that these two mg genes encode unidentified proteins that are responsible for the decolorization of malachite green.     

Genome-Wide Transposon Mutagenesis in Pathogenic Leptospira Species

Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans.     

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 the Pasteurellaceae family cause disease and identify novel live vaccine candidates and new potential antibiotic targets.     

Isolation and characterization of mini-Tn5Km2 insertion mutants of Brucella abortus deficient in internalization and intracellular growth in HeLa cells

Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. To identify genes related to internalization and multiplication in host cells, Brucella abortus was mutagenized by mini-Tn5Km2 transposon that carryied the kanamycin resistance gene, 4,400 mutants were screened, and HeLa cells were infected with each mutant. Twenty-three intracellular-growth-defective mutants were screened and were characterized for internalization and intracellular growth. From these results, we divided the mutants into the following three groups: class I, no internalization and intracellular growth within HeLa cells; class II, an internalization similar to that of the wild type but with no intracellular growth; and class III, internalization twice as high as the wild type but with no intracellular growth. Sequence analysis of DNA flanking the site of transposon showed various insertion sites of bacterial genes that are virulence-associated genes, including virB genes, an ion transporter system, and biosynthesis- and metabolism-associated genes. These internalization and intracellular-growth-defective mutants in HeLa cells also showed defective intracellular growth in macrophages. These results suggest that the virulence-associated genes isolated here contributed to the intracellular growth of both nonprofessional and professional phagocytes.     

Identification of a virulence-associated determinant, dihydrolipoamide dehydrogenase (lpd), in Mycoplasma gallisepticum through in vivo screening of transposon mutants

To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.     

Identification of Actinobacillus pleuropneumoniae genes important for survival during infection in its natural host

Actinobacillus pleuropneumoniae is a strict respiratory tract pathogen of swine and is the causative agent of porcine pleuropneumonia. We have used signature-tagged mutagenesis (STM) to identify genes required for survival of the organism within the pig. A total of 2,064 signature-tagged Tn10 transposon mutants were assembled into pools of 48 each, and used to inoculate pigs by the endotracheal route. Out of 105 mutants that were consistently attenuated in vivo, only 11 mutants showed a >2-fold reduction in growth in vitro compared to the wild type, whereas 8 of 14 mutants tested showed significant levels of attenuation in pig as evidenced from competitive index experiments. Inverse PCR was used to generate DNA sequence of the chromosomal domains flanking each transposon insertion. Only one sibling pair of mutants was identified, but three apparent transposon insertion hot spots were found--an anticipated consequence of the use of a Tn10-based system. Transposon insertions were found within 55 different loci, and similarity (BLAST) searching identified possible analogues or homologues for all but four of these. Matches included proteins putatively involved in metabolism and transport of various nutrients or unknown substances, in stress responses, in gene regulation, and in the production of cell surface components. Ten of the sequences have homology with genes involved in lipopolysaccharide and capsule production. The results highlight the importance of genes involved in energy metabolism, nutrient uptake and stress responses for the survival of A. pleuropneumoniae in its natural host: the pig.     

Construction and comparison of recombinant plasmids encoding type 1 fimbriae of members of the family Enterobacteriaceae.

The genes encoding type 1 fimbriae of Salmonella typhimurium, Enterobacter cloacae, and Serratia marcescens were cloned in Escherichia coli. All transformants possessing recombinant plasmids were shown to be fimbriate and demonstrated mannose-sensitive hemagglutinating activity. A comparison of the physical maps of these plasmids revealed little similarity among them, although plasmids encoding type 1 fimbriae of Escherichia coli and Klebsiella pneumoniae appeared similar with respect to restriction enzyme sites. The fimbrial gene cluster ranged in size from 5.5 to 9.0 kilobase pairs as determined by transposon mutagenesis. Plasmid-containing E. coli strains were shown to produce species-specific fimbrial antigens with little or no cross-reactivity between genera. Therefore, it was presumed that each plasmid contained the gene encoding the fimbrial subunit. Complementation was not detected between nonfimbriate insertion mutants of different species but was seen with mutants of the same species.     

Transposon mutagenesis reveals novel loci affecting tolerance to salt stress and growth at low temperature

Using transposon mutagenesis in the haploid Saccharomyces cerevisiae strain W303-1A we have identified genes required for growth in high salt medium, survival of a hypo-osmotic shock and growth at 15 degrees C. Screening 25,000 transposon insertions revealed a total of 61 insertions that caused salt-sensitivity; and those insertions affected 31 genes. Only 12 of those genes were previously known to be required for salt-tolerance. Among the 61 insertions, three caused general osmo-sensitivity. We identified one single insertion mutant in the already-known gene, FPS1, required for survival of hypo-osmotic shock. A total of 31 insertions caused failure to grow at low temperature. Those identified ten different genes, three of which had previously been reported to affect cold-tolerance. Four genes were identified in both the salt and the cold-sensitivity screen. We found several unusual insertion mutations: (1) insertions in or close to essential genes, (2) insertion in an intergenic region and (3) insertions causing stress-sensitivity in W303-1A, while the deletion mutant in BY4741 did not show such a phenotype. Surprisingly, our mutant set and that reported in the large-scale transposon insertion project (TRIPLES, http://ygacmed.yale.edu/triples/triples.htm) only marginally overlap. We discuss some of the features of transposon mutagenesis in light of the availability of the complete set of yeast deletion mutants and we discuss the possible roles of the genes we identified.     

Identification of Burkholderia cenocepacia genes required for bacterial survival in vivo

Burkholderia cenocepacia (formerly Burkholderia cepacia complex genomovar III) causes chronic lung infections in patients with cystic fibrosis. In this work, we used a modified signature-tagged mutagenesis (STM) strategy for the isolation of B. cenocepacia mutants that cannot survive in vivo. Thirty-seven specialized plasposons, each carrying a unique oligonucleotide tag signature, were constructed and used to examine the survival of 2,627 B. cenocepacia transposon mutants, arranged in pools of 37 unique mutants, after a 10-day lung infection in rats by using the agar bead model. The recovered mutants were screened by real-time PCR, resulting in the identification of 260 mutants which presumably did not survive within the lungs. These mutants were repooled into smaller pools, and the infections were repeated. After a second screen, we isolated 102 mutants unable to survive in the rat model. The location of the transposon in each of these mutants was mapped within the B. cenocepacia chromosomes. We identified mutations in genes involved in cellular metabolism, global regulation, DNA replication and repair, and those encoding bacterial surface structures, including transmembrane proteins and cell surface polysaccharides. Also, we found 18 genes of unknown function, which are conserved in other bacteria. A subset of 12 representative mutants that were individually examined using the rat model in competition with the wild-type strain displayed reduced survival, confirming the predictive value of our STM screen. This study provides a blueprint to investigate at the molecular level the basis for survival and persistence of B. cenocepacia within the airways.     

At Least Four Percent of the Salmonella typhimurium Genome Is Required for Fatal Infection of Mice

Salmonella typhimurium infection of mice is an established model system for studying typhoid fever in humans. Using this model, we identified S. typhimurium genes which are absolutely required to cause fatal murine infection by testing independently derived transposon insertion mutants for loss of virulence in vivo. Of the 330 mutants tested intraperitoneally and the 197 mutants tested intragastrically, 12 mutants with 50% lethal doses greater than 1,000 times that of the parental strain were identified. These attenuated mutants were characterized by in vitro assays which correlate with known virulence functions. In addition, the corresponding transposon insertions were mapped within the S. typhimurium genome and the nucleotide sequence of the transposon-flanking DNA was obtained. Salmonella spp. and related bacteria were probed with flanking DNA for the presence of these genes. All 12 attenuated mutants had insertions in known genes, although the attenuating effects of only two of these were previously described. Furthermore, the proportion of attenuated mutants obtained in this study suggests that mutations in about 4% of the Salmonella genome lead to 1,000-fold or greater attenuation in the mouse typhoid model of infection. Most of these genes appear to be required during the early stages of a natural infection.     

Transposon-mediated mutagenesis of a baculovirus

Spontaneous mutants of insect nuclear polyhedrosis viruses of Autographa californica (AcMNPV) and Galleria mellonella (GmMNPV) were analyzed. These mutants produce few polyhedra in infected cells and have insertions of host cell DNA. All the insertions mapped to two adjacent sites in the genome. The junctions between two host insertions and the viral DNA were sequenced. One of the insertions contained a perfect 7-bp inverted terminal repeat, and had caused a direct duplication of 4 bp of viral DNA at the point of insertion. Therefore, this insertion sequence has properties of a transposon of the host cell Trichoplusia ni.     

A system for generalized mutagenesis of Haemophilus ducreyi.

The lack of a generalized mutagenesis system for Haemophilus ducreyi has hampered efforts to identify virulence factors expressed by this sexually transmitted pathogen. To address this issue, the transposable element Tn1545-delta 3, which encodes resistance to kanamycin, was evaluated for its ability to insert randomly into the H. ducreyi chromosome and produce stable, isogenic mutants. Electroporation of H. ducreyi with 1 microgram of plasmid pMS1 carrying Tn1545-delta 3 resulted in the production of 10(4) kanamycin-resistant transformants; Southern blot analysis of a number of these transformants indicated that insertion of the transposon into the chromosome occurred at a number of different sites. This pMS1-based transposon delivery system was used to produce an H. ducreyi mutant that expressed an altered lipooligosaccharide (LOS). Passage of this mutant in vitro in the presence or absence of kanamycin did not affect the stability of the transposon insertion. To confirm that the observed mutant phenotype was the result of the transposon insertion, a chromosomal fragment containing Tn1545-delta 3 was cloned from this H. ducreyi LOS mutant. Electroporation of the wild-type H. ducreyi strain with this DNA fragment yielded numerous kanamycin-resistant transformants, the majority of which had the same altered LOS phenotype as the original mutant. Southern blot analysis confirmed the occurrence of proper allelic exchange in the LOS-deficient transformants obtained in this backcross experiment. The ability of Tn1545-delta 3 to produce insertion mutations in H. ducreyi should facilitate genetic analysis of this pathogen.     

Identification of motility and autoagglutination Campylobacter jejuni mutants by random transposon mutagenesis

Campylobacter jejuni has been identified as the leading cause of acute bacterial diarrhea in the United States, yet compared with other enteric pathogens, considerably less is understood concerning the virulence factors of this human pathogen. A random in vivo transposon mutagenesis system was recently developed for the purpose of creating a library of C. jejuni transformants. A total of 1,065 C. jejuni transposon mutants were screened for their ability to swarm on motility agar plates and autoagglutinate in liquid cultures; 28 mutants were subsequently identified. The transposon insertion sites were obtained by using random-primed PCR, and the putative genes responsible for these phenotypes were identified. Of these mutants, all 28 were found to have diminished motility (0 to 86% that of the control). Seventeen motility mutants had insertions in genes with strong homology to functionally known motility and chemotaxis genes; however, 11 insertions were in genes of unknown function. Twenty motility mutants were unable to autoagglutinate, suggesting that the expression of flagella is correlated with autoagglutination (AAG). However, four mutants expressed wild-type levels of surface FlaA, as indicated by Western blot analysis, yet were unable to autoagglutinate (Cj1318, Cj1333, Cj1340c, and Cj1062). These results suggest that FlaA is necessary but not sufficient to mediate the AAG phenotype. Furthermore, two of the four AAG mutants (Cj1333 and Cj1062) were unable to invade INT-407 intestinal epithelial cells, as determined by a gentamicin treatment assay. These data identify novel genes important for motility, chemotaxis, and AAG and demonstrate their potential role in virulence.