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PubMed1997

Construction of an 800-kb contig in the near-centromeric region of the rice blast resistance gene Pi-ta2 using a highly representative rice BAC library.

We constructed a rice Bacterial Artificial Chromosome (BAC) library from green leaf protoplasts of the cultivar Shimokita harboring the rice blast resistance gene Pi-ta. The average insert size of 155 kb and the library size of seven genome equivalents make it one of the most comprehensive BAC libraries available, and larger than many plant YAC libraries. The library clones were plated on seven high density membranes of microplate size, enabling efficient colony identification in colony hybridization experiments. Seven percent of clones carried chloroplast DNA. By probing with markers close to the blast resistance genes Pi-ta2(closely linked to Pi-ta) and Pi-b, respectively located in the centromeric region of chromosome 12 and near the telomeric end of chromosome 2, on average 2.2 +/- 1.3 and 8.0 +/- 2.6 BAC clones/marker were isolated. Differences in chromosomal structures may contribute to this wide variation in yield. A contig of about 800 kb, consisting of 19 clones, was constructed in the Pi-ta2 region. This region had a high frequency of repetitive sequences. To circumvent this difficulty, we devised a "two-step walking" method. The contig spanned a 300 kb region between markers located at 0 cM and 0.3 cM from Pi-ta. The ratio of physical to genetic distances (> 1,000 kb/cM) was more than three times larger than the average of rice (300 kb/cM). The low recombination rate and high frequency of repetitive sequences may also be related to the near centromeric character of this region. Fluorescent in situ hybridization (FISH) with a BAC clone from the Pi-b region yielded very clear signals on the long arm of chromosome 2, while a clone from the Pi-ta2 region showed various cross-hybridizing signals near the centromeric regions of all chromosomes.

Centromere↗
PubMed1998

Identification of PhoP-PhoQ activated genes within a duplicated region of the Salmonella typhimurium chromosome.

Salmonellae virulence requires the PhoP-PhoQ two-component regulatory system. PhoP-PhoQ activate the transcription of genes following phagocytosis by macrophages which are necessary for survival within the phagosome environment. Thirteen previously undefined PhoP-activated gene fusions generated by MudJ and TnphoA (pag A, and E-P, respectively) were cloned and sequenced. Most pag products show no similarity to proteins in the database, while others are predicted to encode: a UDP-glucose dehydrogenase (pagA); a protein with similarity to the product of an E. coli aluminium-induced gene (pagH); a protein encoded within a Salmonella-unique region adjacent to the sinR gene (pagN); a protein similar to a product of the Yersinia virulence plasmid (pagO); and a protein with similarity to CrcA which is necessary for resistance of E. coli to camphor (pagP). Of the pag characterized, only pagK, M and O were closely linked. pagJ and pagK were shown to be unlinked but nearly identical in DNA sequence, as each was located within a 1.6 kb DNA duplication. The translations of sequences surrounding pagJ and pagK show similarity to proteins from extrachromosomal elements as well as those involved in DNA transposition and rearrangement, suggesting that this region may have been or is a mobile element. The transcriptional start sites of pagK, M, and J were determined; however, comparison to other known pag gene promoters failed to reveal a consensus sequence for PhoP-regulated activation. DNA sequences hybridizing to a Salmonella typhimurium pagK specific probe were found in S. enteritidis but absent in other Salmonella serotypes and Enterobacteriaceae tested, suggesting that these genes are specific for broad host range Salmonellae that cause diarrhoea in humans. Cumulatively, these data further demonstrate: (1) that PhoP-PhoQ is a global regulator of the production of diverse envelope or secreted proteins; (2) that PhoP-PhoQ regulate the production of proteins of redundant function; and (3) that pag are often located in regions of horizontally acquired DNA that are absent in other Enterobacteriaceae.

Artificial Gene Fusion↗
PubMed1990

Identification, cloning, and sequencing of piv, a new gene involved in inverting the pilin genes of Moraxella lacunata.

Moraxella lacunata is a bacterium that is a causative agent of human conjunctivitis and keratitis. We have previously cloned the Q and I pilin (formerly called beta and alpha pilin) genes of Moraxella bovis and determined that an inversion of 2 kilobases (kb) of DNA determines which pilin gene is expressed. Using an M. bovis pilin gene as a hybridization probe to screen a lambda ZAP library of M. lacunata DNA, we have isolated a clone that not only contains the entire type 4 pilin gene inversion region of M. lacunata but inverts the 2-kb region on a plasmid subclone (pMxL1) in Escherichia coli. Deletion derivatives of pMxL1 yielded some plasmids that still had the entire inversion region but were phase locked into one or the other of the two potential orientations. Similarly, insertions of a 2-kb streptomycin-resistant element (omega) within some regions outside of the inversion also resulted in phase-locked plasmids. These deletions and insertions thus localize a probable invertase necessary for the inversion event. The region was sequenced, and an open reading frame with over 98% DNA sequence homology to an open reading frame that we previously found in M. bovis and called ORF2 appeared to be a strong candidate for the invertase. This conclusion was confirmed when a plasmid containing the M. bovis ORF2 supplied, in trans, the inversion function missing from one of the M. lacunata phase-locked inversion mutants. We have named these putative invertase genes piv(ml) (pilin inversion of M. lacunata) and piv(mb) (pilin inversion of M. bovis). Despite previously noted sequence similarities between the M. bovis sites of inversion and those of the Hin family of invertible segments and a 60-base-pair region within the inversion with 50% sequence similarity to the cin recombinational enhancer, there is no significant sequence similarity of the Piv invertases to the Hin family of invertases.

Amino Acid Sequence↗
PubMed2006

Identification of potential virulence determinants by Himar1 transposition of infectious Borrelia burgdorferi B31.

Lyme disease Borrelia organisms are highly invasive spirochetes that alternate between vertebrate and arthropod hosts and that establish chronic infections and elicit inflammatory reactions in mammals. Although progress has been made in the targeted mutagenesis of individual genes in infectious Borrelia burgdorferi, the roles of the vast majority of gene products in pathogenesis remain unresolved. In this study, we examined the feasibility of using transposon mutagenesis to identify infectivity-related factors in B. burgdorferi. The transformable, infectious strain 5A18 NP1 was transformed with the spirochete-adapted Himar1 transposon delivery vector pMarGent to create a small library of 33 insertion mutants. Single mouse inoculations followed by culture of four tissue sites and serology were used to screen the mutants for infectivity phenotypes. Mutants that appeared attenuated (culture positive at some sites) or noninfectious (negative at all sites) and contained the virulence-associated plasmids lp25 and lp28-1 were examined in more extensive animal studies. Three of these mutants (including those with insertions in the putative fliG-1-encoded flagellar motor switch protein and the guaB-encoded IMP dehydrogenase) were noninfectious, whereas four clones appeared to exhibit reduced infectivity. Serological reactivity in VlsE enzyme-linked immunosorbent assays correlated with the assignment of mutants to the noninfectious or attenuated-infectivity groups. The results of this study indicate that random transposon mutagenesis of infectious B. burgdorferi is feasible and will be of value in studying the pathogenesis of Lyme disease Borrelia.

Animals↗
PubMed2002

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.

Agglutination↗
PubMed2005

Identification of new secreted effectors in Salmonella enterica serovar Typhimurium.

A common theme in bacterial pathogenesis is the secretion of bacterial products that modify cellular functions to overcome host defenses. Gram-negative bacterial pathogens use type III secretion systems (TTSSs) to inject effector proteins into host cells. The genes encoding the structural components of the type III secretion apparatus are conserved among bacterial species and can be identified by sequence homology. In contrast, the sequences of secreted effector proteins are less conserved and are therefore difficult to identify. A strategy was developed to identify virulence factors secreted by Salmonella enterica serovar Typhimurium into the host cell cytoplasm. We constructed a transposon, which we refer to as mini-Tn5-cycler, to generate translational fusions between Salmonella chromosomal genes and a fragment of the calmodulin-dependent adenylate cyclase gene derived from Bordetella pertussis (cyaA'). In-frame fusions to bacterial proteins that are secreted into the eukaryotic cell cytoplasm were identified by high levels of cyclic AMP in infected cells. The assay was sufficiently sensitive that a single secreted fusion could be identified among several hundred that were not secreted. This approach identified three new effectors as well as seven that have been previously characterized. A deletion of one of the new effectors, steA (Salmonella translocated effector A), attenuated virulence. In addition, SteA localizes to the trans-Golgi network in both transfected and infected cells. This approach has identified new secreted effector proteins in Salmonella and will likely be useful for other organisms, even those in which genetic manipulation is more difficult.

Adenylate Cyclase Toxin↗
PubMed2000

Identification and characterization of a thrombomodulin gene mutation coding for an elongated protein with reduced expression in a kindred with myocardial infarction.

Thrombomodulin is an endothelial cell receptor for thrombin. It functions as a natural anticoagulant by greatly accelerating activation of protein C by thrombin. Using a direct gene screening strategy we identified a frameshift insertion mutation, insT 1689, in the thrombomodulin gene of a patient with myocardial infarction. The mutation predicts an elongated gene product because of substitution of the 12 C-terminal amino acids by 61 abnormal residues. Pedigree analysis showed that the mutation was also likely to have been present in a sibling who had had fatal myocardial infarction. Carriers of the mutant allele express significantly lower amounts of thrombomodulin on the surface of their monocytes detected by flow cytometry and have lower levels of soluble thrombomodulin in plasma. Wild type and the mutant thrombomodulin were expressed in COS-7 cells. Cellular distribution of the expressed proteins was evaluated by immunofluorescence microscopy, which showed reduced cell surface expression and intense juxtanuclear localization of the abnormal protein. This suggests impaired translocation through the endoplasmic reticulum/Golgi apparatus. Cells expressing abnormal thrombomodulin had reduced ability ( approximately 2.5-fold) to accelerate the thrombin mediated activation of protein C. This is the first demonstration of reduced expression arising from a natural thrombomodulin gene mutation. The results provide support for the suggestion that gene mutation of thrombomodulin may be important in the pathogenesis of some cases of occlusive thrombotic disease. (Blood. 2000;95:569-576)

Adult↗
PubMed2002

Genetic and phenotypic analysis of Acinetobacter baumannii insertion derivatives generated with a transposome system.

Acinetobacter baumannii is a metabolically versatile pathogen that causes severe infections in compromised patients. However, little is known about the genes and factors involved in its basic physiology and virulence properties. Insertion mutagenesis was used to initiate the identification and characterization of some of these factors and genes in the prototype strain 19606. The utilization of the pLOFKm suicide delivery vector, which harbors a suicide mini-Tn10 derivative, proved to be unsuccessful for this purpose. The EZ::TN Tnp transposome system available from Epicentre was then used in conjunction with electroporation to generate isogenic insertional derivatives of A. baumannii 19606. Replica plating showed that 2% of the colonies that grew after electroporation on agar plates without antibiotics also grew in the presence of 40 micro g of kanamycin per ml. DNA hybridization proved that all of the kanamycin-resistant derivatives contained the EZ::TN insertion element, which was mapped to different genomic locations. Replica plating on Simmons citrate agar and microtiter plate-plastic tube assays identified growth- and biofilm-defective derivatives, respectively. The location of the insertion in several of these derivatives was determined by self-ligation of NdeI- or EcoRI-digested genomic DNA and electroporation of Escherichia coli TransforMax EC100D (pir(+)). Sequence analysis of the recovered plasmids showed that some of the A. baumannii 19606 growth-defective derivatives contain insertions within genes encoding activities required for the generation of energy and cell wall components and for the biosynthesis of amino acids and purines. A gene encoding a protein similar to the GacS sensor kinase was interrupted in four derivatives, while another had an insertion in a gene coding for a hypothetical sensor kinase. A. baumannii 19606 derivatives with defective attachment or biofilm phenotypes had insertions within genes that appear to be part of a chaperone-usher transport system described for other bacteria. DNA hybridization experiments showed that the presence of strain 19606 genes encoding regulatory and attachment or biofilm functions is widespread among other A. baumannii clinical isolates.

Acinetobacter baumannii↗
PubMed2026

Mobilization of blaVIM genes via the Tn6292 transposon among carbapenem-resistant Enterobacter cloacae complex isolates from colonized patients in a Spanish hospital.

UNLABELLED: The aim of this study was to perform molecular characterization of the carbapenem-resistant Enterobacter cloacae complex (ECC) isolates from colonized patients in a hospital using whole-genome sequencing (WGS) technology. As part of routine surveillance for multidrug-resistant bacterial colonization, 21 ECC isolates were recovered from patients at San Carlos Hospital in Madrid (Spain) between December 2020 and November 2024. WGS was used to determine their genetic relatedness. Furthermore, species identification, sequence type (ST), resistome, plasmid content, and flanking mobile genetic elements (MGEs) of the carbapenemase genes were derived from the WGS data. The most prevalent carbapenemase gene identified was blaVIM-1 (n = 18, 85.7%), with other notable genes including blaKPC-2 (n = 1, 4.8%), blaKPC-3 (n = 1, 4.8%), and blaOXA-48 (n = 1, 4.8%). Several blaACT and blaESBL variants were also found among the carbapenem-resistant ECC isolates. All of them carried at least one blaACT gene, with blaACT-7 (11/21) and blaTEM-type (14/21) genes being the most common AmpC and ESBL-encoding genes, respectively. Additionally, two isolates exhibited the presence of the mcr-9 gene. Overall, E. hormaechei subsp. steigerwaltii (ST93), followed by E. hormaechei subsp. hoffmanii (ST78 and ST50), were the predominant species and STs circulating among the carbapenem-resistant ECC strains. The blaVIM-1 gene was part of class 1 integrons located within a Tn3-family transposon, Tn6292. blaKPC and blaOXA-48 were linked to Tn4401 and Tn1999 transposons, respectively. In conclusion, the presence of the blaVIM within a transposon Tn6292 enhances its mobility across bacterial genomes, underscoring the value of high-throughput sequencing in monitoring the spread of carbapenem-resistant ECC isolates. IMPORTANCE: This study highlights why monitoring the spread of antibiotic-resistant bacteria in hospitals is critical. By analyzing the complete DNA of carbapenem-resistant bacteria, antibiotics were considered a last line of treatment. We found that the resistance genes are not isolated. Instead, they are embedded within mobile elements called transposons. This means that they can "jump" between different bacteria, accelerating the spread of resistance. These findings emphasize the importance of high-resolution genomic technologies to track and control the spread of these dangerous bacteria in clinical settings, helping preserve the effectiveness of life-saving treatments.

Humans↗
PubMed1984

Analysis of the trfA region of broad host-range plasmid RK2 by transposon mutagenesis and identification of polypeptide products.

Broad host-range plasmid RK2 is a member of the Escherichia coli incompatibility group P. Unlike most other groups of plasmids, members of the P group are capable of efficient transfer between and maintenance in most gram-negative bacterial species. It is of interest whether this broad host-range results from differences between the mechanism of replication of broad and narrow host-range plasmids. The regions of RK2 required for replication in E. coli have previously been defined as an origin of vegetative replication, oriVRK2 , and a gene, trfA , specifying a positively required trans-acting product. In this study Tn1723 transposon insertions have been used to map the trfA gene and determine its functional gene product. The Tn1723 insertions define the outer limits of the gene, a promoter region, a "leader" region not essential for trfA activity and a coding region. Three polypeptides of 13 X 10(3), 43 X 10(3) and 32 X 10(3) molecular weight are produced from this region and the production of a 32 X 10(3) Mr polypeptide is shown to be correlated with trfA activity in E. coli. Analysis of polypeptides produced from transposon insertion derivatives in which all but 35 base-pairs of inserted DNA is deleted, along with the effect of these insertions on trfA activity, suggest that the 43 X 10(3) and 32 X 10(3) Mr polypeptide coding sequences overlap in the same reading frame and that all three polypeptides (13 X 10(3), 32 X 10(3) and 43 X 10(3) Mr) may be translated from the same initial transcript.

Chromosome Mapping↗
PubMed2004

Rhesus macaque class I duplicon structures, organization, and evolution within the alpha block of the major histocompatibility complex.

The alpha block of the human and chimpanzee major histocompatibility complex (MHC) class I genomic region contains 10 to 11 duplicated MHC class I genes, including the HLA/Patr-A, -G, and -F genes. In comparison, the alpha block of the rhesus macaque (Macaca mulatta, Mamu) has an additional 20 MHC class I genes within this orthologous region. The present study describes the identification and analysis of the duplicated segmental genomic structures (duplicons) and genomic markers within the alpha block of the rhesus macaque and their use to reconstruct the duplication history of the genes within this region. A variety of MHC class I genes, pseudogenes, transposons, and retrotransposons, such as Alu and ERV16, were used to categorize the 28 duplicons into four distinct structural categories. The phylogenetic relationship of MHC class I genes, Alu, and LTR16B sequences within the duplicons was examined by use of the Neighbor-Joining (NJ) method. Two single-duplicon tandem duplications, two polyduplicon tandem duplications with an accompanying inversion product per duplication, eight polyduplicon tandem duplications steps, 12 deletions, and at least two recombinations were reconstructed to explain the highly complex organization and evolution of the 28 duplicons (nine inversions) within the Mamu alpha block. On the basis of the phylogenetic evidence and the reconstructed tandem duplication history of the 28 duplicons, the Mamu/Patr/HLA-F ortholog was the first MHC class I gene to have been fixed without further duplication within the alpha block of primates. Assuming that the rhesus macaque and the chimpanzee/human lineages had started with the same number of MHC class I duplicons at the time of their divergence approximately 24 to 31 MYA, then the number of genes within the alpha block have been duplicated at an approximately three times greater rate in the rhesus macaque than in either the human or chimpanzee.

Animals↗
PubMed1988

Identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis.

Surfactin is a lipopeptide antibiotic produced by the cells of Bacillus subtilis ATCC 21332. A genetic locus responsible for surfactin production (sfp) was transferred from ATCC 21332 to JH642, a derivative of the standard B. subtilis 168. To study the sfp locus at the molecular level, a Tn917 insertion mutant that was blocked in surfactin production (srf) was isolated. The srf::Tn917 mutation was found to be closely linked to sfp, and both loci mapped by PBS1 phage transduction to the chromosomal region between aroI and mtlB. These studies suggest that JH642, a strain which is not a producer of surfactin (genotypically sfp0), contains at least some of the genes encoding surfactin production. Expression of the srf gene(s) was examined in both sfp and sfp0 cells by assaying beta-galactosidase activity encoded by a promoterless lacZ gene that was fused to the srf::Tn917 insertion. In cells of both strains, srf-directed beta-galactosidase activity increased when cells entered the stationary phase of the growth curve, but the activity in sfp cells was higher than that in sfp0 cells. srf-lacZ expression was partially impaired by a mutation in spo0A. In sfp0 cells, this dependence on the spo0A gene product could be entirely bypassed by an abrB suppressor mutation. In the sfp cells, the abrB mutation could not restore the defect conferred by the spo0A mutation. These data suggest that the sfp locus, which is responsible for surfactin production, alters the transcriptional regulation of srf in JH642 cells.

Anti-Bacterial Agents↗
PubMed1995

Genotypic identification of two groups within the species Bacteroides fragilis by ribotyping and by analysis of PCR-generated fragment patterns and insertion sequence content.

Molecular typing allowed the separation of the species Bacteroides fragilis into two genotypically distinct groups. A unique set of 50 strains of B. fragilis carrying the chromosomal metallo-beta-lactamase gene cfiA was subjected to a comparative analysis with respect to sets of up to 250 randomly collected strains devoid of this gene. The two groups were found to be distinct on the basis of the following results: (i) ribotyping, after DNA digestion with AvaI, revealed a practically homogeneous DNA fragment pattern for the cfiA-positive strains and distinct multiple patterns for the cfiA-negative strains; (ii) PCR, arbitrarily primed with an experimentally selected decamer, generated fragment patterns typical for the strains of each group; (iii) the three insertion sequences described to date in the species B. fragilis, i.e., IS4351, IS942, and IS1186, were all but confined to the cfiA-positive group, in which they were capable of providing promoter sequences for the transcription of cfiA; and (iv) the cepA gene, encoding the so-called endogenous cephalosporinase of B. fragilis, was found exclusively in the cfiA-negative group, in which it was present in ca. 70% of the strains. The cfiA-, cepA-negative fraction was not characterized further. In a natural population of 500 randomly selected strains of B. fragilis, the cfiA-positive and cfiA-negative groups represented ca. 3 and 97% of the strains, respectively. Analysis of 82 metabolic traits revealed no difference between the two groups.

Bacterial Proteins↗
PubMed1998

Characterization of glucose-specific catabolite repression-resistant mutants of Bacillus subtilis: identification of a novel hexose:H+ symporter.

Insertional mutagenesis was conducted on Bacillus subtilis cells to screen for mutants resistant to catabolite repression. Three classes of mutants that were resistant to glucose-promoted but not mannitol-promoted catabolite repression were identified. Cloning and sequencing of the mutated genes revealed that the mutations occurred in the structural genes for (i) enzyme II of the phosphoenolpyruvate-glucose phosphotransferase (PtsG), (ii) antiterminator GlcT, which controls PtsG synthesis, and (iii) a previously uncharacterized carrier of the major facilitator superfamily, which we have designated GlcP. The last protein exhibits greatest sequence similarity to the fucose:H+ symporter of Escherichia coli and the glucose/galactose:H+ symporter of Brucella abortus. In a wild-type B. subtilis genetic background, the glcP::Tn10 mutation (i) partially but specifically relieved glucose- and sucrose-promoted catabolite repression, (ii) reduced the growth rate in minimal glucose medium, and (iii) reduced rates of [14C]glucose and [14C]methyl alpha-glucoside uptake. In a delta pts genetic background no phenotype was observed, suggesting that expression of the glcP gene required a functional phosphotransferase system. When overproduced in a delta pts mutant of E. coli, GlcP could be shown to specifically transport glucose, mannose, 2-deoxyglucose and methyl alpha-glucoside with low micromolar affinities. Accumulation of the nonmetabolizable glucose analogs was demonstrated, and inhibitor studies suggested a dependency on the proton motive force. We conclude that B. subtilis possesses at least two distinct routes of glucose entry, both of which contribute to the phenomenon of catabolite repression.

Amino Acid Sequence↗
PubMed1996

Genetic analysis of Rhizobium meliloti bacA-phoA fusion results in identification of degP: two loci required for symbiosis are closely linked to degP.

The function of the Rhizobium meliloti bacA gene, which is a homolog of the Escherichia coli sbmA gene, is required for an intermediate step in nodule development. A strain carrying the bacA386::TnphoA fusion was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, and three mutants that had higher levels of alkaline phosphatase activity were identified. The mutations in these strains were recessive and mapped to the same genetic locus. The gene affected by these mutations was identified and sequenced and was found to be a homolog of the E. coli degP gene, which encodes a periplasmic endopeptidase. Although degP function is important for the virulence of certain intracellular pathogens of mammals, it is not required for the R. meliloti-alfalfa symbiosis. The genetic analyses involving degP were complicated by the presence of a locus immediately upstream of depP that was lethal when present in multiple copies in a DegP- background. R. meliloti derivatives carrying insertion mutations in this locus displayed an N,N,N',N'-tetramethyl-p-phenylenediamine oxidase-negative phenotype, elicited the formation of white cylindrical nodules that did not fix nitrogen, and grew slowly in rich medium, suggesting that the locus was a cyc gene encoding a protein involved in the biosynthesis of a component or components of a respiratory chain. The previously identified fix-382::TnphoA, which similarly causes the formation of white cylindrical nodules that do not fix nitrogen, was shown to affect a gene that is separate from this cyc gene but extremely closely linked to it.

Alkaline Phosphatase↗
PubMed2004

Identification of candidate gammaherpesvirus 68 genes required for virus replication by signature-tagged transposon mutagenesis.

Current methods for determining the role of a given gene product in the gammaherpesvirus 68 (gammaHV68) life cycle require generation of a specific mutation by either homologous recombination in mammalian cells or bacterial artificial chromosome-mediated mutagenesis in Escherichia coli. The mutant virus is then compared to wild-type virus, and the role of the gene in the viral life cycle is deduced from its phenotype. This process is both time-consuming and labor intensive. Here we present the use of random, transposon-mediated signature-tagged mutagenesis for the identification of candidate viral genes involved in virus replication. Pools of viral mutants, each containing a random insertion of a transposon, were generated with a transposon donor library in which each transposon contains a unique sequence identifier. These pools were transfected into mammalian cells, and the ability of each mutant to replicate was assessed by comparing the presence of virus in the output pool to that present in the input pool of viral genomes. With this approach we could rapidly screen up to 96 individual mutants simultaneously. The location of the transposon insertion was determined by sequencing individual clones with a common primer specific for the transposon end. Here we present the characterization of 53 distinct viral mutants that correspond to insertions in 29 open reading frames within the gammaHV68 genome. To confirm the results of the signature-tagged mutagenesis screen, we quantitated the ability of each mutant to replicate compared to wild-type gammaHV68. From these analyses we identified 16 gammaHV68 open reading frames that, when disrupted by transposon insertions, score as essential for virus replication, and six other open reading frames whose disruption led to significant attenuation of virus replication. In addition, transposon insertion in five other gammaHV68 open reading frames did not affect virus replication. Notably, all but one of the candidate essential replication genes identified in this screen have been shown to be essential for the replication of at least one other herpesvirus.

Animals↗
PubMed2001

The Arabidopsis protein SHI represses gibberellin responses in Arabidopsis and barley.

The current model of gibberellin (GA) signal transduction is based on a derepressible system and a number of candidate negative regulators have been identified in Arabidopsis. We previously have reported the identification of the Arabidopsis gene SHORT INTERNODES (SHI) that causes suppression of GA responses when constitutively activated. In this paper, we show by using reporter gene analysis that the SHI gene is expressed in young organs, e.g. shoot apices and root tips. The model predicts a suppressor of GA responses to be active in these tissues to prevent premature growth or development. To study the effect of SHI on GA signaling, we used a functional assay that measures effects of signaling components on a well-defined GA response; the up-regulation of alpha-amylase in barley (Hordeum vulgare) aleurones in response to GA treatment. We found that SHI was able to specifically block the activity of a high-isoelectric point alpha-amylase promoter following GA(3) treatment, which further supports that SHI is a suppressor of GA responses. We have identified two putative loss-of-function insertion alleles of SHI and lines homozygous for either of the new alleles show no phenotypic deviations from wild type. Because SHI belongs to a gene family consisting of nine members, we suggest that SHI and the SHI-related genes are functionally redundant. We also show that a functional ERECTA allele is able to partly suppress the dwarfing effect of the shi gain-of-function mutation, suggesting that the erecta mutation harbored by the Landsberg erecta ecotype is an enhancer of the shi dwarf phenotype.

Alleles↗
PubMed2006

Identification of Salmonella enterica serovar Typhimurium genes important for survival in the swine gastric environment.

Since the stomach is a first line of defense for the host against ingested microorganisms, an ex vivo swine stomach contents (SSC) assay was developed to search for genes important for Salmonella enterica serovar Typhimurium survival in the hostile gastric environment. Initial characterization of the SSC assay (pH 3.87) using previously identified, acid-sensitive serovar Typhimurium mutants revealed a 10-fold decrease in survival for a phoP mutant following 20 min of challenge and no survival for mutants of rpoS or fur. To identify additional genes, a signature-tagged mutagenesis bank was constructed and screened in the SSC assay. Nineteen mutants were identified and individually analyzed in the SSC and acid tolerance response assays; 13 mutants exhibited a 10-fold or greater sensitivity in the SSC assay compared to the wild-type strain, but only 3 mutants displayed a 10-fold or greater decrease in survival following pH 3.0 acidic challenge. Further examination determined that the lethal effects of the SSC are pH dependent but that low pH is not the sole killing mechanism(s). Gas chromatography analysis of the SSC revealed lactic acid levels of 126 mM. Upon investigating the effects of lactic acid on serovar Typhimurium survival in a synthetic gastric fluid, not only was a concentration- and time-dependent lethal effect observed, but the phoP, rpoS, fur, and pnp genes were identified as involved in protection against lactic acid exposure. These studies indicate a role in gastric survival for several serovar Typhimurium genes and imply that the stomach environment is defined by more than low pH.

Animals↗