Genetic rearrangement associated with in vivo mucoid conversion of Pseudomonas aeruginosa PAO is due to insertion elements.

The conversion of Pseudomonas aeruginosa PAO to the mucoid phenotype has been reported for a chronic pulmonary infection model in rats (D. E. Woods, P. A. Sokol, L. E. Bryan, D. G. Storey, S. J. Mattingly, H. J. Vogel, and H. Ceri, J. Infect. Dis. 163:143-149, 1991). This conversion was associated with a genetic rearrangement upstream of the exotoxin A gene. To characterize the genetic rearrangement, the region upstream of the toxA gene was cloned from PAO, PAO-muc (a mucoid strain), and PAO-rev (a nonmucoid revertant strain). The nucleotide sequence of a 4.8-kb fragment from PAO-muc was determined. A+T-rich regions of approximately 2 kb (IS-PA-4) and 0.4 kb (IS-PA-5) were identified in this fragment. DNA probes constructed internal to these regions hybridized to PAO-muc but not to PAO or PAO-rev, suggesting that PAO-muc contains an insertion element. Sequence analysis of the nonmucoid clones indicated that a 2,561-bp fragment corresponding to IS-PA-4 and a 992-bp fragment corresponding to IS-PA-5 were not present in PAO or PAO-rev. Both nonmucoid clones, however, contained in the same location as IS-PA-4, a 1,313-bp region which was not present in PAO-muc. DNA probes complementary to this sequence, designated IS-PA-6, did not hybridize with PAO-muc, indicating that this sequence had been replaced upon conversion to the mucoid phenotype. Between IS-PA-4 and IS-PA-5 there was a 500-bp sequence which was 94% identical to the 500-bp sequence downstream of IS-PA-6. These insertion elements had some DNA sequence similarity to plasmid and transposon sequences, suggesting that they may be of plasmid origin. IS-PA-4 and IS-PA-5 were shown also to be present in two mucoid isolates from cystic fibrosis patients. The insertions occurred in the same location upstream of the toxA gene, suggesting that this type of genetic recombination may also be associated with mucoid conversion in some P. aeruginosa clinical isolates.     

A non-parametric approach to translating gene region heterogeneity associated with phenotype into location heterogeneity

MOTIVATION: The analysis of genetic data poses statistical problems in the form of high dimensionality with small sample sizes. The construction of a composite gene region (sequence pair) heterogeneity measure is one technique for reducing the dimensionality of the problem. This approach however is not without cost, since the contribution of locations to observed gene region differences between groups becomes entangled in this summary measure. This is problematic since it is of scientific interest to identify locations that together depict phenotype. RESULTS: A method is proposed for relating observed gene region heterogeneity back to the location level. In the spirit of a factor analysis-type setting, the approach focuses on identifying a latent variable structure among locations to explain within and between group genetic differences associated with phenotype. The method is flexible for identifying either the additive contribution from individual locations or the additive contribution from a group of locations, to observed gene region heterogeneity, depending upon the weighting scheme used in constructing a gene region heterogeneity measure. The approach is illustrated with clinical trial data, where the problem of altered HIV drug susceptibility is examined through characterizing location contributions to HIV protease gene region differences associated with a phenotypic treatment response. AVAILABILITY: The Splus (MathSoft, Inc. S-Plus 2000, Seattle, WA, 1999) developed menu-driven functions for obtaining results, GENE_ S (J.Kowalski, Harvard School of Public Health, Boston, MA 2001), is available from the author upon request.     

Distribution and sequence analysis of a family of type ill-dependent effectors correlate with the phylogeny of Ralstonia solanacearum strains

In Ralstonia solanacearum, we previously have reported on the characterization of popP1 and popP2 genes. These genes encode type III-dependent pathogenicity effectors related to the large family of AvrRxv/YopJ cysteine proteases that are shared among pathogens of plants and animals. In this study, we identify a third gene, named popP3, that is inactivated in the genome sequence of strain GMI1000 by insertion of a copy of the insertion sequence ISRso13. The three popP genes are localized on two large chromosomal pathogenicity islands, with popP1 and popP2 being present on the same island. Phylogenic analysis demonstrated that the PopP2 and PopP3 proteins are clearly distinct from other effectors of this family previously characterized in plant and animal pathogens. Analysis of the distribution and allelic variations of the three genes in 30 strains representative of the biodiversity of R. solanacearum established that popP genes are distributed widely among strains from two of the three phyla previously defined on the basis of the structure of the core genome. Sequencing of the popP genes from the different strains revealed limited allelic variations at the three loci but did not show evidence of recombination between the popP genes. Limited allelic variation together with occurrence of insertion sequences within or in the close vicinity of popP genes and the presence of gene duplications in these pathogenicity islands suggest that genomic rearrangements might be a major evolutionary driving force controlling evolution of the genes encoded in these regions. The implications of these observations in terms of bacterial evolution, gene acquisition, and horizontal gene transfers are discussed.     

The population genetic test Tajima's D identifies genes encoding pathogen‐associated molecular patterns and other virulence‐related genes in Ralstonia solanacearum

The detection of pathogen‐associated molecular patterns (PAMPs) by plant pattern recognition receptors (PRRs) is an essential part of plant immunity. Until recently, elf18, an epitope of elongation factor‐Tu (EF‐Tu), was the sole confirmed PAMP of Ralstonia solanacearum, the causal agent of bacterial wilt disease, limiting our understanding of R. solanacearum–plant interactions. Therefore, we set out to identify additional R. solanacearum PAMPs based on the hypothesis that genes encoding PAMPs are under selection to avoid recognition by plant PRRs. We calculated Tajima's D, a population genetic test statistic which identifies genes that do not evolve neutrally, for 3003 genes conserved in 37 R. solanacearum genomes. The screen flagged 49 non‐neutrally evolving genes, including not only EF‐Tu but also the gene for Cold Shock Protein C, which encodes the PAMP csp22. Importantly, an R. solanacearum allele of this PAMP was recently identified in a parallel independent study. Genes coding for efflux pumps, some with known roles in virulence, were also flagged by Tajima's D. We conclude that Tajima's D is a straightforward test to identify genes encoding PAMPs and other virulence‐related genes in plant pathogen genomes.     

Histopathology of Susceptible and Resistant Capsicum annuum Cultivars Infected with Ralstonia solanacearum

The vascular colonisation of resistant and susceptible hot chilli (Capsicum annuum) cultivars by Ralstonia solanacearum was examined using transmission electron microscopy. Tap roots of artificially-inoculated plants, grown in sterilised soil were investigated to observe the morphological barriers involved in the restriction of bacterial spread. In the resistant cultivar, several responses induced in response to bacterial infection, were observed. First, a cell wall coating material developed together with swelling of the primary wall of the xylem vessels, limiting the bacterial spread. Second, formation of various types of vesicles in the vascular parenchyma cells, which enveloped the bacterial mass and also partly restricted the pathogen spread. Third, induction of hypersensitive reaction in the xylem vessels resulted in the distortion and lysis of the bacteria. In the susceptible cultivar, vascular coating, production of vesicle and induction of hypersensitive reaction were not observed and bacterial spread was not limited. Rapid vascular colonisation of the susceptible cultivar seemed to be generalised which resulted in the rapid wilting of affected plants. Other reactions involved in both resistant and susceptible cultivars include disorganisation of cytoplasm of parenchyma cells, disintegration of nuclei, and rupturing of xylem vessel walls. The restriction of pathogen spread associated with the resistance in C. annuum to bacterial wilt was mainly attributed to some induced, morphological and physical barriers.     

Changes in mumps virus gene sequence associated with variability in neurovirulent phenotype

Mumps virus is highly neurotropic and, prior to widespread vaccination programs, was the major cause of viral meningitis in the United States. Nonetheless, the genetic basis of mumps virus neurotropism and neurovirulence was until recently not understood, largely due to the lack of an animal model. Here, nonneurovirulent (Jeryl Lynn vaccine) and highly neurovirulent (88-1961 wild type) mumps virus strains were passaged in human neural cells or in chicken fibroblast cells with the goal of neuroadapting or neuroattenuating the viruses, respectively. When tested in our rat neurovirulence assay against the respective parental strains, a Jeryl Lynn virus variant with an enhanced propensity for replication (neurotropism) and damage (neurovirulence) in the brain and an 88-1961 wild-type virus variant with decreased neurotropic and neurovirulent properties were recovered. To determine the molecular basis for the observed differences in neurovirulence and neuroattenuation, the complete genomes of the parental strains and their variants were fully sequenced. A comparison at the nucleotide level associated three amino acid changes with enhanced neurovirulence of the neuroadapted vaccine strain: one each in the nucleoprotein, matrix protein, and polymerase and three amino acid changes with reduced neurovirulence of the neuroattenuated wild-type strain: one each in the fusion protein, hemagglutinin-neuraminidase protein, and polymerase. The potential role of these amino acid changes in neurotropism, neurovirulence, and neuroattenuation is discussed.     

Induction of resistance and expression of defense-related genes in tobacco leaves infiltrated with Ralstonia solanacearum

Ralstonia solanacearum 8107 (8107) is non-pathogenic to tobacco and elicits the hypersensitive response (HR). In Nicotiana tabacum cv. Samsun NN leaves infiltrated with 8107, acquired resistance to challenging tobacco mosaic virus (TMV) was induced 2-6 d after 8107-infiltration. hsr203J and hin1 genes were expressed only in the 8107-infiltrated area. On the other hand, the expression of PR-1a and PR-1b genes was not detected in the 8107-infiltrated area, but in areas other than that developing the HR. Expression of these PR-1 genes was regulated simultaneously and the kinetics of the expression was dependent on the distance from the infiltration area. Therefore, diffusible signal(s) might be produced in HR-causing cells and transmitted to peripheral cells resulting in expression of PR genes. In NahG10 tobacco infiltrated with 8107, the HR was induced but resistance to TMV was not. Analysis using NahG10 tobacco also showed that the salicylic acid (SA)-dependent signal regulated the expression of hsr203J and PR-1a, but not that of hin1 and PR-1b. These results suggest that resistance of tobacco to 8107 is SA-independent and involves a quite different mechanism from acquired resistance to TMV induced by 8107-infiltration which is SA-dependent.     

Specific and sensitive detection of Ralstonia solanacearum in soil with quantitative, real-time PCR assays

Aims:  The aim of this study was to develop a sensitive and an effective method suitable for large-scale detection and quantification of Ralstonia solanacearum in soil.
Methods and Results:  Based on the specific sequence of R. solanacearum strain G1000, the primer pair R.sol1-R.sol2 and the TaqMan probe Rs-pro were designed, and specific and sensitive PCR detection methods were successfully established. The detection limit was 100 fg μl⁻¹ DNA in conventional PCR and 1·2 fg μl⁻¹ in real-time PCR. By combining real-time PCR with the modified protocols to extract DNA from soil, it was possible to achieve real-time detection of R. solanacearum in soil, and the degree of sensitivity was 100 fg μl⁻¹. To detect inhibition in soil samples, an exogenous internal positive control (IPC) was included preventing false negative results, and IPC was successfully amplified from all samples tested. The methodology developed was used to detect the presence of R. solanacearum in tobacco fields in China.
Conclusions:  The real-time PCR combined with the protocol to extract DNA from soil led to the development of a specific, sensitive and rapid detection method for R. solanacearum in soil.
Significance and Impact of the Study:  The real-time PCR improves the detection sensitivity and specificity and provides an important tool for routine detection of R. solanacearum in soil samples and for epidemiological and ecological studies.     

Individual variation in dispersal associated with phenotype influences fine-scale genetic structure in weasels

In general, landscape genetic studies have ignored the potential role that the phenotype of individuals plays in determining fine-scale genetic structure in species. This potential over-simplification ignores an important component that dispersal is both condition- and phenotype-dependent. In order to investigate the relationship between potential dispersal, habitat selection and phenotype, we examined the spatial ecology, body mass and fine-scale genetic structure of weasels (Mustela nivalis) in Bia?owie?a Forest in Poland. Our study population is characterized by an almost three-fold phenotypic variation in adult body mass and weasels were segregated in certain habitats according to size. We detected significant genetic structuring associated with habitat within the studied area and analyses of radio-tracking and re-capture data showed that the maximum extent of movement was achieved by weasels of medium body size, whereas the smallest and largest individuals exhibited higher site fidelity. With the unrestricted movement of the medium-sized individuals across optimal habitat, genetic admixture does occur. However, the presence of a barrier leads to unidirectional gene flow, with larger individuals outcompeting smaller individuals and therefore maintaining the genetic break in the study area. This highlights the importance of considering both intrinsic (phenotype) and extrinsic (environmental) factors in understanding dispersal patterns and ultimately, gene flow in complex landscapes.     

Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA.

We have postulated that chromosomal replication origin regions in eukaryotes have in common clusters of certain modular sequence elements (Benbow, Zhao, and Larson, BioEssays 14, 661-670, 1992). In this study, computer analyses of DNA sequences from six origin regions showed that each contained one or more potential initiation regions consisting of a putative DUE (DNA unwinding element) aligned with clusters of SAR (scaffold associated region), and ARS (autonomously replicating sequence) consensus sequences, and pyrimidine tracts. The replication origins analyzed were from the following loci: Tetrahymena thermophila macronuclear rDNA gene, Chinese hamster ovary dihydrofolate reductase amplicon, human c-myc proto-oncogene, chicken histone H5 gene, Drosophila melanogaster chorion gene cluster on the third chromosome, and Chinese hamster ovary rhodopsin gene. The locations of putative initiation regions identified by the computer analyses were compared with published data obtained using diverse methods to map initiation sites. For at least four loci, the potential initiation regions identified by sequence analysis aligned with previously mapped initiation events. A consensus DNA sequence, WAWTTDDWWWDHWGWHMAWTT, was found within the potential initiation regions in every case. An additional 35 kb of combined flanking sequences from the six loci were also analyzed, but no additional copies of this consensus sequence were found.     

Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria.

We constructed the broad-host-range plasmid pUCD800 containing the sacB gene of Bacillus subtilis for use in the positive selection and isolation of insertion sequence (IS) elements in gram-negative bacteria. Cells containing pUCD800 do not grow on medium containing 5% sucrose unless the sacB gene is inactivated. By using pUCD800, we isolated a 1.4-kilobase putative IS element from Agrobacterium tumefaciens NT1RE by selection for growth on sucrose medium. This putative IS element appears to be unique to Agrobacterium strains.     

An algorithm for identification of bacterial selenocysteine insertion sequence elements and selenoprotein genes

MOTIVATION: Incorporation of selenocysteine (Sec) into proteins in response to UGA codons requires a cis-acting RNA structure, Sec insertion sequence (SECIS) element. Whereas SECIS elements in Escherichia coli are well characterized, a bacterial SECIS consensus structure is lacking. RESULTS: We developed a bacterial SECIS consensus model, the key feature of which is a conserved guanosine in a small apical loop of the properly positioned structure. This consensus was used to build a computational tool, bSECISearch, for detection of bacterial SECIS elements and selenoprotein genes in sequence databases. The program identified 96.5% of known selenoprotein genes in completely sequenced bacterial genomes and predicted several new selenoprotein genes. Further analysis revealed that the size of bacterial selenoproteomes varied from 1 to 11 selenoproteins. Formate dehydrogenase was present in most selenoproteomes, often as the only selenoprotein family, whereas the occurrence of other selenoproteins was limited. The availability of the bacterial SECIS consensus and the tool for identification of these structures should help in correct annotation of selenoprotein genes and characterization of bacterial selenoproteomes.     

New mammalian selenocysteine-containing proteins identified with an algorithm that searches for selenocysteine insertion sequence elements

Mammalian selenium-containing proteins identified thus far contain selenium in the form of a selenocysteine residue encoded by UGA. These proteins lack common amino acid sequence motifs, but 3'-untranslated regions of selenoprotein genes contain a common stem-loop structure, selenocysteine insertion sequence (SECIS) element, that is necessary for decoding UGA as selenocysteine rather than a stop signal. We describe here a computer program, SECISearch, that identifies mammalian selenoprotein genes by recognizing SECIS elements on the basis of their primary and secondary structures and free energy requirements. When SECISearch was applied to search human dbEST, two new mammalian selenoproteins, designated SelT and SelR, were identified. We determined their cDNA sequences and expressed them in a monkey cell line as fusion proteins with a green fluorescent protein. Incorporation of selenium into new proteins was confirmed by metabolic labeling with (75)Se, and expression of SelT was additionally documented in immunoblot assays. SelT and SelR did not have homology to previously characterized proteins, but their putative homologs were detected in various organisms. SelR homologs were present in every organism characterized by complete genome sequencing. The data suggest applicability of SECISearch for identification of new selenoprotein genes in nucleotide data bases.     

Mutations in the lrpE gene of Ralstonia solanacearum affects Hrp pili production and virulence

The Ralstonia solanacearum hrpB-regulated gene lrpE (hpx5/brg24) encodes a PopC-like leucine-rich repeat (LRR) protein that carries 11 tandem LRR in the central region. Defects in the lrpE gene slightly reduced the virulence of R. solanacearum on host plants and changed the bacterial morphology leading to the formation of large aggregates in a minimal medium. The aggregation in the deltalrpE background required the presence of a functional Hrp type III secretion system. In wild-type R. solanacearum, Hrp pili disappeared from the bacterial surface at the end of the exponential growth phase, when the pili form into long bundles. However, even in the late growth phase, bundled Hrp pili were still observed on the cell surface of the deltalrpE mutant. Such bundles were entangled and anchored the mutant cells in the aggregates. In contrast to PopC, LrpE accumulated in bacterial cells and did not translocate into plant cells as an effector protein. The expression levels of hrp genes increased three- to fivefold in the deltalrpE background compared with those in the wild type. We propose that LrpE may negatively regulate the production of Hrp pili on the cell surface of R. solanacearum to disperse bacterial cells from aggregates. In turn, dispersal may contribute to the movement of the pathogen in the plant vascular system and, as a consequence, the pathogenicity of R. solanacearum.     

An Acetobacter xylinum insertion sequence element associated with inactivation of cellulose production.

An insertion sequence (IS) element, IS1031, caused insertions associated with spontaneous cellulose deficient (Cel-) mutants of Acetobacter xylinum ATCC 23769. The element was discovered during hybridization analysis of DNAs from Cel- mutants of A. xylinum ATCC 23769 with pAXC145, an indigenous plasmid from a Cel- mutant of A. xylinum NRCC 17005. An IS element, IS1031B, apparently identical to IS1031, was identified on pAXC145. IS1031 is about 950 bp. DNA sequencing showed that the two elements had identical termini with inverted repeats of 24 bp containing two mismatches and that they generated 3-bp target sequence duplications. The A. xylinum ATCC 23769 wild type carries seven copies of IS1031. Southern hybridization showed that 8 of 17 independently isolated spontaneous Cel- mutants of ATCC 23769 contained insertions of an element homologous to IS1031. Most insertions were in unique sites, indicating low insertion specificity. Significantly, two insertions were 0.5 kb upstream of a recently identified cellulose synthase gene. Attempts to isolate spontaneous cellulose-producing revertants of these two Cel- insertion mutants by selection in static cultures were unsuccessful. Instead, pseudorevertants that made waxlike films in the liquid-air interface were obtained. The two pseudorevertants carried new insertions of an IS1031-like element in nonidentical sites of the genome without excision of the previous insertions. Taken together, these results suggest that indigenous IS elements contribute to genetic instability in A. xylinum. The elements might also be useful as genetic tools in this organism and related species.     

Functional role of C-terminal sequence elements in the transporter associated with antigen processing

TAP delivers antigenic peptides into the endoplasmic reticulum (ER) that are subsequently bound by MHC class I molecules. TAP consists of two subunits (TAP1 and TAP2), each with a transmembrane (TMD) and a nucleotide-binding (NBD) domain. The two TAP-NBDs have distinct biochemical properties and control different steps during the peptide translocation process. We noted previously that the nonhomologous C-terminal tails of rat TAP1 and TAP2 determine the distinct functions of TAP-NBD1 and -NBD2. To identify the sequence elements responsible for the asymmetrical NBD function, we constructed chimeric rat TAP variants in which we systematically exchanged sequence regions of different length between the two TAP-NBDs. Our fine-mapping studies demonstrate that a nonhomologous region containing the alpha6/beta10-loop in conjunction with the downstream switch region is directly responsible for the functional separation of the TAP-NBDs. The alpha6/beta10-loop determines the nonsynonymous nucleotide binding of NBD1 and NBD2, whereas the switch region seems to play a critical role in regulating the functional cross-talk between the structural domains of TAP. Based on our findings, we postulate that these two sequence elements build a minimal functional unit that controls the asymmetry of the two TAP-NBDs.