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191.
Oxidative damage of the gastric mucosa in Helicobacter pylori positive chronic atrophic and nonatrophic gastritis, before and after eradication 总被引:3,自引:0,他引:3
Iacopini F Consolazio A Bosco D Marcheggiano A Bella A Pica R Paoluzi OA Crispino P Rivera M Mottolese M Nardi F Paoluzi P 《Helicobacter》2003,8(5):503-512
Background. Helicobacter pylori is the main cause of gastritis and a primary carcinogen. The aim of this study was to assess oxidative damage in mucosal compartments of gastric mucosa in H. pylori positive and negative atrophic and nonatrophic gastritis. Materials and methods. Five groups of 10 patients each were identified according to H. pylori positive or negative chronic atrophic (Hp‐CAG and CAG, respectively) and nonatrophic gastritis (Hp‐CG and CG, respectively), and H. pylori negative normal mucosa (controls). Oxidative damage was evaluated by nitrotyrosine immunohistochemistry in the whole mucosa and in each compartment at baseline and at 2 and 12 months after eradication. Types of intestinal metaplasia were classified by histochemistry. Results. Total nitrotyrosine levels appeared significantly higher in H. pylori positive than in negative patients, and in Hp‐CAG than in Hp‐CG (p < .001); no differences were found between H. pylori negative gastritis and normal mucosa. Nitrotyrosine were found in foveolae and intestinal metaplasia only in Hp‐CAG. At 12 months after H. pylori eradication, total nitrotyrosine levels showed a trend toward a decrease in Hp‐CG and decreased significantly in Hp‐CAG (p = .002), disappearing from the foveolae (p = .002), but remaining unchanged in intestinal metaplasia. Type I and II of intestinal metaplasia were present with the same prevalence in Hp‐CAG and CAG, and did not change after H. pylori eradication. Conclusions. Oxidative damage of the gastric mucosa increases from Hp‐CG to Hp‐CAG, involving the foveolae and intestinal metaplasia. H. pylori eradication induces a complete healing of foveolae but not of intestinal metaplasia, reducing the overall oxidative damage in the mucosa. 相似文献
192.
Ceccaroli P Saltarelli R Cesari P Pierleoni R Sacconi C Vallorani L Rubini P Stocchi V Martin F 《Fungal genetics and biology : FG & B》2003,39(2):168-175
The metabolism of [1-13C]glucose in the vegetative mycelium of the ectomycorrhizal ascomycete Tuber borchii was studied in order to characterize the biochemical pathways for the assimilation of glucose and amino acid biosynthesis. The pathways were characterized using nuclear magnetic resonance spectroscopy in conjunction with [1-13C]glucose labeling. The enzymes of mannitol cycle and ammonium assimilation were also evaluated. The majority of the 13C label was incorporated into mannitol and this polyol was formed via a direct route from absorbed glucose. Amino acid biosynthesis was also an important sink of assimilated carbon and 13C was mainly incorporated into alanine and glutamate. From this intramolecular 13C enrichment, it is concluded that pyruvate, arising from [1-13C]glucose catabolism, was used by alanine aminotransferase, pyruvate dehydrogenase and pyruvate carboxylase before entering the Krebs cycle. The transfer of 13C-labeled mycelium on [12C]glucose showed that mannitol, alanine, and glutamate carbon were used to synthesize glutamine and arginine that likely play a storage role. 相似文献
193.
Basolateral Ca2+-dependent K+-channels play a key role in Cl- secretion induced by taurodeoxycholate from colon mucosa 总被引:1,自引:0,他引:1
Moschetta A Portincasa P Debellis L Petruzzelli M Montelli R Calamita G Gustavsson P Palasciano G 《Biology of the cell / under the auspices of the European Cell Biology Organization》2003,95(2):115-122
The diarrhea associated with malabsorption of bile salts such as the secondary hydrophobic taurodeoxycholate (TDC) may be partly explained by the TDC-induced increase in colon Cl(-) secretion. We, therefore, investigated the effects of TDC (0.5-8 mM) on electrical parameters and electrolyte transport of rat proximal colon mucosa mounted in Ussing chambers. Colonic secretion, measured as short circuit current (I(SC)), progressively increased on mucosal incubation with TDC ranging 0.5-2 mM; up to TDC 2 mM, a spontaneous recovery toward control values with no changes in epithelial resistance (Rt), and lactate dehydrogenase (LDH) release was observed. In contrast, for TDC > 2 mM, I(SC) increased further and the effect was progressive and associated with a significant decrease in the Rt and increased LDH release, implying a cytolytic effect. Mucosal preincubation with the Cl(-) channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), fully prevented the precytolytic effect of TDC on I(SC). Serosal preincubation with furosemide, a Na(+)/K(+)/2Cl(-) cotransporter inhibitor, significantly reduced TDC-induced increase in I(SC). Inhibition of the basolateral Ca(2+)-dependent K(+) channel-rSK4-with serosal clotrimazole or incubation with mucosal Ca(2+)-free (EGTA) buffer completely prevented precytolytic TDC-induced increase in I(SC). In conclusion, Cl(-) secretion is activated in colon mucosa by TDC low concentrations; while at higher concentrations, a detergent cytotoxic effect intervenes. Activation of the Ca(2+)-dependent basolateral K(+) pathway, through TDC-induced apical Ca(2+) influx, provides the Na(+)/K(+)/2Cl(-) basolateral activation, thereby the driving force for the apical exit of Cl(-) ions. These findings further enhance the knowledge of the pathogenic mechanisms of diarrhea associated with bile salt malabsorption. 相似文献
194.
Lorch SA Banks BA Christie J Merrill JD Althaus J Schmidt K Ballard PL Ischiropoulos H Ballard RA 《Free radical biology & medicine》2003,34(9):1146-1152
Plasma protein levels of 3-nitrotyrosine and 3-chlorotyrosine were measured by LC-MS/MS at 0 and 72 h after the initiation of inhaled nitric oxide (INO) at 20 ppm in 22 prematurely born infants with clinically documented bronchopulmonary dysplasia. Infants were classified at the time of hospital discharge as either "off mechanical ventilation," "on mechanical ventilation," or "expired/organ failure." These outcomes were tested for association with changes in plasma levels of 3-nitrotyrosine and 3-chlorotyrosine and selected clinical risk factors. Infants whose 3-nitrotyrosine levels decreased over the 72 h period were more likely to wean off of mechanical ventilation (p =.03). There was no significant association between changes in 3-chlorotyrosne levels and outcome. After controlling for other variables, an odds ratio of 8.3 (95% CI: 1.3-54.4) for improved outcomes was observed if the 3-nitrotyrosine levels decreased. These data suggest that nitrative and oxidative stress may be related to the severity of lung disease and, consequentially, the overall outcome in this select group of infants with severe bronchopulmonary dysplasia. 相似文献
195.
Coelho RG Di Stasi LC Vilegas W 《Zeitschrift für Naturforschung. C, Journal of biosciences》2003,58(1-2):47-52
The new flavonoid glycoside kaempferol-3-O-alpha-L-rhamnopyranosyl(1-->2)-O-[alpha-L-rhamnopyranosyl(1-->6)]-O-beta-D-galactopyranoside-7-O-alpha-L-rhamnopyranoside was isolated together with (S)-zierin from the leaves of Zollernia ilicifolia (Fabaceae), a medicinal plant used as analgesic and antiulcerogenic effects in Brazilian Tropical Atlantic Rain Forest. The structures were established on the basis of 1H, 13C NMR and 2D NMR (COSY, HMBC, HMQC), UV, MS and IV spectra. The infusion of Zollernia ilicifolia was qualitatively compared to the infusion of the espinheiras-santas (Maytenus aquifolium and Maytenus ilicifolia) by HPLC-DAD. 相似文献
196.
Graziella Chini Zittelli Roberta Pastorelli Mario R. Tredici 《Journal of applied phycology》2000,12(3-5):521-526
Nannochloropsis sp. was grown in a Modular FlatPanel Photobioreactor (MFPP) consisting of sixalveolar panels each with 20.5 L culture volume and3.4 m2 illuminated surface area. The panelsformed a closely-packed unit with illuminationprovided by banks of fluorescent tubes placed betweenthe panels. The whole unit was contained in athermoregulated cabinet. Continuous illumination ofone side of the panels with 115 molphoton m-2 s-1 attained a mean volumetricproductivity of 0.61 g (d. wt) L-1 24 h-1,increasing to 0.97 g (d. wt) L-1 24 h-1 whenthe same irradiance was provided on both sides of thepanels. With 230 mol photon m-2 s-1 onone side of the panel, a mean productivity of 0.85 g(d. wt) L-1 24 h-1 was achieved, whichreached 1.45 g (d. wt) L-1 24 h-1 when bothsides were illuminated. Increasing the amount of lightprovided to the culture (either by increasingirradiance or the illuminated surface area) decreasedpigment and enhanced the total fatty acid content, butdid not change significantly the content ofeicosapentaenoic acid. A MFPP of the presentdimensions could produce sufficient microalgae tosupport a hatchery producing 6 million sea breamfingerlings annually. 相似文献
197.
Maurizio Sorice Tina Garofalo Roberta Misasi Agostina Longo Joanna Mikulak Vincenza Dolo Giuseppe Mario Pontieri Antonio Pavan 《Glycoconjugate journal》2000,17(3-4):247-252
The aim of this study was to further elucidate our previous observation on molecular interaction of GM3, CD4 and p56lck in microdomains of human peripheral blood lymphocytes (PBL). We analyzed GM3 distribution by immunoelectron microscopy and the association between GM3 and CD4-p56lck complex by scanning confocal microscopy and co-immunoprecipitation experiments. Scanning confocal microscopy analysis showed an uneven signal distribution of GM3 molecules over the surface of human lymphocytes. Nearly complete colocalization areas indicated that CD4 molecules were distributed in GM3-enriched plasma membrane domains. Co-immunoprecipitation experiments revealed that CD4 and p56lck were immunoprecipitated by IgG anti-GM3, demonstrating that GM3 tightly binds to the CD4-p56lck complex in human PBL. In order to verify whether GM3 association with CD4 molecules may depend on the presence of p56lck, we analyzed this association in U937, a CD4+and p56lck negative cell line. The immunoprecipitation with anti-GM3 revealed the presence of a 58[emsp4 ]kDa band immunostained with anti-CD4 Ab, suggesting that the GM3-CD4 interaction does not require its association with p56lck. These findings support the view that GM3 enriched-domains may represent a functional multimolecular complex involved in signal transduction and cell activation. 相似文献
198.
Cristina Sotgia Umberto Fascio Roberta Pennati Fiorenza De Bernardi 《Development, growth & differentiation》1998,40(1):75-84
Animal caps isolated from Xenopus laevis embryos at the blastula stage were treated sequentially with NH4 Cl, a known cement gland inducer, and with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a known neural inducer. The two artificial inducers were also used in reverse order to see if they can mimic the natural inducers acting during the progressive determination of the ectodermal organ. Immunofluorescence and whole-mount in situ hybridization were used to study the expression of tubulin, taken to indicate an early step on the pathway of cell elongation, and neural cell adhesion molecule (N-CAM) taken to indicate an early step in the determination of the nervous system. The expression of XCG-1, a marker of early specification of the cement gland, was also studied. The results showed that the two artificial inducers can mimic the effects of the natural inducers in animal cap explants. The TPA behaves like a neural inducer, reducing the number and the extension of the cement gland when added to the medium in addition to NH4 Cl, before or after NH4 Cl treatment. In the process of cement gland/neural induction, it is possible to redirect the ectoderm already specified as cement gland to neural tissue, but it does not seem possible to respecify the neural tissue as cement gland. Moreover, the animal caps were also cut into dorsal and ventral parts and the two halves were treated separately. The results were similar to those obtained with treatment of the entire animal cap, suggesting that a dorsal-ventral pattern is not yet established before the gastrula stage, and that in normal embryos there are boundaries between the effects of different inducers. 相似文献
199.
Evidence for Interspecies Gene Transfer in the Evolution of 2,4-Dichlorophenoxyacetic Acid Degraders
Catherine McGowan Roberta Fulthorpe Alice Wright J. M. Tiedje 《Applied and environmental microbiology》1998,64(10):4089-4092
Small-subunit ribosomal DNA (SSU rDNA) from 20 phenotypically distinct strains of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was partially sequenced, yielding 18 unique strains belonging to members of the alpha, beta, and gamma subgroups of the class Proteobacteria. To understand the origin of 2,4-D degradation in this diverse collection, the first gene in the 2,4-D pathway, tfdA, was sequenced. The sequences fell into three unique classes found in various members of the beta and gamma subgroups of Proteobacteria. None of the α-Proteobacteria yielded tfdA PCR products. A comparison of the dendrogram of the tfdA genes with that of the SSU rDNA genes demonstrated incongruency in phylogenies, and hence 2,4-D degradation must have originated from gene transfer between species. Only those strains with tfdA sequences highly similar to the tfdA sequence of strain JMP134 (tfdA class I) transferred all the 2,4-D genes and conferred the 2,4-D degradation phenotype to a Burkholderia cepacia recipient.Bacteria capable of mineralizing 2,4-dichlorophenoxyacetic acid (2,4-D), a commonly used herbicide, are found in many different phylogenetic groups (2, 3, 7, 11, 22, 23). Evidence suggests that numerous variants of 2,4-D catabolic genes exist and that catabolic operons consist of a near-random mixing of these variants (7). Interspecies gene transfer is a well-documented phenomenon (13), and horizontal gene transfer of the 2,4-D-degrading plasmid pJP4 has been shown (3, 5). However, not all 2,4-D catabolic operons are found on plasmids (10, 11, 16, 20). The extent to which other 2,4-D genes have been exchanged in nature is unknown. The aim of this research was to assess the role of horizontal gene transfer in the evolution of 2,4-D-degrading strains. This article summarizes the results of two aspects of this work—the study of the transfer of the entire 2,4-D pathway by using standard mating experiments and a phylogenetic study of the tfdA gene. The tfdA gene codes for an α-ketoglutarate-dependent 2,4-D dioxygenase which converts 2,4-D into 2,4-dichlorophenol and glyoxylate (6). This 861-bp gene was first sequenced from Ralstonia eutropha JMP134 (19). Two more tfdA genes were cloned from chromosomal locations in Burkholderia strain RASC and Burkholderia strain TFD6 (16, 20). These proved to be identical to each other and 78.5% similar to the original. An alignment of the two variants allowed conserved areas to be identified and primers to be designed for the amplification of tfdA-like genes from other sources (24). Sequence analysis of putative tfdA fragments and the small-subunit ribosomal DNA (SSU rDNA) of the strains carrying them allowed us to construct phylogenies of the genes and their hosts and to look for congruency between them.
Open in a separate windowaThe generus and/or species most similar to the strain is given based on similarities of SSU rDNA sequences. bSymbols: +, able to transfer 2,4-D degradation to B. cepacia D5; (+), able to transfer at very low frequency; −, no transfer detected. cND, not determined. d—, no amplificate was obtained. The disappearance of 2,4-D from the culture medium was monitored by high-performance liquid chromatography. Cells were removed by centrifugation, and the supernatant was filtered through 0.2-μm-pore-size filters. These samples were then analyzed on a Lichrosorb Rp-18 column (Anspec Co., Ann Arbor, Mich.) with 60% methanol–40% 0.1% H3PO4 as the eluant. 2,4-D was detected by measuring light absorption at 230 nm. The presence of tfd genes was detected by hybridizing colony blots with a DNA probe derived from the entire pJP4 plasmid. The identity of the colonies was confirmed by probing with the nptII gene of Tn5 (found in B. cepacia D5). Probes were labeled with random hexanucleotides incorporating [32P]dCTP (3,000 Ci/mmol; New England Nuclear, Boston, Mass.). Hybridizations were done under high-stringency conditions by using 50% formamide and Denhardt’s solution (18) at 42°C. Of the 15 unique strains tested, 9 transferred 2,4-D degradation abilities to D5. This transfer was confirmed by hybridization with pJP4 for eight of these strains. B. cepacia RASC could transfer degradative abilities, but neither it nor the transconjugant hybridized to the pJP4 probe. Work subsequent to this study has confirmed that the genes carried by RASC do not hybridize to those found on pJP4 under high-stringency conditions (7).
Mating experiments.
A collection of 2,4-D degraders containing 15 unique strains as determined by genomic fingerprinting (7) was used as a source of donors in a series of mating experiments (Table (Table1).1). Burkholderia cepacia D5, lacking the ability to grow on 2,4-D and not hybridizing to any tfd genes, was used as a recipient in mating experiments. Strain D5 contains neomycin phosphotransferase genes (nptII) carried on transposon Tn5 and is resistant to 50 μg each of kanamycin, carbenicillin, and bacitracin per ml. All of the 2,4-D strains used were sensitive to these antibiotics. Filter matings were performed with a donor-to-recipient ratio of 1:10. Colonies which grew on selective medium (500 ppm of 2,4-D in mineral salts agar [MMO] [23] including 50 μg of kanamycin, carbenicillin, and bacitracin per ml) were subjected to further tests. Their ability to catabolize 2,4-D was tested in liquid medium (same composition as that described above).TABLE 1
2,4-D-degrading strains, geographic origins, and GenBank accession numbersStrain | GenBank accession no. (SSU rDNA) | Origin | Most similar to genus and/or speciesa | Transferb | tfdA typec | GenBank accession no. (tfdA gene) | Reference or source |
---|---|---|---|---|---|---|---|
JMP134 | AF049542 | Australia | Ralstonia eutropha | + | I | M16730 | 3 |
EML1549 | AF049546 | Oregon | Burkholderia sp. | + | I | 2 | |
TFD39 | AF049539 | Saskatchewan | Burkholderia sp. | + | I | U43197 | 23 |
K712 | AF049543 | Michigan | Burkholderia sp. | + | I | U43276 | 11 |
TFD9 | AF049537 | Saskatchewan | Alcaligenes xylosoxidans | + | I | U43276 | 23 |
TFD41 | AF049541 | Michigan | Ralstonia eutropha | + | I | 23 | |
TFD38 | AF049540 | Michigan | Ralstonia eutropha | + | NDc | 23 | |
TFD23 | AF049536 | Michigan | Rhodoferax fermentans | + | I | U43276 | 23 |
RASC | AF049544 | Oregon | Burkholderia sp. | (+) | II | U25717 | 2 |
TFD6 | AF049546 | Michigan | Burkholderia sp. | − | II | 23 | |
TFD2 | AF049545 | Michigan | Burkholderia sp. | − | II | 23 | |
TFD31 | AF049536 | Saskatchewan | Rhodoferax fermentans | − | III | 23 | |
B6-9 | AF049538 | Ontario | Rhodoferax fermentans | ND | III | U43196 | 9 |
I-18 | U22836 | Oregon | Halomonas sp. | ND | III | U22499 | 15 |
K1443 | AF049531 | Michigan | Sphingomonas sp. | − | —d | 11 | |
2,4-D1 | AF049535 | Montana | Sphingomonas sp. | − | — | R. Sanford | |
B6-5 | AF049533 | Ontario | Sphingomonas sp. | ND | — | 9 | |
B6-10 | AF049534 | Ontario | Sphingomonas sp. | ND | — | 9 | |
EML146 | AF049532 | Oregon | Sphingomonas sp. | − | — | 2 | |
M1 | AF049530 | French Polynesia | Rhodospeudomonas sp. | ND | R. Fulthorpe |
Phylogenetic analyses.
Total genomic DNA was isolated from 20 unique 2,4-D-degrading strains (including all 15 used for mating experiments) grown on 500 ppm of 2,4-D mineral salts medium amended with 50 ppm of yeast extract. SSU rDNA was amplified by using fD1 and rD1 as primers (25). Putative tfdA fragments were amplified by using primers TVU and TVL as previously described (24). PCR products were purified with a Gene Clean kit (Bio 101, La Jolla, Calif.). Sequencing was done with an Applied Biosystems model 373A automatic sequencer (Perkin-Elmer Cetus) by using fluorescently labeled dye termination at the Michigan State University Sequencing Facility. The sequencing primer used for SSU rDNA fragments was 519R (5′ GTA TTA CCG CGG CTG CTG G-3′). For tfdA fragments, the sequencing primers were the same as the amplification primers. GenBank accession numbers for these sequences are given in Table Table11.The SSU rDNA sequences were compared to sequences in GenBank by using the Basic Local Alignment Search Tool (BLAST) (1), and those strains with the highest maximal segment pair scores were retrieved from GenBank and included in the phylogenetic analysis. Sequences were aligned manually with the software SeqEd (Applied Biosystems) and with MacClade (14). Sites where nucleotides were not resolved for all sequences were deleted from the alignment, as were those nucleotides corresponding to the small loop in this region that is absent in the alpha subgroup of the class Proteobacteria. These deletions left 283 unambiguous sites for the construction of the SSU rDNA phylogenies. Phylogenetic trees were constructed by using the neighbor-joining analysis of pairwise Jukes-Cantor distances (4), and the topology was confirmed by using the maximum parsimony method PAUP (21). Desulfomonile tiedjei of the δ-Proteobacteria was used as an outgroup. Bootstrap analysis based on 100 replicates was used to place confidence estimates on the tree. Only bootstrap values of greater than 50 were used.2,4-D degrader diversity.
The 2,4-D degraders in this study were distributed throughout the alpha, beta, and gamma subgroups of the Proteobacteria (Fig. (Fig.1).1). The lack of representation of gram-positive bacteria is likely a reflection of isolation methods, not of the lack of gram-positive 2,4-D degraders. The majority of these strains were members of the beta subgroup of Proteobacteria, five of which were most closely related to the genus Burkholderia, having at least 92% sequence similarity with each other. Three were closely related to Rhodoferax fermentans (close to the class Comamonadaceae), three were related to Ralstonia eutropha, and one was related to Alcaligenes xylosoxidans. TFD39 falls outside any clear cluster. One member of the γ-Proteobacteria, strain I-18, a haloalkaliphile, was found to be closely related to the salt-loving genus Halomonas (15). The remaining six strains all clustered in the alpha branch of Proteobacteria (Fig. (Fig.1).1). Of this subgroup, five were most closely related to the genus Sphingomonas. One member of the α-Proteobacteria, strain M1, which is the most oligotrophic and slow growing of all the strains used in this study, is 97% similar to Rhodopseudomonas palustris. The character of strain M1 correlates well with its phylogenetic placement near the slow-growing genus Bradyrhizobium. Open in a separate windowFIG. 1Neighbor-joining dendrogram (Jukes-Cantor distances) of SSU rDNA from 2,4-D-degrading bacteria (indicated in boldface type) and reference strains (indicated in italic type). Class I (•), class II (▴), and class III (■) types of tfdA genes are indicated. Bootstrap confidence limits (percentages) are indicated above each branch. Scale bar represents a Jukes-Cantor distance of 0.01.tfdA gene fragments.
tfdA gene fragments were successfully amplified and sequenced from 10 strains of β-Proteobacteria and 1 strain of γ-Protobacteria. None of the strains from the α-Proteobacteria gave any amplificates with these primers. These 313 contiguous nucleotides were aligned with additional tfdA sequences from JMP134 and from strain RASC (Fig. (Fig.2).2). Three distinct classes of tfdA gene sequences with slight variations in each class were found. Class I included fragments from JMP134, TFD39, TFD23, K712, and TFD9 that differed from each other by 2 bp at the most. Class I tfdA genes are probably plasmid encoded. All strains with a class I tfdA gene examined so far contained broad-host-range, self-transmissible plasmids containing 2,4-D genes (2, 3, 11, 17). All of the strains with a class I tfdA gene were able to transfer the 2,4-D phenotype in the mating studies reported above. The class II tfdA sequences included identical fragments amplified from RASC, TFD6, and TFD2 which were 76% similar to those in class I. Class III included identical fragments from strains TFD31, B6-9, and I-18 which were 77% similar to class I genes and 80% similar to class II genes. Both class II and III tfdA genes differed from each other and from class I genes in the same nine sites corresponding to the third base pair of the codons. The tfdA phylogenetic tree is a simple one, with three distinct branches that are incongruent with the SSU rDNA-derived phylogeny (Fig. (Fig.3).3). Class I tfdA sequences were found in Burkholderia-like strains, in strains related to the Comamonas-Rhodoferax group, and in the Ralstonia-Acaligenes group, all in the β-Proteobacteria. Class II sequences are less widely distributed, found only in Burkholderia-like branches. However, even in this subgroup, this tfdA variant is found in strains that differ by 7% at the SSU rDNA level (RASC and TFD2). However, the class III sequences were most interesting, being found both in the Comamonas-Rhodoferax group and in a strain of the γ-Proteobacteria, I-18, strains that differ by 24% at the SSU rDNA level. Class III genes have since been found in a collection of randomly isolated non-2,4-D degraders, including gram-positive bacilli, as well as in various gram-negative bacteria, even though the gene is not expressed (10). Open in a separate windowFIG. 2Alignment of 313 nucleotides of internal fragments of tfdA genes from representative strains. Nucleotides identical to tfdA from pJP4 are represented by periods.Open in a separate windowFIG. 3Phylogenetic incongruency of tfdA genes and SSU rDNA from diverse 2,4-D-degrading bacteria. Dendrograms for tfdA and SSU rDNA are indicated. Shading indicates the type of tfdA sequence, either class I, II, or III. Note that branch lengths are not drawn to scale.An interesting result was the detection of two different tfdA gene variants in sibling strains. TFD23 and TFD31 are identical at the ribosomal gene level, but one harbors a class I gene and the other harbors a class III gene. Similarly, TFD6 and EML159 are rRNA siblings that carry a class II and class I gene, respectively.None of the α-Proteobacteria yielded a PCR product when amplified with the conserved tfdA primers. This finding complements our observation that none of these bacteria hybridized to the tfdA gene, even under conditions of low stringency, indicating that any tfdA-like genes in the α-Proteobacteria are likely to be more divergent from the ones sequenced here (7, 11). In addition, none of the Sphingomonas strains in the study hybridized with a whole pJP4 probe, and similarly, no Sphingomonas strains scored positive for transfer of 2,4-D-degrading ability to recipient B. cepacia D5. Together these results suggest a reduced gene flow between members of the α- and β- or γ-Proteobacteria or poor gene expression of β- or γ-derived genes by α-Proteobacteria. Although plasmid pJP4 is a broad-host-range plasmid and has been known to transfer to α-Proteobacteria such as Rhizobium and Agrobacterium species and to γ-Proteobacteria such as Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas aeruginosa, the 2,4-D pathway is not expressed in these strains of the α- or γ-Proteobacteria (3). Phylogenetically limited expression of plasmid-borne 3-chlorobenzoate-degradative genes has also been noted for the pseudomonads (8). Subsequent studies have found divergent but related sequences for the tfdB and tfdC genes in 2,4-D-degrading Sphingomonas strains (7, 12, 24).With the exceptions of the minor differences within the class I pJP4-like tfdA sequences, there were no intermediate tfdA sequences. The most likely explanation of this is that the rate of horizontal transfer of the tfd genes is high relative to the rate at which mutations can accumulate. Examination of sequences of tfdA genes from a greater variety of organisms may turn up more intermediate variation. 相似文献200.