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Lydia V. Rump Peter C. H. Feng Markus Fischer Steven R. Monday 《Applied and environmental microbiology》2010,76(3):945-947
The O-antigen (rfb) operon and related genes of MA6, an O rough:H7 Shiga-toxigenic Escherichia coli strain, were examined to determine the cause of the lack of O157 expression. A 1,310-bp insertion, homologous to IS629, was observed within its gne gene. trans complementation with a functional gne gene from O157:H7 restored O157 antigen expression in MA6.Shiga-toxigenic Escherichia coli (STEC) serotype O157:H7 carries O157 and H7 antigens, so these traits are extensively used in identification (1). Strain MA6, isolated from beef in Malaysia (8), carries the O157:H7 virulence factor genes, including the Shiga toxin 2 gene (stx2), the γ intimin allele (γ-eae), the enterohemolysin gene (ehxA), and the +93 uidA single nucleotide polymorphism (SNP) found only in O157:H7 strains (1). Multilocus sequence typing also showed MA6 to have the most common sequence type (ST-66) for O157:H7 strains. However, and in spite the fact that MA6 had per gene sequences essential for O157 antigen synthesis (2), no O157 antigen is expressed (O rough), and therefore, it is undetectable with serological assays used in O157:H7 analysis.The biosynthesis and assembly of E. coli O antigen are highly complex (9). The rfb operon (12 genes) (16), along with 3 ancillary genes outside of the rfb, is required for the biosynthesis of the 4 sugar nucleotide precursors and the assembly of the O unit (11). This is then linked to the core antigen, comprising an inner and an outer component, which require 3 other operons for biosynthesis and assembly (9). As defects in any of these genes could produce the O-null phenotype (13), we systematically examined these genes (Table (Table1)1) to elucidate the cause of the absence of O157 expression in MA6.
Open in a separate windowaLPS, lipopolysaccharide.PCR and sequencing primers for the individual genes were designed from sequences for the O157:H7 strain EDL933 (GenBank accession no. ). The 50-μl PCR mix contained 5 U of HotStar Taq (Qiagen, Valencia, CA), 1× polymerase buffer, 2.5 to 3.5 mM MgCl2, 400 μM each dNTP, 300 nM of each primer, and ∼100 ng of template DNA from either MA6 or the EDL931 reference strain. The “touchdown” PCR ( AE00517410) consisted of 95°C for 15 min and 10 cycles of 95°C for 30 s, 69 to 60°C (−1°C/cycle) for 20 s, and 72°C for 1.5 min, followed by 35 cycles of 95°C for 30 s, 60°C for 20 s, and 72°C for 1.5 min, with a single step of 72°C for 1 min for final extension. Products were examined on a 1% agarose gel in Tris-borate-EDTA (TBE) buffer. Comparison of amplicons from respective genes from MA6 and EDL931 showed that no gross differences in size were observed for any of the rfb or related genes, suggesting the absence of major insertions or deletions. Consistently, contigs assembled from the MA6 amplicon were identical in sequence to those of EDL933 in GenBank, indicating the absence of base mutations in either the promoter or any of the open reading frames (ORF). One exception was the gne gene, encoding UDP-acetylgalactosamine (GalNAc)-4-epimerase, which is essential for the synthesis of one of the oligosaccharide subunits in the O antigen (14). When PCR primers that bound upstream of the putative promoter and downstream of the gne gene were used, an expected ∼1,400-bp product was obtained from EDL931 (Fig. (Fig.1,1, lane 3), but the MA6 amplicon was ∼2,700 bp (Fig. (Fig.1,1, lane 4). PCR of other O157:H7 strains all yielded the ∼1,400-bp product, while MA6 consistently produced the larger amplicon. Comparison of sequences to that of EDL933 showed the presence of a 1,310-bp insertion within the MA6 gne ORF at +385 that shared 96% homology to the insertion sequence 629 (IS629) (accession no. ) element. Furthermore, the deduced protein sequences for the putative orfA and orfB genes on the insert were 100% and 99% identical to those of the IS629 transposase in O157:H7 strains Sakai (accession no. X51586), and EDL933 and EC4115 (accession no. NC_002695), respectively. NC_011353Open in a separate windowFIG. 1.Agarose gel electrophoresis of gne amplicons derived from EDL931 (O157:H7) and MA6. Lanes: 1, exACTGene (1 kb) plus molecular size ladder (Fisher BioReagents, Pittsburgh, PA); 2, negative control (reaction mix without DNA template); 3, EDL931; 4, MA6.To determine whether gne::IS629 (accession no. ) caused the absence of O157 expression in MA6, the wild-type EDL931 gne ORF was amplified using primers that added BamHI and SacI restriction sites at the 5′ and 3′ termini, respectively. The purified amplicon was digested accordingly, ligated into pTrc99A vector (Stratagene, La Jolla, CA), and electroporated into E. coli DH5α ( GU18313810). Transformants were selected on LB plates with 100 μg/ml ampicillin (Amp). Colonies that were Amp resistant (Ampr) were PCR amplified with vector-specific primers, and those carrying the insert were sequenced to confirm the presence of the wild-type gne insert in the construct (pGNE). For trans-complementation studies, pGNE was electroporated into MA6. Ampr transformants were PCR amplified with vector-specific primers as well as primers that annealed to sequences outside the gne gene and also not present on the vector, to confirm that they carried both pGNE and the gne::IS629 locus. Serological testing with the RIM O157:H7 latex kit (Remel, Lenexa, KS) confirmed that the Ampr MA6 transformants expressed O157 antigen.These results confirmed that gne::IS629 caused the O rough phenotype of MA6. Originally isolated from Shigella sonnei (7), IS629 has since been found, often in multiple copies, to cause gene disruptions in other enteric bacteria (6). fliC::IS629 caused nonmotility of an E. coli O111 strain (17), and wbaM::IS629 resulted in an O rough Shigella boydii strain (15). The IS629 recognition site remains unknown (5), so it is uncertain that there is an IS629 hot spot within the O157:H7 gne ORF. Other bacteria, like O157:H7, also have the gne gene positioned upstream of the rfb operon (12), but no gne::IS629 rough strains of these have been reported. This suggests that the IS629 insertion site within the gne of MA6 may have occurred as a result of a random mutation and that MA6 appears to be the only naturally occurring O rough O157:H7 strain that resulted from the gne::IS629 insertion.The O antigen is not required for growth but does confer protection (9), so the loss of the O antigen has been reported to make pathogens serum sensitive or less virulent (4). If that is so, we would expect MA6 to be less pathogenic than O157:H7; consistent with that speculation, MA6 has not been implicated in illness. Even so, while no O rough O157:H7 strains have caused disease, other O rough STEC strains have caused illnesses (3); hence, the virulence potential of MA6 remains undetermined.In conclusion, the absence of O157 antigen expression by MA6 is caused by gne::IS629. Occurrence of O rough:H7 strains like MA6 in food or clinical samples is of concern, as they are undetectable by the serological assays used to identify O157:H7. However, the IS629 insertion site within the O157:H7 gne ORF appears to have been due to a random mutational event, and therefore, MA6-like O rough mutants of O157:H7 are thus far uncommon. 相似文献
TABLE 1.
rfb operon genes, ancillary genes, and waa cluster genes examined in this studyCategory | General functiona | Gene(s) |
---|---|---|
O-antigen (rfb) operon | Nucleotide sugar transfer | wbdN, wbdO, wbdP, wbdQ, wbdR |
O-unit processing | wzy, wzx | |
Nucleotide sugar synthesis | per, gmd, fcl, manC, manB | |
waa core gene clusters | Structure modification | waaQ, waaP, waaY |
Nucleotide sugar transfer | rfaG, rfaC | |
LPS core biosynthesis enzyme | waaI, waaJ, waaD, waaL | |
Ancillary genes | Nucleotide sugar synthesis | manA |
O-unit processing | wecA | |
Nucleotide sugar synthesis | gne |
85.
John R. Wallace Matthew C. Gordon Lindsey Hartsell Lydia Mosi M. Eric Benbow Richard W. Merritt Pamela L. C. Small 《Applied and environmental microbiology》2010,76(18):6215-6222
Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe necrotizing skin disease that causes significant morbidity in Africa and Australia. Person-to-person transmission of Buruli ulcer is rare. Throughout Africa and Australia infection is associated with residence near slow-moving or stagnant water bodies. Although M. ulcerans DNA has been detected in over 30 taxa of invertebrates, fish, water filtrate, and plant materials and one environmental isolate cultured from a water strider (Gerridae), the invertebrate taxa identified are not adapted to feed on humans, and the mode of transmission for Buruli ulcer remains an enigma. Recent epidemiological reports from Australia describing the presence of M. ulcerans DNA in adult mosquitoes have led to the hypothesis that mosquitoes play an important role in the transmission of M. ulcerans. In this study we have investigated the potential of mosquitoes to serve as biological or mechanical vectors or as environmental reservoirs for M. ulcerans. Here we show that Aedes aegypti, A. albopictus, Ochlerotatus triseriatus, and Culex restuans larvae readily ingest wild-type M. ulcerans, isogenic toxin-negative mutants, and Mycobacterium marinum isolates and remain infected throughout larval development. However, the infections are not carried over into the pupae or adult mosquitoes, suggesting an unlikely role for mosquitoes as biological vectors. By following M. ulcerans through a food chain consisting of primary (mosquito larvae), secondary (predatory mosquito larva from Toxorhynchites rutilus septentrionalis), and tertiary (Belostoma species) consumers, we have shown that M. ulcerans can be productively maintained in an aquatic food web.Infection with Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU) disease, is associated with residence near stagnant and slow-moving water bodies in areas in which the disease is endemic (5, 36, 40, 45, 50). A plasmid-encoded macrolide toxin, mycolactone, is the primary virulence determinant of M. ulcerans (8, 41). Biting aquatic insects, such as several taxa in the Belostomatidae and Naucoridae families (Hemiptera), have been suggested as possible vectors of M. ulcerans in several laboratory experiments (16, 19, 20, 24, 31, 32); however, there is little empirical evidence from field studies to support the contention that these biting insects vector M. ulcerans to humans (2). In Melbourne, Australia, recent epidemiological evidence suggests that mosquitoes may serve as vectors in the transmission of BU disease (10, 11, 12, 34, 35). In this study, 957 pools consisting of over 11,000 mosquitoes of four different species were collected and tested by quantitative PCR (qPCR) for the presence of M. ulcerans DNA, and positive results were obtained from 48 of 957 pools tested (10). Of the 48 positive pools, 13 were positive for PCR directed against two insertion sequences (IS2404 and IS2606) as well as against sequence based on the ketoreductase domain of the mycolactone toxin genes. Because all of these target sequences are present multiple times in the genome, it was difficult to assign genome equivalents to these results. However, data from laboratory experiments suggested that 10 to 100 M. ulcerans isolates per mosquito were present in the positive pools. Epidemiological work also suggested a seasonal relationship between Buruli ulcer and mosquito-vectored diseases in Australia (12). These studies are extremely provocative and raise a number of questions for further work. What is the prevalence of M. ulcerans in other invertebrate taxa in the same environment? What is the infection rate in equal numbers of mosquitoes collected from areas in which the disease is not endemic? Is it possible to obtain physical evidence for the presence of M. ulcerans through microscopy or culture of mosquitoes in areas in which the disease is endemic, and, finally, what can we learn from laboratory studies concerning the interaction between mosquitoes and M. ulcerans?The recent work from Australia suggesting that M. ulcerans is spread by mosquitoes is particularly significant because adult mosquitoes are the most important group of insects in the spread of human disease. They may serve as biological vectors that provide a major environment for pathogen replication, as in malaria or yellow fever, or as mechanical vectors that carry organisms between hosts without serving as a site of replication (1, 4, 7, 9, 38). Larval mosquitoes are common in habitats associated with BU disease, most notably lentic or standing water habitats, and feed by filtering particles in the water using labral head fans (21). Members of some genera (i.e., Anopheles) aggregate at the air-water interface in microlayers near plant stems and algal mats (27, 28, 46), where they feed on microorganisms such as bacteria and algae (47). Because of their collecting-filtering feeding mode, there is potential for larvae to consume M. ulcerans and concentrate mycobacteria through their feeding activities (22, 23).In Ghana, the occurrence of M. ulcerans among invertebrate communities in lentic habitats has been documented from regions in Ga West and Ga East Districts in which the disease is endemic as well as those in which it is not endemic (2, 49) but not in geographically distinct areas in which the disease is not endemic such as the Volta region (49). M. ulcerans has been identified in a suite of environmental samples such as filtered water, biofilms, and algae as well as among a broad spectrum of invertebrate taxa, including both larval and adult mosquitoes (2, 11, 17, 49). However, the replication and trophic movement of M. ulcerans within these environmental samples and invertebrate communities have not been experimentally investigated. Conceptual models have been proposed that assume that the primary consumers of M. ulcerans (e.g., mosquito larvae, cladocerans, and chironomid larvae) may feed on bacteria and algae in biofilms, filter suspended matter from the water column, and then initiate the passage of M. ulcerans through an aquatic food web (2, 22, 31). This model predicts the movement of M. ulcerans through secondary and tertiary consumers and implies a complex trophic relationship in the ecology of M. ulcerans as well as an important role of aquatic invertebrates in the disease ecology of M. ulcerans.In the studies reported here, we have explored the role of mosquitoes as biological or mechanical vectors of M. ulcerans, as well as the potential of mosquito larvae to play a central role in the movement of M. ulcerans through an aquatic food web. In order to investigate the ability of mosquito larvae to ingest and maintain M. ulcerans within their digestive tract as well as to persist throughout the mosquito development cycle, we took advantage of the fact that mosquito larvae naturally feed upon bacteria. Results presented here show that strains of M. ulcerans from Africa and Australia, as well as Mycobacterium marinum, were maintained at high levels in the larval mosquito gut for 6 days. However, neither M. ulcerans nor M. marinum was detected in adult mosquitoes that were infected in the larval stage. These results suggest that mosquitoes are unlikely to serve as biological vectors of M. ulcerans.We further developed a model for following the passage of M. ulcerans through a series of consumers to determine whether M. ulcerans could be passed up a trophic chain from primary to tertiary consumers. In this model, we conducted similar experiments using four species of nonpredatory mosquito larvae, Aedes aegypti (Linnaeus), Aedes albopictus (Skuse), Ochlerotatus triseriatus (Theobald), and Culex restuans (Theobald), as primary consumers. These larvae were infected with isogenic wild-type (WT) and toxin-negative isolates of M. ulcerans and of M. marinum, the closest relative to M. ulcerans (13, 14, 51). We have shown that M. ulcerans in mosquito larvae survive passage through secondary and tertiary consumers, thus providing the first laboratory evidence that M. ulcerans has the potential to move between and be maintained within different species in an aquatic food web. 相似文献
86.
Evidence for different contributions of archaea and bacteria to the ammonia-oxidizing potential of diverse Oregon soils 总被引:8,自引:0,他引:8
Taylor AE Zeglin LH Dooley S Myrold DD Bottomley PJ 《Applied and environmental microbiology》2010,76(23):7691-7698
A method was developed to determine the contributions of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) to the nitrification potentials (NPs) of soils taken from forest, pasture, cropped, and fallowed (19 years) lands. Soil slurries were exposed to acetylene to irreversibly inactivate ammonia monooxygenase, and upon the removal of acetylene, the recovery of nitrification potential (RNP) was monitored in the presence and absence of bacterial or eukaryotic protein synthesis inhibitors. For unknown reasons, and despite measureable NPs, RNP did not occur consistently in forest soil samples; however, pasture, cropped, and fallowed soil RNPs commenced after lags that ranged from 12 to 30 h after acetylene removal. Cropped soil RNP was completely prevented by the bacterial protein synthesis inhibitor kanamycin (800 μg/ml), whereas a combination of kanamycin plus gentamicin (800 μg/ml each) only partially prevented the RNP (60%) of fallowed soils. Pasture soil RNP was completely insensitive to either kanamycin, gentamicin, or a combination of the two. Unlike cropped soil, pasture and fallowed soil RNPs occurred at both 30°C and 40°C and without supplemental NH(4)(+) (≤ 10 μM NH(4)(+) in solution), and pasture soil RNP demonstrated ~ 50% insensitivity to 100 μM allyl thiourea (ATU). In addition, fallowed and pasture soil RNPs were insensitive to the fungal inhibitors nystatin and azoxystrobin. This combination of properties suggests that neither fungi nor AOB contributed to pasture soil RNP and that AOA were responsible for the RNP of the pasture soils. Both AOA and AOB may contribute to RNP in fallowed soil, while RNP in cropped soils was dominated by AOB. 相似文献
87.
Nestor Laurier Engone Obiang Alfred Ngomanda Raymonde Mboma Thomas Nzabi Alfred Ngoye Lydia Atsima Léopold Ndjélé Jean‐Pierre Mate Christophe Lomba Nicolas Picard 《Oikos》2010,119(10):1643-1653
When considering all trees irrespective of their species, natural tropical rain forests typically exhibit spatial patterns that range from random to regular. The regularity is often interpreted as a footprint of tree competition. Using 23 permanent sample plots totalling 61 ha in the rain forests of central Africa, we characterized their spatial patterns and modelled those that exhibited regularity by a Strauss point process. This Strauss process is obtained as a Markov point process whose interaction function is an exponential function of a competition index commonly used in forestry. The parameter of this Strauss process characterizes the strength of competition. The 23 plots in central Africa differed in tree density and basal area, and could be discriminated depending on the type of spatial patterns: plots having a large basal area with respect to their density had a non regular pattern, whereas those having a small basal area with respect to their density had a regular pattern. For those plots that exhibited regularity, average tree size could be used to predict the strength of competition. The parameter of the Strauss process was significantly related to the average size by a linear relationship, such that competition decreases as average tree size increases. This relationship extrapolated to a null value of the Strauss parameter when average tree size reaches 32 cm in diameter. This relationship between average tree size and spatial pattern is a testable feature for future studies on the relationship between competition and spatial pattern in natural forests. 相似文献
88.
Nurit Haspel Mark Moll Matthew L Baker Wah Chiu Lydia E Kavraki 《BMC structural biology》2010,10(Z1):S1
Background
Many proteins undergo extensive conformational changes as part of their functionality. Tracing these changes is important for understanding the way these proteins function. Traditional biophysics-based conformational search methods require a large number of calculations and are hard to apply to large-scale conformational motions.Results
In this work we investigate the application of a robotics-inspired method, using backbone and limited side chain representation and a coarse grained energy function to trace large-scale conformational motions. We tested the algorithm on four well known medium to large proteins and we show that even with relatively little information we are able to trace low-energy conformational pathways efficiently. The conformational pathways produced by our methods can be further filtered and refined to produce more useful information on the way proteins function under physiological conditions.Conclusions
The proposed method effectively captures large-scale conformational changes and produces pathways that are consistent with experimental data and other computational studies. The method represents an important first step towards a larger scale modeling of more complex biological systems.89.
M Alonso N Alonso Rodriguez C Garzelli M Martínez Lirola M Herranz S Samper MJ Ruiz Serrano E Bouza D García de Viedma 《BMC microbiology》2010,10(1):151
Background
The Beijing lineage of Mycobacterium tuberculosis is causing concern due to its global distribution and its involvement in severe outbreaks. Studies focused on this lineage are mainly restricted to geographical settings where its prevalence is high, whereas those in other areas are scarce. In this study, we analyze Beijing isolates in the Mediterranean area, where this lineage is not prevalent and is mainly associated with immigrant cases. 相似文献90.
Pablo García-Gómez Abdellatif Bahaji Samuel Gámez-Arcas Francisco José Muñoz Ángela María Sánchez-López Goizeder Almagro Edurne Baroja-Fernández Kinia Ameztoy Nuria De Diego Lydia Ugena Lukáš Spíchal Karel Doležal Mohammad-Reza Hajirezaei Luis C. Romero Irene García Javier Pozueta-Romero 《Plant, cell & environment》2020,43(10):2551-2570