Complete genome sequences of six strains of the genus methylobacterium

The complete and assembled genome sequences were determined for six strains of the alphaproteobacterial genus Methylobacterium, chosen for their key adaptations to different plant-associated niches and environmental constraints.     

Ifih1 gene dose effect reveals MDA5-mediated chronic type I IFN gene signature, viral resistance, and accelerated autoimmunity

Type I IFNs (IFN-I) are normally produced during antiviral responses, yet high levels of chronic IFN-I expression correlate with autoimmune disease. A variety of viral sensors generate IFN-I in their response, but other than TLRs, it is not fully known which pathways are directly involved in the development of spontaneous immune pathologies. To further explore the link between IFN-I induced by viral pathways and autoimmunity, we generated a new transgenic mouse line containing multiple copies of Ifih1, a gene encoding the cytoplasmic dsRNA sensor MDA5 with proven linkage to diabetes and lupus. We show that MDA5 overexpression led to a chronic IFN-I state characterized by resistance to a lethal viral infection through rapid clearance of virus in the absence of a CD8(+) or Ab response. Spontaneous MDA5 activation was not sufficient to initiate autoimmune or inflammatory pathology by itself, even though every immune cell population had signs of IFN activation. When combined with the lupus-susceptible background of the FcγR2B deficiency, MDA5 overexpression did accelerate the production of switched autoantibodies, the incidence of glomerulonephritis, and early lethality. Thus, MDA5 transgenic mice provide evidence that chronic elevated levels of IFN-I are not sufficient to initiate autoimmunity or inflammation although they might exacerbate an ongoing autoimmune pathology.     

ITAM-coupled receptors inhibit IFNAR signaling and alter macrophage responses to TLR4 and Listeria monocytogenes

ITAM-coupled receptors play an essential role in regulating macrophage activation and function by cross-regulating signaling from heterologous receptors. We investigated mechanisms by which ITAM-associated receptors inhibit type I IFN (IFN-α/β) signaling in primary human macrophages and tested the effects of simultaneous ligation of ITAM-associated receptors and TLR4 on TLR4-induced Jak-STAT signaling that is mediated by autocrine IFN-β. Preligation of ITAM-coupled β2 integrins and FcγRs inhibited proximal signaling by the type I IFN receptor IFNAR. Cross-inhibition of IFNAR signaling by β2 integrins resulted in decreased Jak1 activation and was mediated by partial downregulation of the IFNAR1 subunit and MAPK-dependent induction of USP18, which blocks the association of Jak1 with IFNAR2. Simultaneous engagement of ITAM-coupled β2 integrins or Dectin-1 with TLR4 did not affect TLR4-induced direct activation of inflammatory target genes such as TNF or IL6 but abrogated subsequent induction of IFN response genes that is mediated by autocrine IFN-β signaling. Type I IFNs promote macrophage death postinfection by Listeria monocytogenes. Consequently, attenuation of IFN responses by β2 integrins protected primary human macrophages from L. monocytogenes-induced apoptosis. These results provide a mechanism for cross-inhibition of type I IFN signaling by ITAM-coupled β2 integrins and demonstrate that ITAM signaling qualitatively modulates macrophage responses to pathogen-associated molecular patterns and pathogens by selectively suppressing IFN responses.     

The G-Quadruplex Ligand Telomestatin Impairs Binding of Topoisomerase IIIα to G-Quadruplex-Forming Oligonucleotides and Uncaps Telomeres in ALT Cells

In Alternative Lengthening of Telomeres (ALT) cell lines, specific nuclear bodies called APBs (ALT-associated PML bodies) concentrate telomeric DNA, shelterin components and recombination factors associated with telomere recombination. Topoisomerase IIIα (Topo III) is an essential telomeric-associated factor in ALT cells. We show here that the binding of Topo III to telomeric G-overhang is modulated by G-quadruplex formation. Topo III binding to G-quadruplex-forming oligonucleotides was strongly inhibited by telomestatin, a potent and specific G-quadruplex ligand. In ALT cells, telomestatin treatment resulted in the depletion of the Topo III/BLM/TRF2 complex and the disruption of APBs and led to the segregation of PML, shelterin components and Topo III. Interestingly, a DNA damage response was observed at telomeres in telomestatin-treated cells. These data indicate the importance of G-quadruplex stabilization during telomere maintenance in ALT cells. The function of TRF2/Topo III/BLM in the resolution of replication intermediates at telomeres is discussed.     

First Gene Cassettes of Integrons as Targets in Finding Adaptive Genes in Metagenomes

The first gene cassettes of integrons are involved in the last adaptation response to changing conditions and are also the most expressed. We propose a rapid method for the selection of clones carrying an integron first gene cassette that is useful for finding adaptive genes in environmental metagenomic libraries.Integrons, genetic elements discovered in clinical environments in 1989 (26), are known to carry gene cassettes encoding adaptive proteins in different environmental contexts (17, 20); environmental pressures may thus favor the propagation of cassettes conferring a selective advantage (21, 29). Integrons contain (Fig. ​(Fig.1)1) an integrase gene, intI (6, 7, 18); a recombination site, attI (23); a set of gene cassettes formed by a coding sequence and a recombination site, attC (14, 19); and one or two promoters, allowing gene cassette expression (4, 16). Different classes of integrons were defined according to the intI gene diversity. They were found in metagenomes from various environments (9, 15, 22). New metagenomic studies always discover new integron classes, showing the importance and the diversity of such genetic elements (9, 22).Open in a separate windowFIG. 1.Structure of integrons and positions of the primers used. The intI gene encodes an enzyme, allowing the integration of new gene cassettes at the attI recombination site. Thus, the first gene cassette is the last one integrated. Gene cassettes are formed by a recombination site, attC, and a coding sequence. The promoter Pc allows gene cassette expression. The positions of primers AJH72, ICC21, and ICC48 used in this study are indicated.As integrons are involved in bacterial adaptation, study of integrons would allow the finding of adaptive genes in metagenomes. But the detection of such genes among the huge abundance of gene cassettes associated with integrons is a challenge. The integration of a new gene cassette, catalyzed by the integrase, occurs by recombination between the attC site and the attI site of the integron (6, 8) (Fig. ​(Fig.1).1). The first gene cassette of an integron is, therefore, the last one integrated. As it is the closest gene to the promoter, its expression level is the highest in the integron (4). Thus, this gene cassette is a good target to find new adaptive genes in metagenomes. To amplify the first gene cassettes, a forward primer targeting the intI gene or attI site must be used. In previous studies, the determinations of gene cassette collection from environmental metagenomes did not target first gene cassettes, since they were performed by PCR methods targeting attC sites. Thus, we propose a method to construct gene cassette libraries enriched with first gene cassettes and an associated screening method for the clone selection.The method was developed by using DNA from Xanthomonas campestris ATCC 33913^T, a bacterial strain carrying an integron. DNA was extracted according to the work of Goñi-Urriza et al. (12). Coastal sediments maintained in the laboratory were used to validate this method. Total DNA (metagenomic) was extracted 1 week after addition of oil using the UltraClean soil DNA isolation kit (Mo Bio Laboratories), as previously described (24). PCR amplification, targeting the integrase gene intI in the forward direction (because attI sites are not well conserved enough to allow the good design of a primer) and the attC site in reverse, to amplify integron first gene cassettes was performed (Fig. ​(Fig.1).1). Forward primer AJH72 (10) was used for PCR of X. campestris DNA, and primer ICC48 (intB-inverted primer [25]), targeting the class 1 integron intI, was used for PCR of sediment metagenome. As the intI1 genes from environmental contexts exhibit considerable sequence diversity (11), ICC48 does not cover the entire spectrum of known intI1 genes but was chosen for its proximity to the attI site. Many primers used in previous studies targeting attC sites, such as HS286 (27), were unsuccessfully tested in the studied metagenome. Thus, ICC21, a less-degenerated primer, was designed to target the attC sites from class 1 and 2 integrons with the following sequence: 5′-GTCGGCTTGRAYGAATTGTTAGRC-3′. The PCR mixture contained DNA, 1× PCR buffer, 200 μM of each dNTP (deoxynucleoside triphosphate), 1.5 mM MgCl₂, 0.2 μM of each primer, and 5 U of Taq DNA polymerase (Eurobio). The PCRs consisted of 95°C for 10 min, 40 cycles of amplification (95°C for 45 s, 52°C or 51°C for 45 s, 72°C for 1.5 min), and 72°C for 10 min. PCR products were gel purified with the GFX PCR DNA and gel band purification kit (GE Healthcare). Purified products were cloned with the TOPO TA cloning kit (Invitrogen). Clones carrying first gene cassettes were selected by colony PCR. In order to minimize time spent on and the number of PCRs, the following three primers were concomitantly used: the two TOPO TA M13 primers and the primer targeting the intI gene used in the previous PCR (AJH72 or ICC48), and this primer was fluorescently labeled by HEX (6-carboxyhexafluorescein). PCR products were separated by gel electrophoresis, and the fluorescent DNA fragments were detected with a Typhoon 9200 scanner (Amersham). The selected inserts were sequenced by using the BigDye terminator v1.1 cycle sequencing kit (Applied Biosystems). Sequences were analyzed with ORF Finder (28), BLAST (1), and ProDom (3) algorithms.The X. campestris (ATCC 33913^T) integron possesses 23 gene cassettes (10). Different concentrations of primers were tested to amplify the first gene cassette, but in all cases, several amplified fragments were obtained. Sequence analyses revealed that most of them were gene cassettes other than the first one, and in these cases, the reverse primer was also used in the forward direction. The particular structure of the attC site with inverted repeat sequences, allowing the attC primer to anneal with both strands, may explain this result. As there was no other way to amplify the first gene cassettes, their selection could not be performed by PCR only. Because sequencing all clones would represent too much work when studying metagenomes, a triplex PCR screening method was developed (Fig. ​(Fig.2A).2A). Since the forward primer sequence is found only in the fragment containing the first gene cassette, the corresponding clones produce two PCR products, with one that is labeled (Fig. ​(Fig.2).2). Sequence-labeled inserts confirmed that the integron first gene cassette of X. campestris was selected. As a control, the sequences of unlabeled inserts showed that they contained integron gene cassettes but not the first gene cassettes.Open in a separate windowFIG. 2.Screening strategy for first gene cassette inserts. (A) Schematic of the clone library PCR screening strategy. The inserts are amplified by PCR using three primers. Only inserts carrying a first gene cassette lead to labeled amplified fragments. (B, C) Gel electrophoresis of insert PCR products from clones of X. campestris gene cassettes. (1) Insert carrying the integron first gene cassette of X. campestris; (2, 3) inserts carrying integron gene cassettes of X. campestris other than the first gene cassette; (4) molecular weight marker (SmartLadder; Eurogentec). (B) Detection of fluorescence; (C) detection of all fragments by ethidium bromide staining of the same gel.This method was then applied to coastal mud metagenomes, in which we focused on class 1 integrons, most commonly involved in adaptive responses (13). After PCR products were cloned, among 100 clones screened, 23 fluorescent fragments were detected and sequenced. As the primer targeting intI binds at the beginning of the gene, it was nearly impossible to recognize the intI sequence, except that the intI gene is longer at the 5′ end. On the other side of the sequences, a part of the attC site must be present. Sequence analysis revealed that some fragments showed similarities to characteristic class 1 or 2 integron attC sites, but these sites could not be found in each case because of their large variability (5). A total of 29 open reading frames (ORF) were characterized as potentially transcribed by an integron promoter, but for 16 of the ORF, no similarity with any known amino acid sequences could be found. The 13 other ORF exhibited less than 40% similarity with known sequences, and no putative conserved domains were found. These observations are in accordance with previous studies showing that most of the environmental integron gene cassettes code for proteins with unknown functions (2, 17).The first-gene cassettes of integrons appear to be good candidates to find gene cassettes, which aid bacteria in effecting a rapid adaptive response. We are now able to reveal integron last gene acquisitions of environmental bacterial communities submitted to stressful conditions. This method presents two limiting steps when working with metagenomes, as follows. (i) The primers are critical to cover the largest number of integrons. In this study, we targeted class 1 integrons because they are known to be mobile and to carry adaptive genes (29). (ii) When fragments with a large size disparity are cloned, the smallest fragments are preferentially cloned. In order to obtain a complete library with metagenomes, the cloning should be performed after fragment size separation. The PCR method combined with the screening method leads to 100% of clones carrying a first gene cassette. Thus, this new method allows the focus to be on spreading first gene cassettes in metagenomes after a specific stress.     

Development of an mlrA Gene-Directed TaqMan PCR Assay for Quantitative Assessment of Microcystin-Degrading Bacteria within Water Treatment Plant Sand Filter Biofilms

We report for the first time a quantitative mlrA gene-directed TaqMan PCR assay for the rapid detection of microcystin-degrading bacteria. This was applied, in combination with 16S ribosomal DNA-directed quantitative PCR and denaturing gradient gel electrophoresis, to study virgin sand filter column biofilm development and to correlate mlrA gene abundance with microcystin removal efficiency.It has been predicted that the changing climatic conditions around the world are likely to increase both the occurrence and the intensity of blue-green algal (cyanobacterial) blooms (16). Of particular concern to the water industry are the blooms of Microcystis, Anabaena, Nostoc, and Planktothrix species, which are capable of producing microcystin toxins within surface water storages used for potable water supply (4, 5, 12). In dissolved (extracellular) form, microcystins are not efficiently removed by conventional water treatment processes (6), and more advanced treatment options, such as activated carbon application or ozonation, are usually employed. However, these are expensive alternatives, and removal efficiencies are often compromised by the presence of natural organic matter (15). Biological filtration of microcystins is now recognized as an alternative treatment barrier (1, 7, 8, 14) and is favored by water utilities, as the process is generally low technology, chemical free, and requires little maintenance, where retrofitting of the process into existing water treatment plant (WTP) infrastructure is often feasible.To date, 10 different microcystin-degrading bacteria have been isolated from rivers, lakes, and biofilters (7), and the mlr gene cluster has been demonstrated to encode proteins involved in the initial steps of microcystin biodegradation by such organisms (2, 3). The MlrA protein is responsible for the initial hydrolytic cleavage of the cyclic microcystin structure, and conventional mlrA gene-directed PCR has been employed for qualitative detection of microcystin-degrading bacteria from lakes (18) and within the biofilm of biofilters (1, 7, 8). However, these conventional PCR assays do not allow for accurate quantitation of mlrA gene abundance and have not been designed with degenerate primer sequences to allow for variations that exist between different mlrA homologues. In this study, we report for the first time a quantitative mlrA gene-directed TaqMan PCR assay, including degenerate oligonucleotides targeting conserved DNA regions, for the rapid detection of microcystin-degrading bacteria.Using all available mlrA nucleotide sequences to date (GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"DQ112243","term_id":"70987104","term_text":"DQ112243"}}DQ112243, {"type":"entrez-nucleotide","attrs":{"text":"AF411068","term_id":"345808052","term_text":"AF411068"}}AF411068, {"type":"entrez-nucleotide","attrs":{"text":"AB114203","term_id":"34392799","term_text":"AB114203"}}AB114203, {"type":"entrez-nucleotide","attrs":{"text":"AB161685","term_id":"42415302","term_text":"AB161685"}}AB161685, and {"type":"entrez-nucleotide","attrs":{"text":"AB114202","term_id":"34392797","term_text":"AB114202"}}AB114202), primers qmlrAf (5′-AGCCCKGGCCCRCTGC-3′) and qmlrAr (5′-ATGCCARGCCCACCACAT-3′) and the TaqMan probe qmlrA-tm, which was labeled with 6-carboxyfluorescein (FAM) at the 5′ end and labeled with black hole quencher 1 (BHQ1) at the 3′ end (5′-FAM-TGCCSCAGCTSCTCAAGAAGTTTG-BHQ1-3′), were designed to target highly conserved regions of the mlrA gene for quantitative TaqMan PCR. Reactions resulted in the amplification of a 120-bp product and were carried out in quadruplicate on a Rotor Gene 6000 (Corbett Research, New South Wales, Australia) thermal cycling system. Each 25-μl reaction mixture contained 200 μM of each deoxynucleoside triphosphate, 2.0 mM of MgCl₂, 1× PCR buffer, 0.5 μM of primers qmlrAf and qmlrAr, 0.25 μM of TaqMan probe qmlrA-tm, 400 μg/ml bovine serum albumin, 0.5 U of Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), and 2.5 μl of either a DNA standard or sample template. Thermal cycling conditions consisted of an initial denaturation at 95°C for 2 min followed by 45 cycles of denaturation at 95°C for 5 s and annealing/extension at 62°C for 25 s. Data were collected in the FAM channel (gain set to 6) at the end of each annealing/extension step.Quantitation was performed using DNA standard curves constructed from a serial dilution, in Milli-Q water, of an 807-bp mlrA gene fragment (18) from the two microcystin-degrading bacteria Sphingomonas sp. strain ACM-3962 (2) and Sphingopyxis sp. strain LH21 (7). Results were linear over the range of 1 × 10¹ to 1 × 10⁹ mlrA gene copies/μl with linear coefficient values (R²) for ACM-3962 and LH21 of 0.999 and 0.998 and reaction efficiencies of 0.97 and 0.99, respectively. Specificity of the mlrA TaqMan assay was verified by the absence of signals for genomic DNA from the non-microcystin-degrading bacteria Aeromonas hydrophila (ATCC 7966), Bacillus subtilis (ATCC 10145), Escherichia coli (ATCC 11775), Pseudomonas aeruginosa (ATCC 10145), and Staphylococcus epidermidis (ATCC 12228).Determination of the DNA extraction efficiency, linear range, and the limit of detection of the TaqMan PCR assay under environmentally relevant conditions was achieved by the spiking of known amounts of Sphingopyxis sp. strain LH21 into Myponga reservoir water and Morgan WTP filter sand (containing a biofilm) in log₁₀-fold increments ranging from 0 to 1 × 10⁸ cells/ml or g, respectively. DNA from each water (0.5 ml) and sand preparation (0.5g) was then extracted in triplicate using the UltraClean soil DNA isolation kit (MoBio Laboratories Inc., Carlsbad, CA). For reservoir water, the TaqMan assay was linear over 6 log₁₀ orders of magnitude, with an R² of 0.998 and a limit of detection equivalent to 10² mlrA copies/ml. For sand filter medium, the TaqMan assay was linear over 5 log₁₀ orders of magnitude, with an R² equivalent to 0.998 and a limit of detection equivalent to 10³ mlrA copies/g. These results are comparable to other quantitative PCR (qPCR) data cited in the literature, where the detection limit was 2 × 10² cells/ml for Methylocystis sp. from water (13) and approximately 10³ cells/g for Escherichia coli O157:H7 (11) and Rhodococcus sp. (17) cells from soil.The mlrA TaqMan assay was then applied, in conjunction with 16S rDNA qPCR (10) and 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) cluster analysis (9), to investigate the attachment and subsequent biofilm formation upon virgin sand particles within a laboratory scale sand filter and also to investigate the previously unknown relationship between mlrA gene copy abundance and microcystin removal through biofiltration processes. Virgin sand (effective particle size, 1.25 mm; uniformity coefficient, 1.4; particle density, 1.62 g/cm³) (Riversands Pty Ltd, Carbrook, Queensland, Australia) was washed and sterilized by autoclaving. Sand was then packed into a glass column (length, 30 cm; internal diameter, 2.5 cm) at a bed height of 15 cm, and the column was continually fed with Myponga reservoir water (empty bed contact time, 15 min; UV₂₅₄/cm, 0.412; dissolved organic carbon, 11.8 mg/liter; specific UV absorbance, 3.5 liters/mg-m; and pH, 6.7; South Australia) and spiked daily with microcystin-LR at a target concentration of 5 μg/liter. Full details of the sand column apparatus are described by Ho et al. (8). Influent and effluent water samples were taken at regular intervals for microcystin-LR determination by high-performance liquid chromatography (8). Sand samples (0.5 g) were sampled in triplicate from the top surface of the sand bed, and the biofilm DNA samples were extracted using the UltraClean soil DNA isolation kit (Mo Bio).As shown in Fig. ​Fig.1a,1a, bacterial biofilm development (determined by 16S rDNA qPCR) appeared to begin within the first 24 h of start-up, and following day 3, the 16S bacterial abundance remained steady at approximately 1 × 10⁸ copies/g. Despite the limited change in total bacterial abundance following day 3, 16S rDNA PCR-DGGE cluster analysis revealed a dynamic shift in the overall bacterial community composition throughout the 18-day study (Fig. ​(Fig.2).2). There was also a 3-day period before removals of microcystin were observed through the column (Fig. ​(Fig.1b).1b). At day 4, there was a 12.6% removal of microcystin, although the abundance of mlrA-containing bacteria on day 4 remained below the limit of detection of the TaqMan assay (Fig. ​(Fig.1b).1b). By day 7, the removal of microcystin had increased to 30.8% and was accompanied by the detection of 1.17 × 10³ mlrA gene copies/g of sand. For the remainder of the study, there was a close association between the trend of microcystin removal and the abundance of mlrA gene copies/g of sand (Fig. ​(Fig.1b).1b). These data suggested that the efficiency of microcystin removals by biofiltration processes was directly related to the abundance of microcystin-degrading bacteria within the sand filter biofilm. Within biofilters that have had no preexposure to microcystins, lag periods have been reported to be as short as 2 to 4 days (1, 8), but they have been reported to be up to 211 days for a filter containing virgin sand (19). In most cases, 100% removal of microcystin through biofilters usually occurs rapidly within several days following the lag period (8, 19), although complete removal of microcystin in this study was achieved 15 days following the 3-day lag period (Fig. ​(Fig.1b1b).Open in a separate windowFIG. 1.(a) Abundance of 16S rDNA (closed circles) and mlrA gene copies (closed squares) within the sand filter column biofilm. (b) Abundance of mlrA gene copies (closed squares) within the sand filter column biofilm and the percentage of microcystin-LR removed (open triangles) through the column. Error bars represent standard deviations of the results for triplicate analyses.Open in a separate windowFIG. 2.Cluster analysis of DGGE band profiles for the sand filter column study. Scale bar represents two band differences.A clone library was then constructed to investigate the diversity of mlrA homologues detected by the mlrA TaqMan PCR assay within the sand filter column on day 18. Following sequence analysis of 50 cloned DNA fragments, three unique mlrA gene sequences, MC-A, MC-B, and MC-C, were obtained. Each sequence had greater than 98% similarity to previously described mlrA gene sequences, where MC-A, MC-B, and MC-C represented 22, 72, and 6% of the clone library, respectively.In summary, this study has demonstrated the development of an mlrA gene-directed TaqMan PCR assay for the assessment of microcystin-degrading bacteria within biologically active sand filter biofilm. The data presented here revealed that during the early phase of operation of a sand filter column, the removal of microcystin was directly related to the abundance of microcystin-degrading bacteria within the biofilm, although removals began only once an adequate number of microcystin-degrading bacteria were established. It is envisaged that the TaqMan PCR assay will be valuable for WTP operators who wish to investigate the abundance of microcystin-degrading bacteria within their biofilters and, in turn, assess the capacity of the biofilters for removing microcystin toxins—especially as such episodes are particularly transient in nature.     

From Local Surveys to Global Surveillance: Three High-Throughput Genotyping Methods for Epidemiological Monitoring of Xanthomonas citri pv. citri Pathotypes

Asiatic citrus canker is a major disease worldwide, and its causal agent, Xanthomonas citri pv. citri, is listed as a quarantine organism in many countries. Analysis of the molecular epidemiology of this bacterium is hindered by a lack of molecular typing techniques suitable for surveillance and outbreak investigation. We report a comparative evaluation of three typing techniques, amplified fragment length polymorphism (AFLP) analysis, insertion sequence ligation-mediated PCR (IS-LM-PCR) typing, and multilocus variable-number tandem-repeat analysis (MLVA), with 234 strains originating from Asia, the likely center of origin of the pathogen, and reference strains of pathotypes A, A*, and A^w, which differ in host range. The typing techniques were congruent in describing the diversity of this strain collection, suggesting that the evolution pattern of the bacterium may be clonal. Based on a hierarchical analysis of molecular variance, the AFLP method best described the genetic variation found among pathotypes whereas MLVA best described the variation found among individual strains from the same countries or groups of neighboring countries. IS-LM-PCR data suggested that the transposition of insertion sequences in the genome of X. citri pv. citri occurs rarely enough not to disturb the phylogenetic signal. This technique may be useful for the global surveillance of non-epidemiologically related strains. Although pathological characteristics of strains could be most often predicted from genotyping data, we report the occurrence in the Indian peninsula of strains genetically related to pathotype A* strains but with a host range similar to that of pathotype A, which makes the classification of this bacterium even more complicated.The definition of host range is a central parameter for the understanding and, ultimately, the control of infectious diseases in general and bacterial plant diseases in particular. In phytobacteriology, host range is an important aspect of pathogenicity. Control of diseases can be achieved with resistance genes which reduce host range (50, 64). Epidemiological characteristics are highly dependent on host range, and the emergence of new diseases is sometimes correlated with broadened host ranges (74). Xanthomonads have the particularity of an extremely narrow host range (sometimes reduced to a single plant genus), although a very large number of plant families can be hosts when all members of the genus are considered (33), which led plant pathologists to create the concept of pathovar at an infrasubspecific level. Pathovars were defined as groups of strains sharing several pathological characteristics, such as their host range and the disease facies they cause (18). Based on molecular data, strains classified as a single pathovar usually form a discrete monomorphic or weakly polymorphic cluster, suggesting that strains of a pathovar have a common ancestral origin (3, 56). Xanthomonas citri pv. citri is the causal agent of Asiatic canker, a severe disease infecting most commercial citrus cultivars and some genera in the Rutaceae family in many citrus-producing areas worldwide (6, 60, 61). This pathovar has two types of strains, which differ in their host ranges: pathotype A has a wide host range and a worldwide distribution and is a permanent threat for citriculture (29); in contrast, the more recently characterized pathotype A* causes citrus canker on Mexican lime (Citrus aurantifolia) and has a much less severe impact on citriculture (72). Strains of this pathotype were considered to belong to the pathovar citri because of their phenotypic and genetic relatedness to pathotype A. Their distribution was initially reported to include Saudi Arabia, Oman, Iran, and India and was recently found to extend to southeast Asia, with reports of these strains in Thailand (10) and Cambodia (11). Finally, strains genetically related to pathotypes A and A* but able to infect Mexican lime and Citrus macrophylla naturally were recently detected in Florida and classified as a pathotype designated A^w (68). The molecular basis of the specific interaction of X. citri pv. citri pathotypes A* and A^w with a restricted range of citrus hosts is not known (5). An interaction between a host resistance gene and an avr gene product from the pathogen inducing host-pathogen incompatibility has not yet been demonstrated for the X. citri pv. citri-citrus pathosystem, as it has been previously for other plant pathogenic bacteria (43).No assumption can be made about whether the apparent contemporary emergence of pathotype A* strains is due to a change in virulence or to environmental or human factors. Host range shifts have sometimes been related to modifications in the repertoire of virulence genes by horizontal gene transfer or intragenomic recombinations or mutations (20, 32, 75). A clear understanding of the evolutionary relationships among pathotypes A, A*, and A^w and of the diversity among strains of each pathotype would be helpful for assessing these issues.Due to the extreme difficulty and cost of the complete eradication of Asiatic citrus canker, several canker-threatened citrus-producing regions rely on integrated pest management strategies for control (30). Data derived from the huge effort put into the molecular typing of human bacterial pathogens (46, 63, 67, 69) suggest that an extensive knowledge of populations of plant pathogenic bacteria may improve our understanding of epidemic situations.The tools most often used for the molecular epidemiology of citrus canker have been repetitive-element-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE) (13, 16, 19, 28, 68). The lack of discriminatory power of rep-PCR and the high labor requirement for PFGE make it difficult to use these techniques extensively for outbreak investigations or regional or global surveillance (67). Therefore, alternative high-resolution and high-throughput molecular typing systems for X. citri pv. citri should be developed. Amplified fragment length polymorphism (AFLP) analysis of an Iranian collection of strains causing Asiatic citrus canker suggested previously that this technique has better discriminatory power than the rep-PCR method (39). AFLP has the advantage of generating a large number of randomly located markers over the whole genome. The detected polymorphism may arise from point mutations at the targeted restriction sites or from insertions and/or deletions in the amplified region (73). The determination of the complete sequence of X. citri pv. citri strain 306 (17) should facilitate the development of molecular typing tools well-suited for deciphering taxonomy, evolution, and/or epidemiology. For instance, it gave access to specific primers associated with transposable elements present in this bacterium (45), which were used for typing DNA from herbarium specimens showing canker-like symptoms and originating from different geographical origins. This technique revealed an unexpectedly high degree of genetic diversity. However, this typing scheme requires more than 50 PCRs for the full analysis of unknown DNA. A new insertion sequence ligation-mediated PCR (IS-LM-PCR) scheme (9) also revealed considerable diversity and is less labor-intensive. This technique amplifies DNA fragments between an insertion sequence element and a selected restriction site (9). We also recently developed a multilocus variable-number tandem-repeat analysis (MLVA) approach for this bacterium, a promising technique targeting tandem repeats (minisatellite-like loci) for fine-scale epidemiology with distinctive advantages, such as high discriminatory power, maximal reproducibility of results, and portability of equipment (12). The characteristics of these newly developed techniques need to be subjected to a comparative evaluation in order to determine which methods would be most useful for global surveillance and molecular epidemiology on small spatial scales. In this study, we compared the AFLP, MLVA, and IS-LM-PCR techniques to explore the genetic diversity of a collection of pathotype A, A*, and A^w strains originating from Asia. Furthermore, we sought to determine the genetic diversity and structure of X. citri pv. citri strains, including a large collection of pathotype A* strains for which no extensive characterization study is available at the moment, from the area of origin of the pathogen.     

A Novel Polyamine Acyltransferase Responsible for the Accumulation of Spermidine Conjugates in Arabidopsis Seed

Hydroxycinnamic acid amides are a class of secondary metabolites distributed widely in plants. We have identified two sinapoyl spermidine derivatives, N-((4′-O-glycosyl)-sinapoyl),N′-sinapoylspermidine and N,N′-disinapoylspermidine, which comprise the two major polyamine conjugates that accumulate in Arabidopsis thaliana seed. Using metabolic profiling of knockout mutants to elucidate the functions of members of the BAHD acyltransferase family in Arabidopsis, we have also identified two genes encoding spermidine disinapoyl transferase (SDT) and spermidine dicoumaroyl transferase (SCT) activities. At2g23510, which is expressed mainly in seeds, encodes a spermidine sinapoyl CoA acyltransferase (SDT) that is required for the production of disinapoyl spermidine and its glucoside in Arabidopsis seed. The structurally related BAHD enzyme encoded by At2g25150 is expressed specifically in roots and has spermidine coumaroyl CoA acyltransferase (SCT) activity both in vitro and in vivo.