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1.
We analyzed the temporal and spatial diversity of the microbiota in a low-usage and a high-usage hospital tap. We identified a tap-specific colonization pattern, with potential human pathogens being overrepresented in the low-usage tap. We propose that founder effects and local adaptation caused the tap-specific colonization patterns. Our conclusion is that tap-specific colonization represents a potential challenge for water safety.Humans are exposed to and consume large amounts of tap water in their everyday life, with the tap water microbiota representing a potent reservoir for pathogens (8). Despite the potential impact, our knowledge about the ecological diversification processes of the tap water microbiota is limited (4, 11).The aim of the present work was to determine the temporal and spatial distribution patterns of the planktonic tap water microbiota. We compared the summer and winter microbiota from two hospital taps supplied from the same water source. We analyzed 16S rRNA gene clone libraries by using a novel alignment-independent approach for operational taxonomic unit (OTU) designation (6), while established OTU diversity and richness estimators were used for the ecological interpretations.Tap water samples (1 liter) from a high-usage kitchen and a low-usage toilet cold-water tap in Akershus University Hospital, Lørenskog, Norway, were collected in January and July 2006. The total DNA was isolated and the 16S rRNA gene PCR amplified and sequenced. Based on the sequences, we estimated the species richness and diversity, we calculated the distances between the communities, and trees were constructed to reflect the relatedness of the microbiota in the samples analyzed. Details about these analytical approaches are given in the materials and methods section in the supplemental material.Our initial analysis of species composition was done using the RDPII hierarchical classifier. We found that the majority of pathogen-related bacteria in our data set belonged to the class Gammaproteobacteria. The genera encompassed Legionella, Pseudomonas, and Vibrio (Table (Table1).1). We found a significant overrepresentation of pathogen-related bacteria in the toilet tap (P = 0.04), while there were no significant differences between summer and winter samples. Legionella showed the highest relative abundance for the pathogen-related bacteria. With respect to the total diversity, we found that Proteobacteria dominated the tap water microbiota (representing 86% of the taxa) (see Table S1 in the supplemental material). There was, however, a large portion (56%) of the taxa that could not be assigned to the genus level using this classifier.
TABLE 1.
Cloned sequences related to human pathogensaOpen in a separate windowaThe relatedness between the cloned sequences and potential pathogens was determined by BLAST searches of the NCBI database, carried out using default settings.To obtain a better resolution of the uncharacterized microbiota, we analyzed the data using a clustering approach that is not dependent on a predefined bacterial group (see the materials and methods section in the supplemental material for details). These analyses showed that there were three relatively tightly clustered groups in our data set (Fig. (Fig.1A).1A). The largest group (n = 590) was only distantly related to characterized betaproteobacteria within the order Rhodocyclales. We also identified another large betaproteocaterial group (n = 320) related to Polynucleobacter. Finally, a tight group (n = 145) related to the alphaproteobacterium Sphingomonas was identified.Open in a separate windowFIG. 1.Tap water microbiota diversity, determined by use of a principal component analysis coordinate system. (A) Each bacterium is classified by coordinates, with the following color code: brown squares, kitchen summer; red diamonds, toilet summer; green triangles, kitchen winter; and green circles, toilet winter. (B and C) Each square represents a 1 × 1 (B) or 5 × 5 (C) OTU. PC1, first principal component; PC2, second principal component.The tap-specific distributions of the bacterial groups were investigated using density distribution analyses. A dominant population related to Polynucleobacter was identified for the toilet summer samples, while for the winter samples there was a dominance of the Rhodocyclales-related bacteria. The kitchen summer samples revealed a dominance of Sphingomonas. The corresponding winter samples did not reveal distinct high-density bacterial populations (see Table S2 in the supplemental material).Hierarchical clustering for the 1 × 1 OTU density distribution confirmed the relatively low overlap for the microbiota in the samples analyzed (Fig. (Fig.2).2). We found that the microbiota clustered according to tap and not season.Open in a separate windowFIG. 2.Hierarchical clustering for the density distribution of the tap water microbiota. The density of 1 × 1 OTUs was used as a pseudospecies for hierarchical clustering. The tree for the Cord distance matrix is presented, while the distances calculated using the three distance matrices Cord, Brad Curtis, and Sneath Sokal, respectively, are shown for each branch.We have described the species diversity and richness of the microbiota in Table S3 in the supplemental material. For the low taxonomic level, these analyses showed that the diversity and species richness were greater for the winter samples than for the summer samples. Comparing the two taps, the diversity and richness were greater in the kitchen tap than in the toilet tap. In particular, the winter sample from the kitchen showed great richness and diversity. The high taxonomic level, however, did not reveal the same clear differences as did the low level, and the distributions were more even. Rarefaction analyses for the low taxonomic level confirmed the richness and diversity estimates (see Fig. S1 in the supplemental material).Our final analyses sought to fit the species rank distributions to common rank abundance curves. Generally, the rank abundance curves were best fitted to log series or truncated log normal distributions (see Table S4 in the supplemental material). The log series distribution could be fit to all of the samples except the kitchen summer samples at the low taxonomic level, while the truncated log normal distribution could not be fit to the kitchen samples at the high taxonomic level. Interestingly, however, the kitchen winter sample was best fit to a geometric curve at both the high and the low taxonomic level.Diversifying, adaptive biofilm barriers have been documented for tap water bacteria (7), and it is known that planktonic bacteria can interact with biofilms in an adaptive manner (3). On the other hand, tap usage leads to water flowthrough and replacement of the global with the local water population by stochastic founder effects (1).Therefore, we propose that parts of the local diversity observed can be explained by local adaptation (10) and parts by founder effects (9).Most prokaryote diversity measures assume log normal or log series OTU dominance density distributions (5). The kitchen winter sample, however, showed deviations from these patterns by being correlated to geometric distributions (in addition to the log series and truncated log normal distributions for the high taxonomic level). This sample also showed a much greater species richness than the other samples. A possible explanation is that the species richness of the tap water microbiota can be linked to usage and that the kitchen tap is driven toward a founder microbiota by high usage.Since our work indicates an overrepresentation of Legionella in the low-usage tap, it would be of high interest to determine whether the processes for local Legionella colonization can be related to tap usage. Understanding the ecological forces affecting Legionella and other pathogens are of great importance for human health. At the Akerhus University Hospital, this was exemplified by a Pseudomonas aeruginosa outbreak in an intensive care unit, where the outbreak could be traced back to a single tap (2). 相似文献2.
Paul W. J. J. van der Wielen Gertjan Medema 《Applied and environmental microbiology》2010,76(14):4876-4881
Bacteroidales species were detected in (tap) water samples from treatment plants with three different PCR assays. 16S rRNA gene sequence analysis indicated that the sequences had an environmental rather than fecal origin. We conclude that assays for Bacteroidales 16S rRNA genes are not specific enough to discern fecal contamination of drinking water in the Netherlands.Drinking water in many countries is routinely monitored for recent fecal contamination by testing for fecal indicator organisms Escherichia coli, thermotolerant coliforms, and/or intestinal enterococci to demonstrate microbial safety (13, 21, 42). Although these indicator organisms have been used for many decades, they have some limitations: the number of E. coli/coliform/enterococcus bacteria in feces is relatively low (18, 38), and they sometimes might be able to grow in the environment (10, 11, 14, 27). Consequently, scientists have been searching for alternative indicator organisms to determine fecal contamination of water. In 1967, bacteria belonging to the genus Bacteroides were suggested as alternative indicator organisms (26). Bacteroides spp. might have some advantages over the traditional indicator organisms. The numbers of Bacteroides spp. in the intestinal tract of humans and animals are 10 to 100 times higher than the numbers of E. coli or intestinal enterococci (1, 2, 12, 26). However, the use of Bacteroides spp. as indicator organisms was hampered by the complex cultivation conditions required (1, 2). The introduction of molecular methods made it possible to detect bacterial species that belong to the order Bacteroidales, an order that includes the genus Bacteroides, without cultivation. As a result, real-time PCR methods were developed for the quantitative detection of Bacteroidales in surface and recreation water and the potential of Bacteroidales species as an indication of fecal contamination of recreational waters was demonstrated (6, 12, 16, 19, 20, 29). Bacteroidales species might be useful indicator organisms for fecal contamination of drinking water as well. However, methods to detect fecal contamination in drinking water should be more sensitive, because people ingest more drinking water and the quality assessments and standards for fecal contamination are stricter than for bathing water. Studies exploring real-time PCR for the detection of Bacteroidales genes in drinking water have not been published to our knowledge. The objective of our study was, therefore, to determine if assays for the detection of Bacteroidales 16S rRNA genes can be used to detect fecal contamination in drinking water.Unchlorinated tap water samples were obtained in November 2007 and February 2010 from one or more locations in the distribution systems of nine different drinking water treatment plants (plants A to I; Table Table1)1) that produced unchlorinated drinking water from confined (plants B, C, E, F, and G) and unconfined (plants A, D, H, and I) groundwater. The treatment plants are located in the central part of the Netherlands within 90 km of each other. In addition, untreated groundwater from extraction wells and/or untreated raw groundwater (mixture of groundwater from different extraction wells) was sampled in March 2008 (Table (Table1).1). Water samples (100 ml) were filtered over a 25-mm polycarbonate filter (0.22-μm pore size, type GTTP; Millipore, Netherlands) and a DNA fragment was added as internal control to determine the recovery efficiency of DNA isolation and PCR analysis (2a, 40). DNA was isolated using a FastDNA spin kit for soil (Qbiogene, United States) according to the supplier''s protocol. Primer sets AllBac 296f and AllBac 412r, resulting in a PCR product of 108 bp, were used in combination with TaqMan probe AllBac375Bhqr to quantitatively determine the number of Bacteroidales 16S rRNA gene copies in the water samples using a real-time PCR instrument (20). The PCR cycle after which the fluorescence signal of the amplified DNA was detected (threshold cycle [CT]) was used to quantify the concentration of 16S rRNA gene copies. Quantification was based on comparison of the sample CT value with the CT values of a calibration curve graphed using known copy numbers of the Bacteroidales 16S rRNA gene, as previously described (12, 20). The correlation coefficient of the calibration curve was 0.99, and the efficiency of the PCR 95 to 105%. Finally, the Bacteroidales cell number was calculated by using the recovery rate of the internal standard and assuming five 16S rRNA gene copy numbers per cell (5). The detection limit of this gene assay was 50 Bacteroidales cells 100 ml−1 (corresponding to 10 16S rRNA gene copies per reaction mixture). Furthermore, the 16S rRNA genes that were obtained from several water samples from treatment plant C with the AllBac and TotBac (12) primer sets were sequenced, and the nearest relatives were obtained from the GenBank database using BLAST searches.
Open in a separate windowaValues are the average results and standard deviations from replicate PCRs on the same water sample using the AllBac primer set (20). In November 2007, the distribution systems (tap water) of plants A, B, and G were sampled at three different locations, whereas for the other plants, one location in the distribution system was sampled. In March 2008, raw water of plants A to G was sampled, as well as one (plant E) or three (plant C) different extraction wells. Finally, in February 2010, the distribution systems of plants A, B, C, D, E, and G were sampled again.bMore than one tap water sample from a treatment plant means that samples were taken at different locations in the distribution system.The Bacteroidales 16S rRNA gene, quantified with the AllBac primer set, was detected in all tap water samples in November 2007 and February 2010. The number of cells varied between 154 and 7,862 Bacteroidales cells 100 ml−1, and the numbers in tap water of each plant were similar in 2007 and 2010 (Table (Table1).1). The Bacteroidales counts were high compared to the number of E. coli that are occasionally observed in fecally contaminated drinking water (17a) but low compared to numbers observed in surface water (4, 20, 22). Water from the extraction wells and raw water used for unchlorinated drinking water production were analyzed, and Bacteroidales species were detected in 10 out of 15 samples (Table (Table1).1). These results would imply that the extracted groundwater, raw water, and tap water were fecally contaminated. According to the Dutch drinking water decree (2b), both raw and tap water from the nine different treatment plants are regularly analyzed for fecal contamination by monitoring for E. coli, F-specific RNA phages, and somatic coliphages. For at least the last 10 years, these indicator organisms have not been detected in these waters.Additional qualitative PCR analyses using TotBac and BacUni primer sets (12, 19) targeting other parts of the Bacteroidales 16S rRNA gene were performed to confirm the presence of Bacteroidales species in the water samples of November 2007 and March 2008. Nine or 10 of the 11 samples that were positive with the AllBac primer set were also positive with the TotBac and BacUni primer sets (data not shown). The BacUni primer set has a higher detection limit (30 gene copies per PCR; 19), which could explain the difference from the results with the AllBac primer set. The TotBac primer set has the same detection limit as the AllBac primer set (12), but small differences in PCR efficiencies might have resulted in different results, since some water samples showed Bacteroidales 16S rRNA gene copy numbers around the detection limit (Table (Table1).1). Nevertheless, the additional PCR analyses demonstrated that the detection of Bacteroidales species in tap, raw, and extracted well water with the AllBac primer set was not an artifact. The primer sets used were developed in three different studies (12, 19, 20) but have been applied in a number of recent studies to detect fecal contamination of surface water (3, 4, 16, 22, 33, 34). The results from most of these studies showed that 16S rRNA genes of Bacteroidales were present in all surface water samples tested. Only Sinigalliano et al. (34) observed that 2 out of 4 water samples were negative with the TotBac primer set. However, the detection limit of the assay was not specified in that study.The nine different treatment plants tested in our study produce unchlorinated drinking water from groundwater, which is considered to be of high hygienic quality. In addition, the extraction wells are protected from fecal contamination by a protection zone where no activities related to human waste or animal manure are allowed. In the Netherlands, this protection zone is based on a 60-day residence time of the water. Previous studies have demonstrated that a residence time of 60 days is highly effective in removing fecal bacteria and viruses (30, 31, 39). Moreover, the Bacteroidales numbers in tap water in November 2007 were significantly higher than the numbers in raw groundwater in March 2008 (Mann-Whitney U test; P < 0.01). Because the recovery efficiency of the internal control was the same between raw water and tap water samples, this result demonstrates that Bacteroidales cell numbers increased during treatment and/or drinking water distribution. This result could suggest that the water was fecally contaminated during drinking water treatment and/or distribution. However, it is unlikely that the integrity of nine different treatment trains and/or supply systems was affected in the sampling period. The statutory monitoring did not show the presence of E. coli at these sites. Another hypothesis is that the increase of Bacteroidales cell numbers in tap water was caused by the growth of Bacteroidales species in (drinking) water systems. In summary, it is unexpected that the majority of the tap water, raw water, and extracted groundwater samples were fecally contaminated. These unexpected observations raise the question of whether the PCR methods detect only fecal Bacteroidales species and, thus, if the gene assays are suitable to discern fecal contamination in drinking water in the Netherlands.Sequence analyses of the Bacteroidales 16S rRNA genes were performed to determine the relatedness of sequences from the different sampling sites to sequences from the nearest relatives in the GenBank database. All sequences contained the primer regions, indicating that nonspecific amplification had not occurred in the PCRs. Because the PCR product from the AllBac primer set was small (108 bp), many 16S rRNA gene sequences (100 to 5,000) in the GenBank database were identical to the Bacteroidales 16S rRNA gene sequences obtained from groundwater and unchlorinated tap water samples from plant C. These identical 16S rRNA gene sequences were in general obtained from fecal sources, but some of them came from environmental rather than fecal sources (Table (Table2).2). The AllBac 16S rRNA gene sequences from tap water and groundwater had relative high similarities (96.3 to 100%) to sequences from bacterial species of the genera Bacteroides, Prevotella, and Tannerella (Table (Table2),2), which all belong to the order Bacteroidales.
TABLE 1.
Numbers of Bacteroidales cells in extraction wells, raw groundwater, and unchlorinated tap water of nine different groundwater plants in the NetherlandsaPlant | Source of sample | No. (100 ml−1) of Bacteroidales cells in: | ||
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2007 | 2008 | 2010 | ||
A | Tap water 1b | 5,948 ± 950 | ||
Tap water 2 | 2,682 ± 1,459 | 1,254 ± 216 | ||
Tap water 3 | 4,362 ± 947 | 439 ± 136 | ||
Raw water | 96 ± 15 | |||
B | Tap water 1 | 3,553 ± 981 | 5,302 ± 2,952 | |
Tap water 2 | 4,487 ± 391 | 2,119 ± 1,367 | ||
Tap water 3 | 7,862 ± 4,588 | 3,896 ± 3,003 | ||
Raw water | 3,209 ± 833 | |||
C | Tap water 1 | 661 ± 75 | 386 ± 199 | |
Tap water 2 | 1,051 ± 626 | |||
Tap water 3 | 831 ± 584 | |||
Tap water 4 | 1,254 ± 216 | |||
Extraction well 1 | 1,126 ± 262 | |||
Extraction well 2 | 2,666 ± 51 | |||
Extraction well 3 | <50 | |||
Raw water | 90 ± 44 | |||
D | Tap water | 1,103 ± 29 | 1,254 ± 216 | |
Raw water | 48 ± 16 | |||
E | Tap water | 1,302 ± 222 | 1,254 ± 216 | |
Extraction well 1 | 671 ± 97 | |||
F | Tap water | 1,317 ± 198 | ||
Raw water | <50 | |||
G | Tap water 1 | 675 ± 92 | 439 ± 300 | |
Tap water 2 | 216 ± 65 | 249 ± 98 | ||
Tap water 3 | 154 ± 6 | 322 ± 137 | ||
Raw water | <50 | |||
H | Tap water | 7,073 ± 845 | ||
Raw water | 511 ± 254 | |||
I | Tap water | 1,577 ± 176 | ||
Raw water | 420 ± 66 |
TABLE 2.
Nearest relatives in GenBank to the Bacteroidales 16S rRNA gene sequences obtained from groundwater and unchlorinated tap water from plant C using different primer setsaOpen in a separate windowaPrimer sets AllBac (20) and TotBac (12) were used in PCRs of samples, and GenBank was searched for relatives using BLAST.bOTUs are indicated by the values in parentheses (number of sequences belonging to the OTU/total number of sequences analyzed).cNumber of base pairs identical in both sequences/total number of base pairs in sequences.16S rRNA gene sequences obtained with the TotBac primer set were longer (∼370 bp) and did not show 100% similarity with the nearest relatives in the GenBank database (Table (Table2).2). Sequences from the GenBank database that showed the highest similarity (91.6% to 99.7%) with the 16S rRNA gene sequences from tap water and groundwater from plant C were in general isolated from environmental sources (Table (Table2).2). The 16S rRNA gene sequences from cultivated bacterial species that showed the highest similarity to the 16S rRNA gene sequences obtained in our study belonged to different genera (Table (Table2).2). Some of these genera (Salinimicrobium, Xanthobacillum, and Psychroserpens) did not belong to the order Bacteroidales. However, the 16S rRNA gene sequences from bacterial species of these genera showed low similarities with the sequences obtained in this study (83.2% to 90.1%) and six mismatches to the TotBac primers. Thus, it is unlikely that DNA from bacterial species belonging to Salinimicrobium, Xanthobacillum, and Psychroserpens was amplified in the gene assay. More importantly, the majority of the nearest environmental clone sequences retrieved from the GenBank database showed no or a single mismatch with the AllBac and TotBac primer and probe sequences. Thus, these primer sets are capable of amplifying 16S rRNA genes from bacteria that have been observed in ecosystems outside the intestinal tract of humans and animals.16S rRNA gene sequences related to Prevotella species were commonly observed in extracted groundwater, raw water, and tap water (Table (Table2).2). The isolation of Prevotella paludivivens from rice roots in a rice field soil (35) demonstrated the environmental nature of some Prevotella species. In addition, primer sequences developed for the detection of fecal Bacteroidales species (8, 12, 19, 20, 25, 29) showed no or a single mismatch with 16S rRNA gene sequences from P. paludivivens, Xylanibacterium oryzae, Paludibacter propionicigenes, Proteiniphilum acetatigenes, and Petrimonas sulfuriphila that are present in the GenBank database. These five Bacteroidales species have all been isolated from ecosystems other than the gastrointestinal tract. Consequently, primer sets for 16S rRNA genes of Bacteroidales species cannot always be used to discern fecal contamination in water.A number of 16S rRNA gene sequences observed in groundwater and tap water fell in the genus Bacteroides. The presence of Bacteroides 16S rRNA gene sequences in groundwater and tap water might also suggest that some Bacteroides species are capable of growth in the environment. However, until now, type strains of Bacteroides species growing outside the animal intestinal tract have not been published. Another possible explanation is that the observed 16S rRNA gene sequences originate from Bacteroides species that inhabit the anoxic intestinal tract of insects. Previous studies have shown that bacterial species belonging to the genus Bacteroides are common inhabitants of the hindguts of insects (15, 23, 24, 28, 32). Some of the 16S rRNA gene sequences obtained with the AllBac primer set in our study showed 100% similarity to 16S rRNA gene sequences from the hindgut of insects. Moreover, a number of 16S rRNA gene sequences isolated from the hindguts of insects (15, 23, 24, 32) showed no or a single mismatch with the TotBac and AllBac primer and probe sequences. In conclusion, these primer sets are capable of detecting Bacteroides species from the hindgut of insects as well. Water insects are normal inhabitants of groundwater and drinking water distribution systems (7, 41) and might be a source of Bacteroides species in water. Bacteroides species from insect feces do not indicate fecal pollution by warm-blooded animals, and insects do not normally shed human fecal pathogenic microorganisms. Bacteroides species from insect feces, therefore, can hamper Bacteroides gene assays developed for the detection of water fecally contaminated by warm-blooded animals. Additional cultivation techniques in combination with molecular tools are required to demonstrate the persistence or growth of Bacteroides bacteria in groundwater and drinking water or whether Bacteroides bacteria are present in water insects. However, these experiments were beyond the scope of our study.The three extraction wells of plant C are located close to each other and extract water from the same aquifer. Subsequently, extracted water from the three wells is mixed and enters the treatment plant as raw water. We hypothesize that if a fecal source in the vicinity of the extraction field of plant C contaminated the groundwater, water from the extraction wells and raw water should (partly) have the same Bacteroidales species. Although a relatively limited amount of clones was sequenced per sample (16), the diversity of Bacteroidales operational taxonomic units (OTU) within a sample was low (Table (Table2).2). In contrast, unique 16S rRNA gene sequences were observed between the different water types (e.g., extracted groundwater, raw water, and tap water) and sequence overlap between water types was low. These results demonstrate that the Bacteroidales 16S rRNA gene sequences at the sampling locations were not from the same fecal source and imply once again that Bacteroidales species were environmental rather than fecal.Finally, we hypothesized that if the Bacteroidales species observed in tap water were of nonfecal origin, human- and/or bovine-specific Bacteroidales strains should not be present in tap water. We tested for the presence of human- or bovine-specific Bacteroidales strains by using source-specific 16S rRNA gene assays (5) on tap water samples from February 2010. The results showed that human- and bovine-specific Bacteroidales 16S rRNA genes could not be detected in tap water, whereas a PCR product was always detected with the positive control. Again, these results indicate that the Bacteroidales species observed in tap water were of nonfecal origin.Overall, the results from our study indicate that gene assays for Bacteroidales detected environmental rather than fecal Bacteroidales species in groundwater and tap water from treatment plants in the Netherlands. First, Bacteroidales 16S rRNA gene sequences obtained from water samples taken at plant C showed (high) similarity to clone sequences that were isolated from environmental sources. The majority of these clone sequences and several Bacteroides clone sequences from the hindguts of insects showed no or a single mismatch with AllBac, TotBac, and BacUni primer and probe sequences. Second, the primer and probe sequences used for the gene assays have no or a single mismatch with 16S rRNA gene sequences of environmental Bacteroidales species P. paludivivens, X. oryzae, P. propionicigenes, P. acetatigenes, and/or P. sulfuriphila (9, 17, 35-37). Third, Bacteroidales 16S rRNA gene sequences from raw water and water from extraction wells were unique, and sequence overlap between water types was low. It is expected that in the case of fecal contamination of groundwater, different water types from the same groundwater area have similar Bacteroidales species. Fourth, the quantitative assays for Bacteroidales 16S rRNA genes commonly used to detect fecal contamination (3, 4, 12, 16, 19, 20, 22, 33, 34) detected Bacteroidales species in deep groundwater and tap water that have no history of fecal contamination. Fifth, Bacteroidales gene copy numbers were significantly higher in tap water than in raw groundwater, demonstrating an increase or growth of Bacteroidales species during the treatment and/or distribution of drinking water. Finally, human- and bovine-specific Bacteroidales strains were not detected in tap water. Consequently, (quantitative) assays for general Bacteroidales 16S rRNA genes are not suitable to discern fecal contamination in groundwater and unchlorinated drinking water in the Netherlands.Nucleotide sequence accession numbers.The 16S rRNA gene sequences obtained in this study were deposited in the GenBank database under accession numbers . GQ169588 to GQ169609相似文献3.
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To determine whether and which spirochetes are cleared from Ixodes ricinus ticks during feeding on ruminants, ticks were removed from goats and cattle grazing on tick-infested pastures. Although about a quarter of ticks questing on the pasture were infected by spirochetes, no molted ticks that had previously engorged to repletion on ruminants harbored Lyme disease spirochetes. Borrelia miyamotoi spirochetes, however, appear not to be eliminated. Thus, the more subadult ticks are diverted from reservoir-competent hosts to zooprophylactic ruminants, the smaller the risk of infection by Lyme disease spirochetes is.Various vertebrates serve as reservoir hosts for the tick-borne agents of Lyme disease. A competent reservoir host acquires Lyme disease spirochetes when an infected tick feeds on it and maintains them to become and remain infectious for feeding ticks (10). It appears that each of the seven genospecies of Borrelia burgdorferi sensu lato prevalent in Central Europe is associated with particular reservoir hosts. Whereas rodents serve as a reservoir for B. afzelii and the recently differentiated but not yet validated “Borrelia bavariensis,” birds maintain B. garinii and B. valaisiana (3, 4). B. lusitaniae and B. spielmanii, on the other hand, seem to be limited to lizards and dormice, respectively (9, 12, 13). Ticks harboring rodent-associated spirochetes from their larval blood meal may lose the infectious burden when feeding as nymphs on a bird and vice versa (5). It appears that solely B. burgdorferi sensu stricto constitutes an intermediate position, since it may be perpetuated by birds and rodents (10, 11). As a generalist, B. burgdorferi sensu stricto appears to be less efficiently adapted to rodents than is the specialist B. afzelii. A host that is competent for one genospecies seems less competent or incompetent for another.The Central European vector tick, Ixodes ricinus, not only feeds on small animals. Wild ruminants, such as red, roe, and fallow deer, are frequently infested by all three stages of this tick (2, 6, 15). Interestingly, virtually no spirochetes were detected microscopically in ticks recovered from shot deer. On pastures, where domesticated ruminants graze at an extensive density, spirochetal infection in questing ticks is less prevalent than in nearby nonpastured sites (8). These ruminants appear to exert a zooprophylactic effect. Ruminants, although feeding numerous ticks, appear to be incompetent hosts for Lyme disease spirochetes. It is not known whether the incompetence of ruminants eliminates spirochetes in the feeding tick and whether it extends to each of the Lyme disease genospecies.To determine whether and which spirochetes are cleared from ticks feeding on ruminants, ticks were removed from goats and cattle grazing on tick-infested pastures and examined at various developmental stages for Lyme disease genospecies and B. miyamotoi. Infection rates in ruminant-derived ticks were compared to that in ticks questing on the pastures.The cattle study site was located southwest of the city of Flensburg, Germany, at the German-Danish border. The former training area of the German armed forces is used as low-intensity pasture, covering about 400 ha. Galloway cattle, in herds of mother cows, and Konik horses are allowed to graze year-round and are rotated on grazing patches. Most cattle which were examined for feeding ticks grazed in a 40-ha area which has been pastured since October 2004 and from which cattle and horses are excluded each year from April through June to permit rare plants to bloom and seed. The approximate grazing density of 0.25 livestock units (LU)/ha throughout the rest of the year fails to keep the vegetation short. The goat site was located about 50 km southeast of Stuttgart, Germany, near the village of Gruibingen in the Swabian highlands. Beech and juniper heath characterize the southern-facing mountain slopes, where goats were allowed to graze in a rotating regime during the vegetation period. The sites were in use as pastures for different lengths of time, with the oldest dating back to 2004.To obtain feeding ticks from cattle and goats, two approaches were used. For the yearly blood sampling in the spring, cattle were corralled into squeeze chutes. The head of each animal was examined for ticks. Feeding ticks were carefully removed with forceps, and replete ticks were gently rubbed off onto a sheet of fabric positioned under the cow''s head. From April through October 2006 and 2007 and in May of 2008, tame goats were examined individually for feeding ticks monthly and feeding ticks were removed with forceps. Ticks recovered from an individual animal were confined in screened vials and stored at 22°C to permit molting and/or until they were examined for spirochetes. Questing ticks were collected monthly from April through October 2008 in the cattle site and from April through October 2005 through 2007 at the goat site. They were collected by means of a flannel flag, identified to stage and species by microscopy, and preserved in 80% ethanol. To detect and identify the various spirochetes that may be present in questing or host-derived ticks, DNA from individual ticks was isolated, and a 600-nucleotide fragment of the gene carrying the 16S rRNA gene was amplified by nested PCR and sequenced as described previously (12). This method detects as little as a single spirochete even in the presence of tick and ruminant DNA. Each resulting sequence was compared with sequences of the same gene fragment representing various spirochetal genospecies. The following sequences were used for comparison: GenBank accession numbers and X85196 for B. burgdorferi sensu stricto, X85203, X85190, and X85192 for B. afzelii, X85194, X85193, and X85199 for B. garinii, M64311 and X98228 for B. lusitaniae, X98229 and X98232 for B. valaisiana, X98233 for B. spielmanii, and AY147008 for B. miyamotoi. A complete match, permitting no more than two nucleotide changes, was required.Ticks removed while feeding on cattle or goats were examined for spirochetal DNA by nested PCR. Nineteen larvae were obtained during their feeding on goats, but none of the 17 engorged larvae and 2 resulting nymphs contained spirochetal DNA (Table AY253149(Table1).1). Of the 416 nymphal ticks that were obtained from 80 goats, only 9% developed to the adult stage, because most of the nymphs were only partially fed. None of the 37 resulting adults contained spirochetal DNA. However, three partially fed nymphal ticks were infected by Lyme disease spirochetes (0.8%), one each by B. afzelii, B. valaisiana, and B. lusitaniae. In three additional nymphs (0.8%), DNA of B. miyamotoi was detected. Of the 415 engorged nymphal ticks obtained from 42 cattle, as many as 319 (77%) molted to the adult stage, because mostly replete ticks had been collected from the cattle''s heads. None of the cattle-derived molted ticks harbored DNA of Lyme disease spirochetes. Four ticks, a nymph and three adults (one male and two females), contained DNA of B. miyamotoi. Of 306 partially engorged females removed from 68 goats, spirochetal DNA was detected in 9 females (2.9%); three harbored B. afzelii, four B. miyamotoi, and one each B. garinii and B. lusitaniae. In addition, 30 females which had fully engorged on cattle were tested for spirochetal DNA after egg-laying. None of these contained spirochetal DNA. Although DNA of Lyme disease spirochetes was detected in a rare partially fed tick, no molted tick that had previously engorged to repletion on a ruminant was infected by Lyme disease spirochetes. In contrast, B. miyamotoi appears to be present in ruminant-fed ticks regardless of their feeding state.
Open in a separate windowaLD, Lyme disease.bafz, B. afzelii; gar, B. garinii; val, B. valaisiana; lus, B. lusitaniae; miy, B. miyamotoi.cValue is not the sum of the above numbers because individual hosts were infested by various tick stages.The prevalence of spirochetal infection was determined in questing ticks collected on the pastures on which the cattle or the goats had roamed. A third of the nymphs and nearly a fifth of the adult ticks that quested on the cattle pasture in northern Germany contained spirochetal DNA (Table (Table2).2). The majority of these nymphs and half the infected adults were infected by B. afzelii. About a fifth of the nymphs and a quarter of the adult ticks questing on the goat pastures in southern Germany were infected by spirochetes. B. afzelii and B. lusitaniae infected most of these ticks. Thus, the cattle and goats in the study sites must have been exposed to numerous vector ticks infected by spirochetes.
Open in a separate windowaafz, B. afzelii; gar, B. garinii; val, B. valaisiana; bur, B. burgdorferi; lus, B. lusitaniae; spi, B. spielmanii; bis, B. bissettii-like (1, 7); miy, B. miyamotoi.Ticks infected by Lyme disease spirochetes appear to lose their infection when feeding on goats or cattle. If the blood meal on ruminants had no effect on the spirochetal burden, about 130 and 70 of the analyzed nymphs derived from cattle and goats, respectively, should have contained spirochetal DNA. The two infected cattle-derived ticks harbored solely spirochetes not related to those causing Lyme disease. Most of the ticks removed from goats were partially fed and appeared to be somewhat more likely to contain spirochetal DNA. Whether the detected DNA indicates viable spirochetes is not known. Either feeding on goats fails to eliminate spirochetes as effectively as does a blood meal on cattle or, more plausibly, engorgement to repletion is required for a complete elimination of DNA from Lyme disease spirochetes. If no spirochetal DNA is detected, the tick cannot contain viable spirochetes and thus is not infectious in its host-seeking stage.Wild and domestic ruminants appear to be reservoir incompetent for Lyme disease spirochetes. They do not constitute reservoirs for this pathogen, because no larval tick feeding on them acquires Lyme disease spirochetes. Of 176 engorged I. ricinus larvae or resulting nymphs that had been collected from roe, fallow, red deer, and wild sheep in a Central European site in an earlier study, spirochetes were detected by dark-field microscopy in only two ticks (6). Similarly, only 2 of nearly 200 Ixodes dammini nymphs resulting from larvae that had engorged on white-tailed deer in northeast America contained spirochetes detectable by direct immunofluorescence (15). No spirochetes were detected by phase-contrast microscopy in more than 200 Swedish nymphs that derived from roe-deer-fed larvae (2). Considering that B. miyamotoi morphologically resembles Lyme disease spirochetes, it is likely that all of the spirochetes detected microscopically in ruminant-derived ticks during these earlier studies were not related to B. burgdorferi sensu lato. Not only do larvae fail to acquire Lyme disease spirochetes from ruminants, but infected nymphs also appear to lose their spirochetal load when feeding on these animals, as the present study demonstrates. In ticks that had fully engorged on cattle, the only spirochetal DNA that was detected was that of B. miyamotoi. Also, the American strain of B. miyamotoi was discovered in larvae resulting from field-collected adult females that had routinely been fed on sheep (14). The previous observation that the prevalence of B. miyamotoi in a cattle pasture was not significantly reduced compared to that in the nonpastured site nearby further exemplifies the differential effect of ruminants on these two kinds of spirochetes (8). Whereas Lyme disease spirochetes are eliminated when their tick vector feeds on a ruminant, B. miyamotoi appears not to be affected by such a blood meal.Ruminants reduce the prevalence of infected ticks on a pasture. For the present study, sites were chosen that had only recently come into use as pastures and where cattle were excluded during the peak season of tick activity. The spirochetal prevalence was thus similar to that in the surrounding areas where no domestic ruminants roamed (data not shown) and permitted us to compare infection rates before and after the blood meal on ruminants. The effect of the grazing schedule and of the grazing duration that is required to result in reduced prevalence still needs to be determined. Domestic ruminants employed in landscape management appear to exert their zooprophylactic effect in multiple ways, by eliminating spirochetes from vector ticks feeding on them and by reducing the ecotonal vegetation, thereby limiting coverage and food sources of reservoir hosts while simultaneously rendering the microclimate less suitable for vector ticks. This study''s observations indicate that Lyme disease spirochetes are eliminated from the tick during its blood meal on a ruminant. The mechanism by which Lyme disease spirochetes are cleared during the tick''s blood meal is under investigation. Evidently, Lyme disease spirochetes are destroyed in a way that renders their DNA no longer detectable by means of nested PCR. A simulation model indicates that the availability of incompetent hosts for subadult tick stages would reduce the prevalence of infection (16). Therefore, the more subadult ticks are diverted from reservoir competent birds or mice to incompetent ruminants, the smaller the risk of infection with the agent of Lyme disease is. 相似文献
TABLE 1.
Borrelia genospecies detected in I. ricinus ticks that had engorged as larvae, nymphs, or adults on goats or cattleHost | No. of host animals | Ticks | % of infected ticks harboring Borrelia sp.b | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Stage | State | No. examined | % infected | % infected by LDa spirochete | afz | gar | val | lus | miy | ||
Goats | 10 | Larva | Engorged | 17 | 0.0 | 0.0 | |||||
2 | Nymph | Molted | 2 | 0.0 | 0.0 | ||||||
80 | Nymph | Engorged | 379 | 1.6 | 0.8 | 16.7 | 0.0 | 16.7 | 16.7 | 50.0 | |
22 | Adult | Molted | 37 | 0.0 | 0.0 | ||||||
68 | Adult | Engorged | 306 | 2.9 | 1.6 | 33.3 | 11.1 | 0.0 | 11.1 | 44.4 | |
Cattle | 36 | Nymph | Engorged | 96 | 1.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 100 |
42 | Adult | Molted | 319 | 0.9 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 100 | |
10 | Adult | Engorged | 30 | 0.0 | 0.0 | ||||||
Total | 144c | 1,186 | 1.4 | 0.7 | 23.5 | 5.9 | 5.9 | 11.8 | 52.9 |
TABLE 2.
Relative prevalences of Borrelia genospecies in questing nymphal and adult I. ricinus ticks sampled on goat or cattle pastures in GermanyGrazing animals on pasture | Ticks | % of infected ticks harboring Borrelia sp.a | ||||||||||
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Stage | No. examined | % infected | afz | gar | val | bur | lus | spi | bis | miy | >1 genospecies | |
Goats | Nymph | 557 | 17.2 | 41.7 | 6.3 | 5.2 | 4.2 | 36.5 | 0.0 | 0.0 | 8.3 | 2.1 |
Adult | 511 | 25.0 | 13.3 | 7.0 | 6.3 | 3.9 | 61.7 | 0.8 | 0.0 | 10.2 | 3.1 | |
Cattle | Nymph | 413 | 32.4 | 90.3 | 0.7 | 0.7 | 0.0 | 0.0 | 0.0 | 4.5 | 6.0 | 2.2 |
Adult | 67 | 17.9 | 50.0 | 16.7 | 8.3 | 0.0 | 0.0 | 0.0 | 16.7 | 8.3 | 0.0 |
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F. Joseph Pollock Pamela J. Morris Bette L. Willis David G. Bourne 《Applied and environmental microbiology》2010,76(15):5282-5286
A real-time quantitative PCR-based detection assay targeting the dnaJ gene (encoding heat shock protein 40) of the coral pathogen Vibrio coralliilyticus was developed. The assay is sensitive, detecting as little as 1 CFU per ml in seawater and 104 CFU per cm2 of coral tissue. Moreover, inhibition by DNA and cells derived from bacteria other than V. coralliilyticus was minimal. This assay represents a novel approach to coral disease diagnosis that will advance the field of coral disease research.Vibrio coralliilyticus has recently emerged as a coral pathogen of concern on reefs throughout the Indo-Pacific. It was first implicated as the etiological agent responsible for bleaching and tissue lysis of the coral Pocillopora damicornis on Zanzibar reefs (2). More recently, V. coralliilyticus has been identified as the causative agent of white syndrome (WS) outbreaks on several Pacific reefs (14). WS is a collective term describing coral diseases characterized by a spreading band of tissue loss exposing white skeleton on Indo-Pacific scleractinian corals (16). V. coralliilyticus is an emerging model pathogen for understanding the mechanisms linking bacterial infection and coral disease (13) and therefore provides an ideal model for the development of diagnostic assays to detect coral disease. Current coral disease diagnostic methods, which are based primarily upon field-based observations of macroscopic disease signs, often detect disease only at the latest stages of infection, when control measures are least effective. The development of diagnostic tools targeting pathogens underlying coral disease pathologies may provide early indications of infection, aid the identification of disease vectors and reservoirs, and assist managers in developing strategies to prevent the spread of coral disease outbreaks. In this paper, we describe the development and validation of a TaqMan-based real-time quantitative PCR (qPCR) assay that targets a segment of the V. coralliilyticus heat shock protein 40-encoding gene (dnaJ).Nucleotide sequences of the dnaJ gene were retrieved from relevant Vibrio species, including V. coralliilyticus (LMG 20984), using the National Center for Biotechnology Information''s (NCBI) Entrez Nucleotide Database search tool (http://www.ncbi.nlm.nih.gov/). Gene sequences of strains not available in public databases (V. coralliilyticus strains LMG 21348, LMG 21349, LMG 21350, LMG 10953, LMG 20538, LMG 23696, LMG 23691, LMG 23693, LMG 23692, and LMG 23694) were obtained through extraction of total DNA using a Promega Wizard Prep DNA Purification Kit (Promega, Sydney, Australia), PCR amplification, and sequencing using primers and thermal cycling parameters described by Nhung et al. (8). A 128-bp region (nucleotides 363 to 490) containing high concentrations of single nucleotide polymorphisms (SNPs), which were conserved within V. coralliilyticus strains but differed from non-V. coralliilyticus strains, was identified, and oligonucleotide primers Vc_dnaJ_F1 (5′-CGG TTC GYG GTG TTT CAA AA-3′) and Vc_dnaJ_R1 (5′-AAC CTG ACC ATG ACC GTG ACA-3′) and a TaqMan probe, Vc_dnaJ_TMP (5′-6-FAM-CAG TGG CGC GAA G-MGBNFQ-3′; 6-FAM is 6-carboxyfluorescein and MGBNFQ is molecular groove binding nonfluorescent quencher), were designed to target this region. The qPCR assay was optimized and validated using DNA extracted from V. coralliilyticus isolates, nontarget Vibrio species, and other bacterial species grown in marine broth (MB) (Table (Table1),1), under the following optimal conditions: 1× TaqMan buffer A, 0.5 U of AmpliTaq Gold DNA polymerase, 200 μM deoxynucleotide triphosphates (with 400 μM dUTP replacing deoxythymidine triphosphate), 0.2 U of AmpErase uracil N-glycosylase (UNG), 3 mM MgCl2, 0.6 μM each primer, 0.2 μM fluorophore-labeled TaqMan, 1 μl of template, and sterile MilliQ water for a total reaction volume to 20 μl. All assays were conducted on a RotoGene 300 (Corbett Research, Sydney, Australia) real-time analyzer with the following cycling parameters: 50°C for 120 s (UNG activation) and 95°C for 10 min (AmpliTaq Gold DNA polymerase activation), followed by 40 cycles of 95°C for 15 s (denaturation) and 60°C for 60 s (annealing/extension). During the annealing/extension phase of each thermal cycle, fluorescence was measured in the FAM channel (470-nm excitation and 510-nm detection).
Open in a separate windowaOrigin, host organism, and dnaJ gene sequence accession numbers are shown for V. coralliilyticus strains.bStrain designations beginning with LMG were derived from the Belgian Coordinated Collections of Microorganisms, ATCC strains are from the American Type Culture Collection, DSM strains are from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH culture collection, AIMS strains are from the Australian Institute of Marine Science culture collection, and C1 and C2 were provided by Pamela Morris.c†, amplification in one of three reactions; ††, amplification in two of three reactions; NA, no amplification.dIsolated from seawater above coral.The qPCR assay specifically detected 12 out of 13 isolated V. coralliilyticus strains tested in this study (Table (Table1).1). The exception was one Caribbean strain (C2), which failed to give specific amplification despite repeated attempts. Positive detection of the target gene segment was determined by the increase in fluorescent signal beyond the fluorescence threshold value (normalized fluorescence, 0.010) at a specific cycle, referred to as the threshold cycle (CT). Specific detection was further confirmed by gel electrophoresis, which revealed a PCR product of the correct theoretical size (128 bp) (data not shown), and DNA sequencing, which confirmed the target amplified product to be a segment of the dnaJ gene. No amplification with the assay was detected for 13 other closely related Vibrio strains, including the closely related Vibrio neptunius and two non-Vibrio species (Table (Table1).1). A total of five other Vibrio strains and one non-Vibrio strain (Shewanella sp.) exhibited CT values less than the cutoff of 32 cycles. However, CT values for these strains (mean ± standard error of the mean [SEM], 27.96 ± 2.40) were all much higher than those for V. coralliilyticus strains (12.30 ± 1.52), and no amplicons were evident in post-qPCR gel electrophoresis (data not shown).The detection limit for purified V. coralliilyticus genomic DNA was 0.1 pg of DNA, determined by performing 10-fold serial dilutions (100 ng to 0.01 pg per reaction), followed by qPCR amplification. Similarly, qPCR assays of serial dilutions of V. coralliilyticus (LMG 23696) cells cultured overnight in MB (108 CFU ml−1 to extinction) were able to detect as few as 104 CFU (Fig. (Fig.1).1). Standard curves revealed a strong linear negative correlation between CT values and both DNA and cell concentrations of V. coralliilyticus over several orders of magnitude, with r2 values of 0.998 and 0.953 for DNA and cells, respectively (Fig. (Fig.11).Open in a separate windowFIG. 1.Standard curves delineating threshold (CT) values of fluorescence for indicators of pathogen presence: (A) concentration of V. coralliilyticus DNA and (B) number of V. coralliilyticus cells in pure culture. Error bars indicate standard error of the mean for three replicate qPCRs.Little interference of the qPCR assay was observed when purified V. coralliilyticus (LMG 23696) DNA (10 ng) was combined with 10-fold serial dilutions (0.01 to 100 ng per reaction) of non-V. coralliilyticus DNA (i.e., Vibrio campbellii [ATCC 25920T]). Over the entire range of nontarget DNA concentrations tested, the resulting CT values (mean ± SEM, 17.76 ± 0.53) were not significantly different from those of a control treatment containing 10 ng of V. coralliilyticus DNA and no nonspecific DNA (16.75 ± 0.18; analysis of variance [ANOVA], P = 0.51) (Table (Table2).2). Detection of V. coralliilyticus (LMG 23696) bacterial cells (104, 105, 106, 107, or 108 CFU per ml) in a background of non-V. coralliilyticus cells (i.e., V. campbellii [ATCC 25920T] at 0, 10, 104, or 107 CFU per ml) showed little reduction in assay sensitivity (see Fig. S1 in the supplemental material). For example, when V. coralliilyticus was seeded at 107 cells with similarly high concentrations of nontarget cells, little inhibition of the assay was observed.
Open in a separate windowaV. coralliilyticus (LMG 23696) DNA (10 ng) free of nontarget DNA and cells served as positive controls.bA qPCR mixture containing no bacterial DNA served as a no-template, or negative, control (NTC).The assay''s detection limit in seawater was tested by inoculating 10-fold serial dilutions of V. coralliilyticus (LMG 23696) cultures (grown overnight in MB medium, pelleted at 14,000 rpm for 10 min, and washed twice with sterile phosphate-buffered saline [PBS]) into 1 liter of seawater (equivalent final concentrations were 106 to 1 CFU ml−1). The entire volume of V. coralliilyticus-seeded seawater was filtered through a Sterivex-GP filter (Millipore), and DNA was extracted using the method described by Schauer et al. (11). The lowest detection limit for V. coralliilyticus cells seeded into seawater was 1 CFU ml−1 (Fig. (Fig.2),2), with no detection in a 1-liter volume of an unseeded seawater negative control. Standard curves revealed a strong correlation between CT values and the concentrations of V. coralliilyticus bacteria seeded into the seawater over several orders of magnitude (r2 of 0.968) (Fig. (Fig.22).Open in a separate windowFIG. 2.Standard curves showing CT values of the fluorescent signal versus the number of V. coralliilyticus cells per ml seawater (▿), and cells per cm2 of M. aequituberculata tissue, with (○) or without (·) enrichment. Each dot represents an independent experiment. Error bars indicate standard error of the mean for three replicate qPCR runs.The detection limit in seeded coral tissue homogenate was determined by seeding 10-fold dilutions (1010 to 103 CFU ml−1) of pelleted, PBS-washed and resuspended (in 10 ml of sterile PBS) V. coralliilyticus cells onto healthy fragments (∼10 cm2) of the coral Montipora aequituberculata collected from Nelly Bay (Magnetic Island, Australia). Corals were collected in March 2009 and maintained in holding tanks supplied with flowthrough ambient seawater. Resuspended cells were inoculated onto M. aequituberculata fragments, each contained in an individual 3.8-liter plastic bag, allowed to sit at room temperature for 30 min, and then air brushed with compressed air until only white skeleton remained. One-milliliter aliquots of the resulting slurry (PBS, bacteria, and coral tissue) was vortexed for 10 min at 14,000 rpm, and DNA was extracted using a PowerPlant DNA Isolation Kit (Mo Bio, Carlsbad, CA). The lowest detection limits for V. coralliilyticus cells seeded onto coral fragments was 104 CFU per cm2 of coral tissue (Fig. (Fig.2).2). Again, standard curves revealed a strong correlation between CT values and the concentrations of seeded bacteria over several orders of magnitude (r2 of 0.981) (Fig. (Fig.2).2). When a 1-ml aliquot of the slurry was also inoculated into 25 ml of MB and enriched for 6 h at 28°C (with shaking at 170 rpm), the detection limit increased by 1 order of magnitude, to 103 CFU of V. coralliilyticus per cm2 of coral tissue (Fig. (Fig.2).2). The slope of the standard curve reveals some inhibition, particularly at the highest V. coralliilyticus concentrations, which could result from lower replication rates in the cultures with the highest bacterial densities (i.e., 109 CFU). However, since this effect is most pronounced only at the highest bacterial concentrations, the detection limit is still valid. In all trials, unseeded coral fragments and enrichment cultures derived from uninoculated coral fragments served as negative controls.The current study describes the first assay developed to detect and quantify a coral pathogen using a real-time quantitative PCR (qPCR) approach. While previous studies have utilized antibodies or fluorescent in situ hybridization (FISH) to detect coral pathogens (1, 6), the combination of high sensitivity and specificity, low contamination risk, and ease and speed of performance (5) make qPCR technology an ideal choice for rapid pathogen detection in complex hosts, such as corals. The assay developed is highly sensitive for V. coralliilyticus, detecting as few as 1 CFU ml−1 of seawater and 104 CFU cm−2 of coral tissue (103 CFU cm−2 of coral tissue with a 6-h enrichment). These detection limits are likely to be within biologically relevant pathogen concentrations. For example, antibodies for specific detection of the coral bleaching pathogen Vibrio shiloi showed that bacterial densities reached 8.4 × 108 cells cm−3 1 month prior to maximum visual bleaching signs on the coral Oculina patagonica (6). Each seeded seawater and coral (enriched and nonenriched) dilution assay was performed in triplicate. The linearity of the resulting standard curves indicates consistent extraction efficiencies over V. coralliilyticus concentrations spanning 6 orders of magnitude (Fig. (Fig.2)2) and provides strong support for the robustness of the assay. In addition, the presence of competing, non-V. coralliilyticus bacterial cells and DNA had a minimal impact on the detection of V. coralliilyticus. This is an important consideration for accurate detection within the complex coral holobiont, where the target organism is present within a matrix of other microbial and host cells.V. coralliilyticus, like V. shiloi (10), is becoming a model pathogen for the study of coral disease. Recent research efforts have characterized the organism''s genome (W. R. Johnson et al., submitted for publication), proteome (N. E. Kimes et al., submitted for publication), resistome (15), and metabolome (4) and enhanced our understanding of the genetic (7, 9) and physiological (7, 13) basis of its virulence. Before effective management response plans can be formulated, however, continuing research on the genetic and cellular aspects of V. coralliilyticus must be complemented with knowledge of the epidemiology of this pathogen, including information on its distribution, incidence of infection, and rates of transmission throughout populations. The V. coralliilyticus-specific qPCR assay developed in this study will provide important insights into the dynamics of pathogen invasion and spread within populations (6) while also aiding in the identification of disease vectors and reservoirs (12). These capabilities will play an important role in advancing the field of coral disease research and effective management of coral reefs worldwide. 相似文献
TABLE 1.
Species, strain, and threshold cycle for all bacterial strains testedaSpecies | Strainb | Origin | Host organism | CT ± SEMc | dnaJ gene sequence accession no. | Reference |
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Vibrio coralliilyticus | LMG 23696 | Nelly Bay, Magnetic Island, Australia | Montipora aequituberculata | 12.43 ± 0.20 | HM215570 | 14 |
LMG 23691 | Majuro Atoll, Republic of Marshall Islands | Acropora cytherea | 14.07 ± 1.33 | HM215571 | 14 | |
LMG 23693 | Nikko Bay, Palau | Pachyseris speciosa | 10.83 ± 2.76 | HM215572 | 14 | |
LMG 23692 | Nikko Bay, Palau | Pachyseris speciosa | 9.40 ± 0.36 | HM215573 | 14 | |
LMG 23694 | Nikko Bay, Palau | Pachyseris speciosad | 12.54 ± 0.24 | HM215574 | 14 | |
LMG 20984T | Indian Ocean, Zanzibar, Tanzania | Pocillopora damicornis | 12.80 ± 0.71 | HM215575 | 2 | |
LMG 21348 | Red Sea, Eilat, Israel | Pocillopora damicornis | 13.81 ± 0.49 | HM215576 | 3 | |
LMG 21349 | Red Sea, Eilat, | Pocillopora damicornis | 12.98 ± 0.94 | HM215577 | 3 | |
LMG 21350 | Red Sea, Eilat, | Pocillopora damicornis | 11.49 ± 0.19 | HM215578 | 3 | |
LMG 10953 | Kent, United Kingdom | Crassostrea gigas (oyster) larvae | 10.53 ± 0.40 | HM215579 | 3 | |
LMG 20538 | Atlantic Ocean, Florianópolis, Brazil | Nodipecten nodosus (bivalve) larvae | 12.13 ± 0.50 | HM215580 | 3 | |
C1 | Caribbean Sea, La Parguera, Puerto Rico | Pseudopterogorgia americana | 14.53 ± 0.28 | HM215568 | 15 | |
C2 | Caribbean Sea, La Parguera, Puerto Rico | Pseudopterogorgia americana | NA | HM215569 | 15 | |
Vibrio alginolyticus | ATCC 17749 | 33.74 ± 0.33 | ||||
Vibio brasiliensis | DSM 17184 | 37.84† | ||||
Vibrio calviensis | DSM 14347 | 27.06 ± 0.52 | ||||
Vibrio campbellii | ATCC 25920T | 39.10† | ||||
Enterovibrio campbellii | LMG 21363 | 37.33 ± 2.41 | ||||
Alliivibrio fischeri | DSM 507 | 31.36 ± 1.42 | ||||
Vibrio fortis | DSM 19133 | NA | ||||
Vibrio furnissii | DSM 19622 | NA | ||||
Vibrio harveyi | DSM 19623 | NA | ||||
Vibrio natriegens | ATCC 14048 | 28.56 ± 0.60 | ||||
Vibrio neptunius | LMG 20536 | NA | ||||
Vibrio ordalii | ATCC 33509 | 25.56 ± 0.41 | ||||
Vibrio parahaemolyticus | ATCC 17802 | NA | ||||
Vibrio proteolyticus | ATCC 15338 | 30.00 ± 0.89†† | ||||
Vibrio rotiferianus | LMG 21460 | NA | ||||
Vibrio splendidus | ATCC 33125 | 32.31 ± 0.82 | ||||
Vibrio tubiashii | ATCC 19109 | NA | ||||
Vibrio xuii | LMG 21346 | NA | ||||
Escherichia coli | ATCC 25922 | NA | ||||
Psychrobacter sp. | AIMS 1618 | NA | ||||
Shewanella sp. | AIMS C041 | 25.34 ± 0.45 |
TABLE 2.
Effect of nontarget bacterial DNA on the detection of 10 ng of purified V. coralliilyticus DNAAmt of nontarget DNA (ng) | CT (mean ± SEM) |
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100 | 16.97 ± 0.33 |
10 | 16.9 ± 0.08 |
1 | 16.74 ± 0.10 |
0.1 | 17 ± 0.09 |
0.01 | 16.37 ± 0.43 |
0a | 16.75 ± 0.18 |
NTCb | 35.04 ± 0.02 |
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Cheng-Jie Duan Jun-Liang Liu Xi Wu Ji-Liang Tang Jia-Xun Feng 《Applied and environmental microbiology》2010,76(14):4867-4870
Endoglucanase C5614-1 comprises a catalytic module (CM) and an X module (XM). The XM showed no significant homology with known carbohydrate-binding modules (CBMs). Recombinant full-length endoglucanase could bind Avicel, whereas the CM could not. The XM could bind various polysaccharides. The results demonstrated that the XM was a new CBM.Most cellulases are modular proteins that comprise two or more discrete modules, such as catalytic modules (CMs) and carbohydrate-binding modules (CBMs), each of which can function independently (9). CBMs are classified into 59 families based on their amino acid similarity in the CAZY database (http://www.cazy.org/fam/acc_CBM.html). The main functions of CBMs are to recognize and bind polysaccharides and to increase the hydrolytic activities of the enzymes against insoluble and soluble substrates (3). Endoglucanase C5614-1 (GenBank accession no. ), which was identified from the metagenome of the contents of buffalo rumen ( ACA611405), is a modular enzyme comprising an N-terminal signal peptide (amino acids [aa] 1 to 20), a CM belonging to the glycosyl hydrolase family 5 (aa 40 to 334), and a C-terminal X module (XM) of unknown function (aa 335 to 537) (Fig. (Fig.1A).1A). No linker region rich in Ser/Pro/Thr was found between the CM and the XM. In this study, we aimed to ascertain the function of the XM in the endoglucanase C5614-1.Open in a separate windowFIG. 1.Endoglucanase C5614-1 and its derivatives. (A) Modular organization of C5614-1 and its truncated derivatives. Abbreviations: SP, signal peptide; GHF5, GHF5 catalytic module; X, the X module (XM). (B) SDS-PAGE of purified recombinant proteins. Protein samples were analyzed on a 10% gel. Lane 1, protein molecular mass standard (molecular masses are shown on the left); lane 2, rC5614-1; lane 3, rGHF5; lane 4, rX.The XM showed no significant homology to known CBMs. The XM shared 25% to 33% identities and 41% to 45% similarities with about 200 amino acids at the C terminus of a xylanase (GenBank accession no. ) from the ruminal bacterium Prevotella ruminicola, uncultured ruminal microbial cellulases ( AAC36862, ABX76045, ACA61132, ACA61135, and ACA61137), and an uncultured bacterial bifunctional mannanase-xyloglucanase ( ABB46200). None of these homologous polypeptides was confirmed to show carbohydrate-binding activity. An alignment of XM with these homologous sequences using ClustalW ( ADA62505http://www.ebi.ac.uk/Tools/clustalw) is shown in Fig. Fig.2.2. Seven conserved aromatic amino acid residues were found in the respective sequences. Aromatic amino acid residues in CBMs play critical roles in recognizing and binding polysaccharides (4).Open in a separate windowFIG. 2.Multiple sequence alignment of the XM (203 aa) in endoglucanase C5614-1 (aa 335 to 537), with its homologous sequences. The identical and similar amino acid residues are indicated by asterisks and dots, respectively, below the alignment. The conserved aromatic amino acid residues are indicated by arrows. GenBank accession no. , Prevotella ruminicola xylanase (aa 376 to 584); AAC36862, ruminal uncultured bacterium endoglycosidase precursor protein (aa 719 to 917); ABB46200, ruminal uncultured microorganism endo-1,4-beta- ABX76045d-glucanase (aa 342 to 552); , ruminal uncultured bacterium bifunctional mannanase-xyloglucanase (aa 721 to 919); ADA62505, ruminal uncultured microorganism cellulase C29-2 (aa 341 to 553); ACA61132, ruminal uncultured microorganism cellulase C35-2 (aa 337 to 552); ACA61135, ruminal uncultured microorganism cellulase C67-1 (aa 335 to 546). The amino acid numbers in each of the parentheses above define the range of the homologous region in each sequence.A PCR-based approach was used to produce constructs expressing C5614-1 derivatives (Fig. ACA61137(Fig.1A).1A). Plasmid C5614 (5), carrying the endoglucanase gene C5614-1, was used as a PCR template. The primer pairs used to amplify the portions of C5614-1 encoding C5614-1 amino acids 20 to 537 (recombinant C5614-1 [rC5614-1], comprising the CM and the XM), 20 to 348 (rGHF5, containing the CM only), and 349 to 537 (rX, containing the XM only) are, respectively, as follows: C5614-1F (5′CAGCCATGGAGGCACAAGATTTTGAGACTGCTACCGAA-3′) and C5614-1R (5′-GACCTCGAGTTGTGCTATGTATTTTTTGCCGTTCTGG-3′), C5614-1F and C5614-1CM (5′-GGGCTCGAGGGTTAATGTCTCAGCCAGGTCAGGCTG-3′), and C5614-1X (5′-GGGCCATGGCCAAAGCCTATCATGGCAGCGCGTTC-3′) and C5614-1R. The underlined sequences in the primers are NcoI and XhoI restriction sites.The digested PCR products were ligated into the same digested expression vector, pET-30a(+) (Novagen, Madison, WI). The recombinant plasmids were transformed into Escherichia coli Rosetta(DE3)pLysS (Novagen), and the cloned fragments were expressed as proteins with 6×His tags at both the N and C termini. The recombinant C5614-1 derivatives were purified by affinity chromatography with Cobalt immobilized metal chromatography resin (Clontech, Palo Alto, CA), according to the user manual. Each of the purified proteins produced a single band on an SDS-PAGE gel (8), and their molecular sizes were in agreement with those of the deduced polypeptides (Fig. (Fig.1B1B).The hydrolytic activities of rC5614-1 and rGHF5 toward carboxymethyl cellulose (CMC) were determined essentially as described by Duan et al. (5). The pH profiles of the enzymatic reactions of rC5614-1 and rGHF5 were similar, and both showed maximum activities at pH 5.0 (data not shown). However, rC5614-1 and rGHF5 showed different temperature profiles (Fig. (Fig.3A).3A). The rGHF5 showed narrower pH stability and lower temperature stability profiles than those of rC5614-1 (Fig. 3B and C). These results indicated that the XM is required for the stability of rC5614-1.Open in a separate windowFIG. 3.Effects of pH and temperature on the activities and stability of rC5614-1 and rGHF5. (A) Influence of temperature on the activities of rC5614-1 and rGHF5. Cellulase activity was measured at pH 5.0 in citrate-phosphate buffer at the indicated temperatures for 10 min. Values are expressed as percentages of maximal activity at 50°C and 35°C for rC5614-1 and rGHF5, respectively. (B) Influence of pH on rC5614-1 and rGHF5 stability. Purified enzyme was first incubated in citrate-phosphate buffer (pH 2.5 to 7.0), 0.1 M Tris-HCl buffer (pH 7.5 to 8.5), or 0.1 M glycine-NaOH buffer (pH 9.0 to 12) at 4°C for 24 h, and activity was measured under optimal conditions for 10 min. Values are expressed as percentages of maximal activity when the sample was incubated under pH 5.5 and pH 6.0 for rC5614-1 and rGHF5, respectively. (C) Thermal stability of recombinant rC5614-1 and rGHF5. Purified enzymes were first incubated at the indicated temperatures for 1 h; activity was then measured under optimal conditions for 10 min. Values are expressed as percentages of untreated-sample activity.The hydrolytic activities of rC5614-1 toward particle substrates, including birch wood xylan, lichenan, and acid-swollen cellulose (ASC), were about three times greater than those of rGHF5; however, rC5614-1 and rGHF5 showed similar hydrolytic activities toward soluble substrates (Table (Table1).1). This result indicated that the presence of the XM enhanced the hydrolytic activity of the enzyme toward insoluble substrates but not toward soluble substrates. Similar phenomena were reported for Irpex lacteus exocellulase I (7), Clostridium thermocellum Xyn10C (1), and Clostridium stercorarium Xyn10B (2).
Open in a separate windowaOne unit of enzyme activity was defined as the amount of enzyme releasing 1 μmol of glucose equivalent or p-nitrophenol per min from substrates.Binding of rC5614-1 and rGHF5 to Avicel was determined quantitatively, as described in the supplemental material. As shown in Table Table2,2, rC5614-1 bound to Avicel, and the binding capability of rC5614-1 increased with increasing Avicel concentration and with prolonged incubation time. Its binding capability was also influenced by the pH of the mixture solution. Approximately 90% of rC5614-1 bound to 4% cellulose in the mixture solution at pH 5.0 after incubation for 5 h, whereas the proportion of the enzyme bound to cellulose dropped slightly to 83% at pH 4.0 and dropped significantly to less than 65% at pHs ≥6. Bovine serum albumin (BSA) in the mixture solution only slightly affected the binding of rC5614-1 to Avicel, suggesting that its binding to Avicel was specific. However, 100% of rGHF5 remained in the supernatant of the binding mixture, showing that rGHF5 could not bind Avicel. These results demonstrated that the XM is absolutely required for the binding of rC5614-1 to Avicel, suggesting that the XM is a CBM.
Open in a separate windowaUnless otherwise stated, the mixture solution was at pH 5.0.bND, not determined.To further confirm the XM as a CBM, the binding of purified rX to insoluble polysaccharides was investigated by incubating the polypeptide with various polysaccharides and comparing the proteins in the supernatant fraction (unbound protein) and in the precipitated fraction (bound protein) by SDS-PAGE, as described in the supplemental material. rX could bind to the insoluble polysaccharides Avicel, ASC, chitin, lichenan, xylan from sugarcane bagasse, powder of sugarcane bagasse, and raw cassava starch. It bound slightly to agarose and Sephadex G-100 (Fig. (Fig.4,4, top). The affinities of rX for soluble polysaccharides were examined by native-affinity PAGE. The migration of rX was strongly retarded by inclusion of methylcellulose, 2-hydroxyethylcellulose, and barley glucan in gels, whereas it was only slightly affected by the presence of birch wood xylan and CMC (Fig. (Fig.4,4, bottom) and not affected by the inclusion of laminarin and soluble starch (data not shown). The affinities of the XM in C5614-1 to insoluble substrates were similar to those of CBM37 from Ruminococcus albus (10) and CBM54 from Clostridium thermocellum (6), which could also bind various insoluble substrates. However, the binding of CBM54 and CBM37 to soluble substrates was either negative or not tested. We propose that the X module in endoglucanase C5614-1 is a novel CBM.Open in a separate windowFIG. 4.Binding of rX to insoluble (top) and soluble (bottom) polysaccharides. In the experiment whose results are shown in the top panel, purified rX was incubated with insoluble polysaccharides, including Avicel (lanes A), ASC (lanes B), chitin (lanes C), lichenan (lanes D), agarose (lanes E), Sephadex G-100 (lanes F), xylan from sugarcane bagasse (lanes G), the powder of sugarcane bagasse (lanes H), and raw cassava starch (lanes I). CK is a control (the amount of protein used in the binding assay). After centrifugation, proteins in the precipitate (lanes 1) and the supernatant (lanes 2) were analyzed by SDS-PAGE. In the experiment whose results are shown in the bottom panel, purified rX and bovine serum albumin (BSA) were separated in nondenaturing polyacrylamide gels containing 0.1% (wt/vol) soluble polysaccharides, including methylcellulose (B), 2-hydroxyethylcellulose (C), barley glucan (D), birch wood xylan (E), and CMC (F). A gel without polysaccharide served as a reference (A). Lanes M contained BSA as a control. 相似文献
TABLE 1.
Specific activities of rC5614-1 and rGHF5 toward various substratesTest substrate | Sp act (U/mg protein)a | |
---|---|---|
rC5614-1 | rGHF5 | |
Barley glucan | 126.1 ± 4.5 | 319.8 ± 19.9 |
Carboxymethyl cellulose | 72.5 ± 2.1 | 62.8 ± 4.4 |
Lichenan | 57.2 ± 3.1 | 20.6 ± 0.8 |
2-Hydroxyethyl cellulose | 24.6 ± 0.8 | 20.0 ± 0.1 |
Methyl cellulose | 8.8 ± 0.7 | 13.6 ± 0.03 |
Birch wood xylan | 6.7 ± 0.7 | 1.9 ± 0.16 |
Acid-swollen cellulose | 1.7 ± 0.05 | 0.5 ± 0.005 |
p-Nitrophenyl-d-cellobioside | <0.005 | <0.002 |
Avicel (β-1,4-glucan) | 0 | 0 |
Laminarin | 0 | 0 |
p-Nitrophenyl-d-glucopyranoside | 0 | 0 |
TABLE 2.
Adsorption properties of rC5614-1 to AvicelAdditive(s) (pH)a | Binding to Avicel (%) after incubation for: | |
---|---|---|
1 hb | 5 h | |
1% Avicel | 25.0 ± 1.3 | 47.8 ± 1.8 |
2% Avicel | 38.1 ± 2.8 | 72.7 ± 1.2 |
4% Avicel | 55.3 ± 1.7 | 92.1 ± 1.2 |
8% Avicel | 65.1 ± 3.8 | 96.2 ± 1.2 |
4% Avicel + 0.08% BSA | ND | 79.8 ± 1.1 |
4% Avicel + 0.01% BSA | ND | 81.5 ± 1.9 |
4% Avicel (4.0) | ND | 83.6 ± 0.4 |
4% Avicel (6.0) | ND | 63.5 ± 3.2 |
4% Avicel (7.0) | ND | 62.0 ± 3.5 |
13.
14.
Yongchao Li Diana C. Irwin David B. Wilson 《Applied and environmental microbiology》2010,76(8):2582-2588
Amino acid modifications of the Thermobifida fusca Cel9A-68 catalytic domain or carbohydrate binding module 3c (CBM3c) were combined to create enzymes with changed amino acids in both domains. Bacterial crystalline cellulose (BC) and swollen cellulose (SWC) assays of the expressed and purified enzymes showed that three combinations resulted in 150% and 200% increased activity, respectively, and also increased synergistic activity with other cellulases. Several other combinations resulted in drastically lowered activity, giving insight into the need for a balance between the binding in the catalytic cleft on either side of the cleavage site, as well as coordination between binding affinity for the catalytic domain and CBM3c. The same combinations of amino acid variants in the whole enzyme, Cel9A-90, did not increase BC or SWC activity but did have higher filter paper (FP) activity at 12% digestion.Cellulases catalyze the breakdown of cellulose into simple sugars that can be fermented to ethanol. The large amount of natural cellulose available is an exciting potential source of fuels and chemicals. However, the detailed molecular mechanisms of crystalline cellulose degradation by glycoside hydrolases are still not well understood and their low efficiency is a major barrier to cellulosic ethanol production.Thermobifida fusca is a filamentous soil bacterium that grows at 50°C in defined medium and can utilize cellulose as its sole carbon source. It is a major degrader of plant cell walls in heated organic materials such as compost piles and rotting hay and produces a set of enzymes that includes six different cellulases, three xylanases, a xyloglucanase, and two CBM33 binding proteins (12). Among them are three endocellulases, Cel9B, Cel6A, and Cel5A (7, 8), two exocellulases, Cel48A and Cel6B (6, 19), and a processive endocellulase, Cel9A (5, 7).T. fusca Cel9A-90 (Uniprot and P26221) is a multidomain enzyme consisting of a family 9 catalytic domain (CD) rigidly attached by a short linker to a family 3c cellulose binding module (CBM3c), followed by a fibronectin III-like domain and a family 2 CBM (CBM2). Cel9A-68 consists of the family 9 CD and CBM3c. The crystal structure of this species (Fig. YP_290232(Fig.1)1) was determined by X-ray crystallography at 1.9 Å resolution (Protein Data Bank [PDB] code 4tf4) (15). Previous work has shown that E424 is the catalytic acid and D58 is the catalytic base (11, 20). H125 and Y206 were shown to play an important role in activity by forming a hydrogen bonding network with D58, an important supporting residue, D55, and Glc(−1)O1. Several enzymes with amino acid changes in subsites Glc(−1) to Glc(−4) had less than 20% activity on bacterial cellulose (BC) and markedly reduced processivity. It was proposed that these modifications disturb the coordination between product release and the subsequent binding of a cellulose chain into subsites Glc(−1) to Glc(−4) (11). Another variant enzyme with a deletion of a group of amino acids forming a block at the end of the catalytic cleft, Cel9A-68 Δ(T245-L251)R252K (DEL), showed slightly improved filter paper (FP) activity and binding to BC (20).Open in a separate windowFIG. 1.Crystal structure of Cel9A-68 (PDB code 4tf4) showing the locations of the variant residues, catalytic acid E424, catalytic base D58, hydrogen bonding network residues D55, H125, and Y206, and six glucose residues, Glc(−4) to Glc(+2). Part of the linker is visible in dark blue.The CBM3c domain is critical for hydrolysis and processivity. Cel9A-51, an enzyme with the family 9 CD and the linker but without CBM3c, had low activity on carboxymethyl cellulose (CMC), BC, and swollen cellulose (SWC) and showed no processivity (4). The role of CBM3c was investigated by mutagenesis, and one modified enzyme, R557A/E559A, had impaired activity on all of these substrates but normal binding and processivity (11). Variants with changes at five other CBM3c residues were found to slightly lower the activity of the modified enzymes, while Cel9A-68 enzymes containing either F476A, D513A, or I514H were found to have slightly increased binding and processivity (11) (see Table Table1).1). In the present work, CBM3c has been investigated more extensively to identify residues involved in substrate binding and processivity, understand the role of CBM3c more clearly, and study the coordination between the CD and CBM3c. An additional goal was to combine amino acid variants showing increased crystalline cellulose activity to see if this further increased activity. Finally, we have investigated whether the changes that improved the activity of Cel9A-68 also enhanced the activity of intact Cel9A-90.
Open in a separate windowaThe target percent digestion could not be reached; activity was calculated using 1.5 μM enzyme.bDEL refers to deletion of T245 to L251 and R252K. 相似文献
TABLE 1.
Activities of Cel9A-68 CBM3c variant enzymes and CD variant enzymes used to create the double variantsEnzyme | Activity (% of wild type) on: | % Processivity | % BC binding | Reference | |||
---|---|---|---|---|---|---|---|
CMC | SWC | BC | FPa | ||||
Wild type | 100 | 100 | 100 | 100 | 100 | 15 | This work |
R378K | 98 | 91 | 103 | 93 | 139 | 20 | 11 |
DELb | 98 | 101 | 101 | 128 | 96 | 20 | |
F476A | 97 | 105 | 79 | 100 | 145 | 21 | 11 |
D513A | 100 | 115 | 121 | 107 | 119 | 20 | 11 |
I514H | 104 | 91 | 112 | 104 | 110 | 23 | 11 |
Y520A | 108 | 78 | 33a | 79 | 87 | 14 | 11 |
R557A | 103 | 98 | 60a | 93 | 90 | This work | |
E559A | 86 | 90 | 30a | 70 | 94 | This work | |
R557A+E559A | 90 | 75 | 15a | 75 | 106 | 15 | 11 |
Q561A | 103 | 56 | 51a | 78 | 74 | This work | |
R563A | 97 | 70 | 52a | 93 | 129 | 20 | 11 |
15.
C. P. A. de Haan R. Kivist? M. L. H?nninen 《Applied and environmental microbiology》2010,76(20):6942-6943
Cj0859c variants fspA1 and fspA2 from 669 human, poultry, and bovine Campylobacter jejuni strains were associated with certain hosts and multilocus sequence typing (MLST) types. Among the human and poultry strains, fspA1 was significantly (P < 0.001) more common than fspA2. FspA2 amino acid sequences were the most diverse and were often truncated.Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide and responsible for more than 90% of Campylobacter infections (7). Case-control studies have identified consumption or handling of raw and undercooked poultry meat, drinking unpasteurized milk, and swimming in natural water sources as risk factors for acquiring domestic campylobacteriosis in Finland (7, 9). Multilocus sequence typing (MLST) has been employed to study the molecular epidemiology of Campylobacter (4) and can contribute to virulotyping when combined with known virulence factors (5). FspA proteins are small, acidic, flagellum-secreted nonflagellar proteins of C. jejuni that are encoded by Cj0859c, which is expressed by a σ28 promoter (8). Both FspA1 and FspA2 were shown to be immunogenic in mice and protected against disease after challenge with a homologous strain (1). However, FspA1 also protected against illness after challenge with a heterologous strain, whereas FspA2 failed to do the same at a significant level. Neither FspA1 nor FspA2 protected against colonization (1). On the other hand, FspA2 has been shown to induce apoptosis in INT407 cells, a feature not exhibited by FspA1 (8). Therefore, our aim was to study the distributions of fspA1 and fspA2 among MLST types of Finnish human, chicken, and bovine strains.In total, 367 human isolates, 183 chicken isolates, and 119 bovine isolates (n = 669) were included in the analyses (3). PCR primers for Cj0859c were used as described previously (8). Primer pgo6.13 (5′-TTGTTGCAGTTCCAGCATCGGT-3′) was designed to sequence fspA1. Fisher''s exact test or a chi-square test was used to assess the associations between sequence types (STs) and Cj0859c. The SignalP 3.0 server was used for prediction of signal peptides (2).The fspA1 and fspA2 variants were found in 62.6% and 37.4% of the strains, respectively. In 0.3% of the strains, neither isoform was found. Among the human and chicken strains, fspA1 was significantly more common, whereas fspA2 was significantly more frequent among the bovine isolates (Table (Table1).1). Among the MLST clonal complexes (CCs), fspA1 was associated with the ST-22, ST-45, ST-283, and ST-677 CCs and fspA2 was associated with the ST-21, ST-52, ST-61, ST-206, ST-692, and ST-1332 CCs and ST-58, ST-475, and ST-4001. Although strong CC associations of fspA1 and fspA2 were found, the ST-48 complex showed a heterogeneous distribution of fspA1 and fspA2. Most isolates carried fspA2, and ST-475 was associated with fspA2. On the contrary, ST-48 commonly carried fspA1 (Table (Table1).1). In our previous studies, ST-48 was found in human isolates only (6), while ST-475 was found in both human and bovine isolates (3, 6). The strict host associations and striking difference between fspA variants in human ST-48 isolates and human/bovine ST-475 isolates suggest that fspA could be important in host adaptation.
Open in a separate windowaIn 0.3% of the strains, neither isoform was found. NF, not found.bNA, not associated.A total of 28 isolates (representing 6 CCs and 13 STs) were sequenced for fspA1 and compared to reference strains NCTC 11168 and 81-176. All isolates in the ST-22 CC showed the same one-nucleotide (nt) difference with both NCTC 11168 and 81-176 strains, resulting in a Thr→Ala substitution in the predicted protein sequence (represented by isolate FB7437, GenBank accession number ; Fig. HQ104931Fig.1).1). Eight other isolates in different CCs showed a 2-nt difference (isolate 1970, GenBank accession number ; Fig. HQ104932Fig.1)1) compared to strains NCTC 11168 and 81-176, although this did not result in amino acid substitutions. All 28 isolates were predicted to encode a full-length FspA1 protein.Open in a separate windowFIG. 1.Comparison of FspA1 and FspA2 isoforms. FspA1 is represented by 81-176, FB7437, and 1970. FspA2 is represented by C. jejuni strains 76763 to 1960 (GenBank accession numbers ). Scale bar represents amino acid divergence.In total, 62 isolates (representing 7 CCs and 35 STs) were subjected to fspA2 sequence analysis. Although a 100% sequence similarity between different STs was found for isolates in the ST-21, ST-45, ST-48, ST-61, and ST-206 CCs, fspA2 was generally more heterogeneous than fspA1 and we found 13 predicted FspA2 amino acid sequence variants in total (Fig. HQ104933 to HQ104946(Fig.1).1). In several isolates with uncommon and often unassigned (UA) STs, the proteins were truncated (Fig. (Fig.1),1), with most mutations being ST specific. For example, all ST-58 isolates showed a 13-bp deletion (isolate 3074_2; Fig. Fig.1),1), resulting in a premature stop codon. Also, all ST-1332 CC isolates were predicted to have a premature stop codon by the addition of a nucleotide between nt 112 and nt 113 (isolate 1960; Fig. Fig.1),1), a feature shared with two isolates typed as ST-4002 (UA). A T68A substitution in ST-1960 (isolate T-73494) also resulted in a premature stop codon. Interestingly, ST-1959 and ST-4003 (represented by isolate 4129) both lacked one triplet (nt 235 to 237), resulting in a shorter FspA2 protein. SignalP analysis showed the probability of a signal peptide between nt 22 and 23 (ACA-AA [between the underlined nucleotides]). An A24C substitution in two other strains, represented by isolate 76580, of ST-693 and ST-993 could possibly result in a truncated FspA2 protein as well.In conclusion, our results showed that FspA1 and FspA2 showed host and MLST associations. The immunogenic FspA1 seems to be conserved among C. jejuni strains, in contrast to the heterogeneous apoptosis-inducing FspA2, of which many isoforms were truncated. FspA proteins could serve as virulence factors for C. jejuni, although their roles herein are not clear at this time. 相似文献
TABLE 1.
Percent distributions of fspA1 and fspA2 variants among 669 human, poultry, and bovine Campylobacter jejuni strains and their associations with hosts, STs, and CCsHost or ST complex/ST (no. of isolates) | % of strains witha: | P valueb | |
---|---|---|---|
fspA1 | fspA2 | ||
Host | |||
All (669) | 64.3 | 35.4 | |
Human (367) | 69.5 | 30.0 | <0.001 |
Poultry (183) | 79.2 | 20.8 | <0.001 |
Bovine (119) | 25.2 | 74.8 | <0.0001 |
ST complex and STs | |||
ST-21 complex (151) | 2.6 | 97.4 | <0.0001 |
ST-50 (76) | NF | 100 | <0.0001 |
ST-53 (19) | NF | 100 | <0.0001 |
ST-451 (9) | NF | 100 | <0.0001 |
ST-883 (11) | NF | 100 | <0.0001 |
ST-22 complex (22) | 100 | NF | <0.0001 |
ST-22 (11) | 100 | NF | <0.01 |
ST-1947 (9) | 100 | NF | 0.03 |
ST-45 complex (268) | 99.3 | 0.7 | <0.0001 |
ST-11 (7) | 100 | NF | NA |
ST-45 (173) | 99.4 | 0.6 | <0.0001 |
ST-137 (22) | 95.5 | 4.5 | 0.001 |
ST-230 (14) | 100 | NF | <0.0001 |
ST-48 complex (18) | 44.4 | 55.6 | NA |
ST-48 (7) | 100 | NF | NA |
ST-475 (8) | NF | 100 | <0.001 |
ST-52 complex (5) | NF | 100 | <0.01 |
ST-52 (4) | NF | 100 | 0.02 |
ST-61 complex (21) | NF | 100 | <0.0001 |
ST-61 (11) | NF | 100 | <0.0001 |
ST-618 (3) | NF | 100 | 0.04 |
ST-206 complex (5) | NF | 100 | <0.01 |
ST-283 complex (24) | 100 | NF | <0.0001 |
ST-267 (23) | 100 | NF | <0.0001 |
ST-677 complex (59) | 100 | NF | <0.0001 |
ST-677 (48) | 100 | NF | <0.0001 |
ST-794 (11) | 100 | NF | <0.001 |
ST-692 complex (3) | NF | 100 | 0.04 |
ST-1034 complex (5) | NF | 80 | NA |
ST-4001 (3) | NF | 100 | 0.04 |
ST-1287 complex/ST-945 (8) | 100 | NF | NA |
ST-1332 complex/ST-1332 (4) | NF | 100 | 0.02 |
Unassigned STs | |||
ST-58 (6) | NF | 100 | <0.01 |
ST-586 (6) | 100 | NF | NA |
16.
Evolutionary Strata in a Small Mating-Type-Specific Region of the Smut Fungus Microbotryum violaceum
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DNA sequence analysis and genetic mapping of loci from mating-type-specific chromosomes of the smut fungus Microbotryum violaceum demonstrated that the nonrecombining mating-type-specific region in this species comprises ∼25% (∼1 Mb) of the chromosome length. Divergence between homologous mating-type-linked genes in this region varies between 0 and 8.6%, resembling the evolutionary strata of vertebrate and plant sex chromosomes.EVOLUTION of mating types or sex-determining systems often involves the suppression of recombination around the primary sex-determining or mating-type-determining locus. In animals and plants, it is often an entire or almost entire chromosome (Y or W in male or female heterogametic species, respectively) that ceases to recombine with its homologous (X or Z) chromosome (Charlesworth and Charlesworth 2000; Charlesworth 2008). Self-incompatibility loci in plants are also thought to be located in regions of suppressed recombination (Charlesworth et al. 2005; Kamau and Charlesworth 2005; Kamau et al. 2007; Li et al. 2007; Yang et al. 2007). Regardless of the phylogenetic position of a species, such nonrecombining regions are known to follow similar evolutionary trajectories. The nonrecombining region on the sex-specific chromosome expands in several steps, forming evolutionary strata—regions of different X/Y (or Z/W) divergence (Lahn and Page 1999; Handley et al. 2004; Sandstedt and Tucker 2004; Nicolas et al. 2005)—and genes in the nonrecombining regions gradually accumulate deleterious mutations that eventually render them dysfunctional (Charlesworth and Charlesworth 2005; Charlesworth 2008).Fungal mating-type systems are very diverse, with the number of mating types varying from two to several hundred (Casselton 2002). Like sex chromosomes in several animals and plants, suppressed recombination has evolved in regions near fungal mating-type loci, including in Ustilago hordei (Lee et al. 1999), Cryptococcus neoformans (Lengeler et al. 2002), and Neurospora tetrasperma (Menkis et al. 2008). These species have two mating types, but no morphologically distinct sexes. The mating-type locus (the region of suppressed recombination) of C. neoformans is small (∼100 kb) compared with known sex chromosomes and contains only ∼20 genes that, unlike many sex chromosomes (Y or W chromosomes), show no obvious signs of genetic degeneration (Lengeler et al. 2002; Fraser et al. 2004). Judging from the divergence between the homologous genes on the two mating-type-specific chromosomes, C. neoformans started to evolve sex chromosomes a long time ago because silent divergence between the two mating types in the most ancient region exceeds 100% (Fraser et al. 2004). Genes in the younger mating-type-specific region are much less diverged between the two sex chromosomes, suggesting that the evolution of the sex locus in C. neoformans might have proceeded through several steps. The nonrecombining region around the mating-type locus of N. tetrasperma is much larger than in C. neoformans (at least 6.6 Mb), and silent divergence between homologous genes on the mating-type-specific chromosomes ranges from zero to 9%, demonstrating that these mating-type-specific chromosomes evolved recently (Menkis et al. 2008).M. violaceum, which causes anther smut disease in Silene latifolia and other species in the family Caryophyllaceae, has two mating types, A1 and A2 (reviewed by Giraud et al. 2008), which are determined by the presence of mating-type-specific chromosomes (hereafter A1 and A2 chromosomes, or sex chromosomes) in the haploid stage of the life cycle (Hood 2002; Hood et al. 2004). The A1 and A2 chromosomes are distinguishable by size in pulsed-field electrophoresis, and it is possible to isolate individual chromosomes electrophoretically (Hood et al. 2004). Random fragments of A1 and A2 chromosomes have previously been isolated from mating-type-specific bands of pulsed-field separated chromosomes of M. violaceum (Hood et al. 2004). These fragments were assumed to be linked to mating type. The same method was used to isolate fragments of non-mating-type-specific chromosomes. On the basis of the analysis of their sequences, (Hood et al. 2004) proposed that mating-type-specific chromosomes in M. violaceum might be degenerate because they contained a lower proportion of protein-coding genes than other chromosomes. However, it was not determined whether the sequences isolated from the mating-type chromosomes originated from the mating-type-specific or from the recombining regions (Hood et al. 2004), and the relative sizes of these regions are not known for these M. violaceum chromosomes. We tested the mating-type specificity of 86 of these fragments and demonstrate that fewer than a quarter of these loci are located in the mating-type-specific region, suggesting that the nonrecombining region on the A1 and A2 chromosomes is quite small, while the rest of the chromosome probably recombines (like pseudoautosomal regions of sex chromosomes) and is therefore not expected to undergo genetic degeneration. Genetic mapping confirms the presence of two pseudoautosomal regions in the M. violaceum mating-type-specific chromosomes.As these chromosomes are mating type specific in the haploid stage of M. violaceum, mating-type-specific loci (or DNA fragments) can be identified by testing whether they are present exclusively in A1 or A2 haploid strains. We therefore prepared haploid A1 and A2 M. violaceum cultures from S. latifolia plants from two geographically remote locations (accessions Sl405 from Sweden and Sl127 from the French Pyrenees). Haploid sporidial cultures were isolated by a standard dilution method (Kaltz and Shykoff 1997; Oudemans and Alexander 1998). Mating types were determined by PCR amplification of each culture with primers designed for A1 and A2 pheromone receptor genes linked to A1 and A2 mating types (Yockteng et al. 2007). The primers were as follows: 5′-TGGCATCCCTCAATGTTTCC-3′ and 5′-CACCTTTTGATGAGAGGCCG-3′ for the A1 pheromone receptor (GenBank accession no. ) and 5′-TGACGAGAGCATTCCTACCG-3′ and 5′-GAAGCGGAACTTGCCTTTCT-3′ for the A2 pheromone receptor (GenBank accession no. EF584742). Cultures with PCR product amplified only from an A1 or A2 pheromone receptor gene were selected for further use. The mating types of the cultures were verified by conjugating them in all combinations.The GenBank nucleotide database was searched using BLAST for sequences similar to those isolated by EF584741Hood et al. (2004). Sequences with similarity to transposable elements (TE) and other repeats were excluded. The resulting set of nonredundant sequences was used to design PCR primers for 98 fragments. Half of these were originally isolated from the A1 and half from the A2 chromosomes and are hereafter called A1-NNN or A2-NNN (where NNN is the locus number; supporting information, Table S1), which does not imply that these loci are A1 or A2 specific, but merely indicates that they were originally isolated from the A1 or A2 chromosomes. Amplification of these regions from new A1 and A2 M. violaceum cultures, independently isolated by ourselves, revealed that only 5 of the 49 loci isolated from the A1 chromosome are indeed A1 specific and only 6 of 49 isolated from the A2 chromosome are A2 specific. All other loci amplified from both A1 and A2 cultures. Figure 1 illustrates some of these results from the Swedish sample (Sl405).Open in a separate windowFigure 1.—Testing of mating-type specificity for loci isolated from A1 and A2 chromosomes. (a) PCR amplifications from haploid cultures from Sl405 using primers designed from six A1-originated loci. Loci in which a PCR product could be amplified only from A1 cultures (boxed) were classified as specific to mating type A1. (b) PCR tests of six A2-originated loci on the same set of haploids as in a. Loci in which a PCR product amplified only from A2 cultures (boxed) were classified as specific to mating type A2. Loci amplified from both A1 and A2 cultures are not mating type specific.The fragments that amplified from both A1 and A2 mating types may be in recombining regions, or they could be present in mating-type-specific regions on both A1 and A2 chromosomes. If they are in recombining regions, the A1- and A2-linked homologs should not be diverged from each other, but if they are in nonrecombining, mating-type-specific regions, the divergence of the A1- and A2-linked homologs should be roughly proportional to the time since recombination stopped in the region. We therefore sequenced and compared PCR fragments amplified from the two mating types of Sl405 or Sl127 cultures (GenBank accession nos. – FI855822). Sequencing of PCR products showed that 12 (4 A1 and 8 A2) loci have more than one copy, and they were excluded from further analysis. Sequences of 61 loci were identical between the A1 and A2 strains, and four loci demonstrated low total divergence (0.24–0.61%) between the two mating types ( FI856001otintseva and D. Filatov, unpublished results). Thus, these loci might be located in the recombining part of the mating-type-specific chromosomes. Ten of 75 loci that amplified in both mating types demonstrated multiple polymorphisms fixed between the mating types rather than between the locations. Given that the strains that we used in the analysis originated from two geographically distant locations, it is highly unlikely that multiple polymorphisms distinguishing the A1 and A2 sequences arose purely by chance; thus, these loci are probably located in the nonrecombining mating-type-specific region of the M. violaceum A1 and A2 chromosomes.
Open in a separate windowaA1, loci originated from the A1 sex chromosome; A2, loci originated from the A2 sex chromosome.bThe number of loci used for genetic map construction is in parentheses.To confirm the mating-type-specific or pseudoautosomal locations of the loci with and without A1/A2 divergence, we conducted genetic mapping in a family of 99 individuals, 50 of which were of mating type A1 and 49 of mating type A2. The family was generated by a cross between A1 and A2 M. violaceum strains from S. latifolia accessions Sl405 (Sweden) and Sl127 (France), respectively. The choice of strains from geographically distant locations was motivated by the hope of maximizing the number of DNA sequence differences between them that can be used as molecular genetic markers in segregation analysis. We inoculated S. latifolia seedlings with sporidial cultures of both mating types. For inoculation, petri dishes with 12-day-old seedlings of S. latifolia were flooded with 2.5 ml of inoculum suspension. Inoculum suspension consisted of equal volumes of the A1 and A2 sporidial cultures that were mixed and conjugated overnight at 14° under rotation (Biere and Honders 1996; Van Putten et al. 2003). Seedlings were potted 3 days after inoculation. Two months later, teliospores were collected from the flowers of the infected plant and grown in petri dishes on 3.6% potato dextrose agar medium. Haploid sporidia formed after meiosis were isolated and grown as separate cultures for DNA extraction. The mating types of single sporidia cultures were identified as described above. The loci analyzed in the segregation analysis were sequenced in the two parental haploid strains and in 99 (50 A1 and 49 A2) haploid strains that were generated in the cross. Single nucleotide differences between the parental strains were used as molecular genetic markers for segregation analysis in the progeny. The genetic map was constructed using MAPMAKER/EXP v3.0 (Lincoln et al. 1992) and MapDisto v1.7 (http://mapdisto.free.fr/).The resulting genetic map is shown in Figure 2. As expected, no recombination was observed between the 10 loci with diverged A1- and A2-linked copies. In addition, one marker with no A1/A2 divergence, A2-397, was also completely linked to the loci with significant A1/A2 divergence. This locus either may be very tightly linked to the nonrecombining mating-type-specific region or may have been added to that region more recently than the loci that had already accumulated some divergence between the alleles in the two mating types. The mating-type-specific pheromone receptor locus (Devier et al. 2009) and 11 mating-type-specific loci are also located in this nonrecombining region (Figure 2). Interestingly, the cluster of nonrecombining markers is flanked on both sides with markers that recombine in meiosis, demonstrating that there are pseudoautosomal regions on both ends of the mating-type-specific chromosomes.Open in a separate windowFigure 2.—Genetic map of the mating-type-determining chromosome in M. violaceum. Genetic distance (in centimorgans) and the relative positions of the markers are shown to the left and the right of the chromosome, respectively. The position of the nonrecombining region corresponds to the cluster of linked markers shown on the right of the figure. Total A1/A2 divergence is shown in parentheses. Eleven mating-type-specific markers (for which sequences are available from only one mating type), located in the nonrecombining mating-type-specific region, are not shown.Our results demonstrate that although the loci reported by Hood et al. (2004) were isolated from the A1 and A2 chromosomes, most of these loci are not located in the nonrecombining mating-type-specific regions. In fact, the nonrecombining region might be relatively small: of 86 tested fragments, only 21 appeared to be either mating type specific or linked to the mating-type locus. Assuming that these loci represent a random set of DNA fragments isolated from the A1 and A2 chromosomes, it is possible to estimate the size of the nonrecombining region using the binomial distribution: the nonrecombining region is expected to be 24.4% (95% CI: 16.7–33.6%) of the chromosome length. As the sizes of the A1 and A2 chromosomes are ∼3.4 and 4.2 Mb long (Hood 2002; Hood et al. 2004), the nonrecombining region might be ∼1 Mb long.Interestingly, total A1/A2 divergence for the 11 loci with A1- and A2-linked copies mapped to the nonrecombining region varied from 0% to 8.6% (Figure 2). In addition, 11 loci amplified from only one mating type. These genes could represent degenerated genes, some of which degenerated in A1 strains, and some in A2 strains. Alternatively, they might be highly diverged genes, such that the PCR primers amplify only one allele, and not the other. Variation in divergence may be the result of the stepwise cessation of recombination between the A1 and A2 chromosomes in M. violaceum, resembling the evolutionary strata reported for human, chicken, and white campion sex chromosomes (Lahn and Page 1999; Handley et al. 2004; Bergero et al. 2007). However, only the differences between the most and the least diverged loci are statistically significant (Devier et al. 2009), the M. violaceum mating-type region has at least three strata: one oldest stratum, including the pheromone receptor locus; a younger stratum with ∼5–9% A1/A2 divergence; and the youngest stratum with 1–4% divergence between the two mating types. There may also be an additional very recently evolved stratum containing the locus named A2-397, which is also present in all A1 strains tested, with no fixed differences between the A1 and A2 strains (No. of sites analyzed Within A1
Within A2
Fixed differences between A1 and A2 A1/A2 divergence (%) Locia Sb total S π (%)c S π (%)c A1/A2 divergence <1% A1-236 456 3 0 0 2 0.44 1 0.44 A1-045 654 4 0 0 0 0 4 0.61 A2-568 413 2 2 0.48 2 0.48 0 0.24 A2-411 480 2 1 0.21 0 0 1 0.31 A1/A2 divergence >1% A1-217 667 9 0 0 0 0 9 1.35 A1-128 569 9 0 0 1 0.18 8 1.49 A1-199 618 13 0 0 1 0.16 12 2.02 A2-422 344 9 0 0 0 0 9 2.62 A2-516 470 14 0 0 0 0 14 2.98 A2-404 508 20 0 0 3 0.59 17 3.64 A2-435 506 22 2 0.39 2 0.39 18 3.95 A2-473 457 23 1 0.22 1 0.22 21 4.81 A2-457 303 17 1 0.33 0 0 16 5.54 A2-575 503 47 5 0.99 3 0.59 39 8.55
TABLE 1
Loci from mating-type-specific chromosomes of M. violaceum used for PCR analysis and genetic map constructionWith nonzero A1/A2 divergenceb
| |||||
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Loci | Mating type specific | <1% | >1% | With zero A1/A2 divergenceb | Total |
A1a | 5 | 2 (1) | 3 (3) | 35 (3) | 45 (7) |
A2a | 6 | 2 (0) | 7 (7) | 26 (3) | 41 (10) |
Subtotal | 4 (1) | 10 (10) | |||
Total | 11 | 14 (11) | 61 (6) | 86 (17) |