EcoRI Restriction Fragment Research Articles

Molecular Characterization of Fusarium oxysporum Causing Chickpea Wilt by Internal Transcribed Spacer (ITS) Marker

Fusarium wilt caused by Fusarium oxysporum f. sp. ciceris is one of the serious disease causes tremendous loss to crop throughout the world and has assumed serious proportions in the recent years. Wilt affected chickpea plant roots samples were collected from forty eight different regions of Maharashtra. The collected samples were characterized by designed ITS primers. The Fusarium oxysporum f. sp. ciceris (Foc) showed about 302 bp amplicon when subjected to PCR with these primers. Further the amplified product was digested with five restriction enzymes viz. AluI, EcoRI, HaeIII, MboI and MspI and restriction fragment length polymorphism (RFLP) pattern were analysed. A dendrogram derived from ITS-RFLP analysis of the rDNA region divided the Foc isolates into four clusters. The maximum genetic distance 0.75 exhibited by five Foc isolates viz. Foc-30, Foc-36, Foc-24, Foc-25 and Foc-48 belonging to Shahartakli, Shelur, Nashik, Satara, Kolhapur regions respectively and found to be more diverse among the other Foc isolates. Whereas isolate no. Foc-20, Foc-21 and Foc-8 from Ambajogai, Karjat and Rahata shown minimum genetic distance (0.13). This showed the genetic variability among the Foc isolates collected from different regions of Maharashtra.

Comparative Study between Greeks and Albanians with the Use of Restriction Fragment Length Polymorphisms of Apolipoprotein B Gene

Aim: The aim of this study was to compare the genetic polymorphisms of apolipoprotein B gene between two different populations, Greeks and Albanians living in Greece and to investigate the possibility of discriminating the two populations by using these polymorphisms. Methodology: Restriction fragment length polymorphisms at codons 2488 (XbaI) and 4154 (EcoRI) of the apolipoprotein B gene were investigated in the above populations, in order to determine if there are differences between them. Two specific DNA regions, each containing the polymorphic site, were amplified by polymerase chain reaction. The products were digested and electrophoresis on 2% agarose gel was followed. A total number of 160 unrelated individuals from each population were randomly collected. Results: The allelic frequencies of the samples from Greeks and Albanians showed variability patterns for the XbaI and EcoRI Restriction Fragment Length Polymorphisms. For Greeks and Original Research Article Ampati et al.; ARRB, 7(2): 75-83, 2015; Article no.ARRB.2015.108 76 Albanians the presence of E+/+ genotype was almost the same (67.5% and 70.6% respectively), without statistical significant differences and the E-/genotype showed low common presence (6.3% and 2.5% respectively). The presence of X-/genotype had almost the same ratio for the two populations (48.1% for Greeks and 39.4% for Albanians) and the presence of X+/+ genotype was low enough for both of them. Conclusion: The study of the two populations (Greeks and Albanians) did not show any statistically significant differences concerning the frequency of the genotypes of XbaI and EcoRI polymorphisms of the APOB gene.

Genetic Diversity of Trichomonas vaginalis Clinical Isolates Determined by EcoRI Restriction Fragment Length Polymorphism of Heat-Shock Protein 70 Genes

Restriction fragment length polymorphism (RFLP) analysis using a multilocus heat-inducible cytoplasmic heat-shock protein 70 (Hsp70) hybridization probe with EcoRI-digested genomic DNA was used in molecular typing of 129 Trichomonas vaginalis isolates. Results indicate that Trichomonas organisms exhibit considerable polymorphism in their Hsp70 RFLP patterns. Analysis of seven American Type Culture Collection reference strains and 122 clinical isolates, including 84 isolates from Jackson, Mississippi, 18 isolates from Atlanta, Georgia, and 20 isolates from throughout the United States, showed 105 distinct Hsp70 RFLP pattern subtypes for Trichomonas. Phylogenetic analysis of the Hsp70 RFLP data showed that the T. vaginalis isolates were organized into two clonal lineages. These results illustrate the substantial genomic diversity present in T. vaginalis and indicate that a large number of genetically distinct Trichomonas isolates may be responsible for human trichomoniasis in the United States.

T cell receptor gene rearrangements and cytotoxic activities of clones isolated from tumour-infiltrating lymphocytes (TIL) from melanoma patients.

The lymphocytes which infiltrate tumours and are grown in vitro to be used in adoptive immunotherapy are often characterized by dominant rearrangement of their T cell receptor (TCR) genes. To investigate the frequency and function of cells contributing to the 'dominant' rearrangement, we have cloned two bulk cell lines of TIL derived from melanoma patients (TIL-1 and TIL-5). These IL-2-propagated TIL cell lines had a CD8+ phenotype and exerted strong cytotoxic activity against autologous melanoma cells, but not against the natural killer (NK)-sensitive K-562 cell line or LAK targets such as Daudi cells. We derived 40 clones from TIL-1 and 23 from TIL-5. All tested clones were CD3+, CD4-, CD8+ and expressed the alpha/beta TCR. From TIL-1, 27 of 40 clones, and 13/19 of the TIL-5 clones lysed autologous tumour cells. In contrast to the NK-negative bulk cultures, K-562 killing was detected in 21 of the TIL-1 clones and 17 of the TIL-5 clones. TIL-1 contained eight clones and TIL-5 two clones with lytic capacity against neither autologous tumour cells nor the K562 cell line, although these clones possessed lytic potential as evidenced in a lectin-mediated lysis assay. LAK activity was not detected in most clones. Cytotoxic activity against autologous tumour could be inhibited by preincubation with anti-CD3 or anti-HLA class I MoAbs. Of the 34 TIL-1 clones analysed, 15 shared a rearranged TCR beta EcoRI restriction fragment of approximately 9.5 kb with the bulk culture. Clones sharing the EcoRI 10.5-kb dominant band present in TIL-5 bulk culture were also isolated. When the pattern of TCR beta rearrangement was compared with the cytotoxic functions, the following conclusions could be drawn: (i) clones contributing to the dominant band had heterogeneous functions. Most killed autologous tumour cells, but clones with no cytotoxic activity or even with no proliferative capacity in response to autologous tumour cells were also detected among those contributing rearrangement; (ii) some clones that share an apparently identical rearranged band different from the 'dominant' rearrangement, may demonstrate the same cytotoxic function. In addition, our data suggest that many of the clones that share the dominant rearrangement originated from diverse progenitors. The high frequency of clonally diverse anti-tumour reactive TIL is likely to be a reflection of the in vivo selection of the TCR repertoire at the site of tumour.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation and characterization of the ATP-binding cassette (ABC) transporter system genes from loofah witches' broom phytoplasma

A clone containing a 3903 bp EcoRI-restriction fragment was obtained from a lambda(ZAP) genomic library of loofah witches' broom (LfWB) phytoplasma by plaque hybridization using a PCR fragment as a probe. Sequence analysis revealed that this fragment contained three open reading frames (ORFs). The deduced amino acid sequences of ORF 1 and ORF 2 showed a high homology with the ATP-binding proteins of the ABC transporter system genes of prokaryotes and eukaryotes, and encoded proteins with a molecular mass of 36 and 30 kDa, respectively. Based on amino acid sequence similarity, secondary structure, hydrophilicity and a signal peptide sequence at the N-terminus, we predicted that ORF 3 might encode a specific solute-binding prolipoprotein of the ABC transporter system with a molecular mass of 62 kDa. The cleavage site of this prolipoprotein signal peptide was similar to those of gram-positive bacteria. In addition to nutrient uptake, ABC transporter systems of bacteria also play a role in signal transduction, drug-resistance and perhaps virulence. The possible implications of the system to the survival and the pathogenesis of phytoplasma were discussed.

Organization of the interferon-inducible 2',5'-oligoadenylate-dependent ribonuclease L (RNase L) gene of mouse.

Interferons (IFNs) induce a 2',5'-oligoadenylate (2-5A)-dependent ribonuclease L (RNase L) following virus-infection of mammalian cells. RNase L degrades both viral and cellular RNAs and restricts virus-proliferation. We have studied organization of RNase L gene in genomic DNA from the mouse liver by Southern blot analysis. Several BamHI, BglII, EcoRI, HincII, HindIII, NcoI, PstI, SacI, and XbaI restriction fragments hybridized to (32)P-labeled mouse RNase L cDNA and the 5'-proximal exon probes. Mouse RNase L gene exists as a single copy (>16 kb DNA) gene. A 5 kb HindIII and a 2.5 kb EcoRI DNA were detected as 5'-upstream DNA of the gene which may contain mouse RNase L promoter. Our results will help studying mouse RNase L gene promoter in further details.

Location and phylogenetic analysis of the region immediately upstream of the granulin gene of the Clostera anachoreta granulovirus

The region immediately upstream of the granulin gene from Clostera anachoreta granulovirus ( ClanGV) was identified from hybridization experiments and sequenced. The sequence of 5122 nt EcoRI restriction fragment was presented and compared with the equivalent area in other GVs. Database searches showed that this region contained three open reading frames (ORFs) similar to the baculovirus genes ( egt, fgf and me53, respectively) and four ORFs unique to ClanGV genome. Phylogenetic trees of the baculovirus genes egt and me53 were constructed. These analyses indicated that ClanGV genes may be more closely related to CfGV, CpGV, ClGV, PoGV and AoGV than to PxGV and XcGV.

Identification of Subtypes of CYP2D Gene Rearrangements among Carriers of CYP2D6 Gene Deletion and Duplication

Cytochrome P450 2D6 (CYP2D6) is one of the best-known polymorphic drug-metabolizing enzymes. Rapidly evolving genotyping techniques permit the identification of single-nucleotide polymorphisms (SNPs) and thereby a prediction of individual metabolic capacities for CYP2D6 substrates. A considerable part of interindividual variability in CYP2D6 enzyme activity, however, is not related to SNPs but to gene deletions and duplications. Currently used genotyping methods assume that these gene rearrangements are homogeneous. We analyzed the interindividual variability in CYP2D6 gene arrangements in genomic DNA from 740 Caucasian individuals by allele-specific PCR to identify common SNPs of the CYP2D6 gene that correspond to the variant alleles CYP2D6*3, *4, and *9. We investigated the presence and variability of CYP2D6*5 (gene deletion), CYP2D6x2 (gene duplication), and CYP2D6xn (gene amplification) by EcoRI and XbaI restriction fragment length polymorphism analyses and by long PCR plus KpnI and BamHI digestion. The presence of new mutations at the CYP2D locus was analyzed by sequencing. CYP2D6 gene rearrangements were present in >12% of individuals. Variability in the rearrangements regarding both gene deletion and gene duplication existed, and one of the unusual arrangements led to incorrect phenotype prediction. The frequency for carriers of unusual gene rearrangements was <0.3% (95% confidence interval, 0%-0.6%) in the population studied. Heterogeneity in CYP2D6 gene rearrangement exists, but the allele frequency indicates that the risk for an erroneous phenotype prediction related to such variability is extremely low and that this risk can be neglected in routine analyses.

Analysis of DNA, lipopolysaccharide structure, and some cultural and morphological properties in closely related strains of Azospirillum brasilense

We studied closely related Azospirillum brasilense strains Sp7 and Cd. For probing of their genomes, the fragments of 85-MDa (p85) and 120-MDa (p120) from A. brasilense Sp245 plasmids were hybridized with 115-MDa (p115) and 90-Mda (p90) plasmids of strain Sp7, respectively. Strain Cd was found to lose the 115-Mda plasmid and one of the two EcoRI restriction fragments of the total DNA (localized within p115 and the chromosome) that was homologous to an EcoRI-generated p85 fragment of 2.4 kb. On the contrary, in the total DNA of strain Sp7-S, in spite of the previously established disappearance of the 115-Mda replicon, two fragments homologous to p85 were revealed, as with strain Sp7. It is suggested that the Sp7-S genome contains the total p115 DNA or at least a certain part of it. Strains Sp7 and Cd were found to differ in size and morphology of colonies on solid and semisolid media, in the levels of resistance to a cation surfactant cetavlon, and in the antigen structure of lipopolysaccharides.

COL1A2 (type I collagen) polymorphisms in the Colorado Indians of Ecuador

EcoRI, MspI and RsaI restriction fragment length polymorphisms (RFLPs) of the COL1A2 (type I collagen) gene are proving to be extremely informative markers for describing human populations; therefore they hold considerable potential for anthropogenetic research. The objective of this study was to characterize at the DNA level the Colorado Indians from Ecuador, for whom only blood group frequency information is available, and to investigate their relationships with the Cayapa-another Ecuadoran Native American group belonging to the same linguistic affiliation-and other world populations. Colorado Indians (n = 80) were analysed for the three anthropologically informative RFLPs of the COL1A2 gene. To better define the genetic relationship between this group and other populations, principal component analysis (PCA) was performed and genetic distances were estimated. Population genetic structure was tested through analysis of molecular variance (AMOVA) by comparing haplotype frequencies. COL1A2 allele and haplotype frequencies showed a certain degree of heterogeneity between the two Chibchan populations of Ecuador. The AMOVA test detected a significant level of differentiation (Fst = 0.034, p = 0.0049) between Colorado and Cayapa Indians. PC and genetic distance analyses showed a clear-cut separation between African and non-African populations; within the latter, the two Native American groups were differentiated from each other. The present findings suggest the presence of a low level of genetic relatedness between the Colorado and the Cayapa, despite their supposed common ethnogenesis. This confirms what has been inferred from other genetic data about the high degree of heterogeneity among Native Americans, even within the same linguistic branch, thus supporting the existence of genetic sub-structure within the central and southern American populations.

Four Variants of the Citrobacter freundii AmpC-Type Cephalosporinases, Including Novel Enzymes CMY-14 and CMY-15, in a Proteus mirabilis Clone Widespread in Poland

Twenty-nine Proteus mirabilis isolates from 17 Polish hospitals were analyzed. The isolates were resistant to a variety of antimicrobials, and their patterns of resistance to beta-lactams resembled those of the constitutive class C cephalosporinase (AmpC) producers. Indeed, beta-lactamases with a pI of approximately 9.0 were found in all of the isolates, and they were subsequently identified as four AmpC-type cephalosporinases, CMY-4, -12, -14, and -15, of which the two last ones were novel enzyme variants. The enzymes were of Citrobacter freundii origin and were closely related to each other, with CMY-4 likely being the evolutionary precursor of the remaining ones. The bla(CMY) genes were located exclusively in chromosomal DNA, within EcoRI restriction fragments of the same size of approximately 10 kb. In the CMY-12- and -15-producing isolates, an additional fragment of approximately 4.5 kb hybridized with the bla(CMY) probe as well, which could have arisen from a duplication event during the evolution of the genes. In all of the isolates, the ISEcp1 mobile element, which most probably is involved in mobilization of the C. freundii ampC gene, was placed at the same distance from the 5' ends of the bla(CMY) genes, and sequences located between them were identical in isolates carrying each of the four genes. These data suggested that a single chromosome-to-chromosome transfer of the ampC gene from C. freundii to P. mirabilis could have initiated the spread and evolution of the AmpC-producing P. mirabilis in Poland. The hypothesis seems to be confirmed by pulsed-field gel electrophoresis typing, which revealed several cases of close relatedness between the P. mirabilis isolates from distant centers and showed an overall similarity between the majority of the multiresistant isolates.

Multifocal detection of multidrug-resistant Pseudomonas aeruginosa producing the PER-1 extended-spectrum beta-lactamase in Northern Italy.

Forty-four nonreplicate clinical isolates of Pseudomonas aeruginosa that were resistant to extended-spectrum cephalosporins (ceftazidime and cefepime) and aztreonam, that putatively produced an acquired extended- spectrum beta-lactamase (ESBL), according to the results of a double-disk synergy test, and that had been involved in nosocomial outbreaks were obtained from six different hospitals in northern Italy and screened for the presence of bla(PER) ESBL determinants. Twenty isolates, associated with nine independent outbreaks that occurred in five hospitals in the Milan area and its surroundings during 1995-2000, were found to carry an acquired bla(PER-1) gene. PER-1 producers representative of the nine outbreaks exhibited a multidrug resistance (MDR) phenotype, including resistance to extended-spectrum cephalosporins, aztreonam, meropenem, aminoglycosides, and in most cases, imipenem and ciprofloxacin. An analysis of macrorestriction profiles of their genomic DNAs by pulsed-field gel electrophoresis revealed an overall clonal diversity of the PER-1 producers, although interhospital clonal spread was also observed. The bla(PER-1) gene was not transferable and appeared to be chromosomally located. An analysis of the EcoRI and EcoRV restriction fragment length polymorphisms of the bla(PER-1) locus revealed identical patterns for all isolates, and the characterization of a 1.9-kb region containing bla(PER-1) revealed a conserved structure in representatives of the various clonal lineages. The present findings indicate that MDR P. aeruginosa clones producing the PER-1 ESBL are endemic to this area of northern Italy, where they have been circulating since the mid-1990s and have been associated with several nosocomial outbreaks.

Characterization of the heterochromatin of the darkling beetle Misolampus goudoti: cloning of two satellite DNA families and digestion of chromosomes with restriction enzymes.

The darkling beetle Misolampus goudoti Er. has 58% of C-banded chromosome material. In this paper we deal with the study of the heterochromatin of this insect both by molecular and cytogenetical methods. Two different satellite DNA families have been characterized in Misolampus goudoti by agarose gel electrophoresis of EcoRI and PstI restriction fragments, respectively. The EcoRI family is composed of a monomeric unit of 196 bp (64.3% A-T rich) DNA sequence, representing about 120,000 copies per haploid genome. The presence of frequent intermediate-size satellite variants and an internal direct repetition of 61 bp in the EcoRI repetitive main monomer suggest that the evolution of this satellite proceeded by unequal crossing-over, occurring both within and between the 196 bp unit. Another highly repetitive sequence, defined by digestion of genomic DNA with PstI, has a more complex unit of 1.2 kb with about 70,000 copies per haploid genome. In situ digestion of M. goudoti chromosomes with restriction enzymes shows a non-specific chromosome DNA extraction from pericentromeric positions with EcoRI and chromosome specific extraction of DNA with PstI and HinfI. This is discussed in relation to the chromosomal location of both satellites.

Sequence mapping of the Californian MSW strain of Myxoma virus.

Partial sequence mapping of the MSW Californian strain of Myxoma virus was performed by cloning EcoRI and SalI restriction fragments of viral DNA and sequencing the ends of these. In this way, regions of 74 MSW open reading frames were sequenced and mapped onto the complete genome sequences of the related leporipoxviruses South American Myxoma virus and Rabbit fibroma virus to form a partial map of the MSW strain. In general, gene locations and sequences were conserved between the three viruses. However the Californian Myxoma virus was more closely related to South American myxoma virus than to Rabbit fibroma virus based on sequence comparisons and the presence of three genes that have been lost from the Rabbit fibroma virus genome. Compared to the other two viruses, the main difference found in the MSW genome was that the terminal inverted repeats were extended with the duplication of 5 complete open reading frames (M151R, M152R, M153R, M154L, M156R) and partial duplication of one open reading frame (M150R). This rearrangement was associated with the loss of the majority of the M009L open reading frame. Three known virulence genes, including the serine proteinase inhibitor (SERPIN) genes M151R and M152R and leukemia associated protein (LAP) gene M153R, and the potential virulence gene M156R are now present in two copies.

Rab Coupling Protein (RCP), a Novel Rab4 and Rab11 Effector Protein

Rab4 and Rab11 are small GTPases belonging to the Ras superfamily. They both function as regulators along the receptor recycling pathway. We have identified a novel 80-kDa protein that interacts specifically with the GTP-bound conformation of Rab4, and subsequent work has shown that it also interacts strongly with Rab11. We name this protein Rab coupling protein (RCP). RCP is predominantly membrane-bound and is expressed in all cell lines and tissues tested. It colocalizes with early endosomal markers including Rab4 and Rab11 as well as with the transferrin receptor. Overexpression of the carboxyl-terminal region of RCP, which contains the Rab4- and Rab11-interacting domain, results in a dramatic tubulation of the transferrin compartment. Furthermore, expression of this mutant causes a significant reduction in endosomal recycling without affecting ligand uptake or degradation in quantitative assays. RCP is a homologue of Rip11 and therefore belongs to the recently described Rab11-FIP family.

Characterization of the replicator region of megaplasmid pTAV3 of Paracoccus versutus and search for plasmid-encoded traits The GenBank accession number for the sequence reported in this paper is AF390867.

The replicon of the pTAV3 megaplasmid (approx. 400 kb) of Paracoccus versutus has been localized to a 4center dot3 kb EcoRI restriction fragment and its entire nucleotide sequence determined. The G+C content of the entire sequence is 66 mol%, which is within the range (62-66 mol%) previously determined for P. versutus total DNA. ORF1 encodes a replication initiation protein Rep (47.2 kDa), which shares substantial similarity with putative proteins of the Coxiella burnetii plasmids QpH1 and QpDV, and the replication protein of Pseudomonas syringae plasmid pPS10. ORF2, located in the opposite transcriptional orientation to ORF1, encodes a putative protein that shares similarity to a subfamily of ATPases involved in plasmid partitioning. The highest similarity was observed with homologous proteins (RepA) encoded by the repABC family of replicons found in several plasmids of Agrobacterium, Rhizobium and Paracoccus spp. The predicted product of ORF3 was similar to AcoR, Nif and NtrC transcriptional activators. A strong incompatibility determinant (inc) was localized between ORF1 (rep) and ORF2 (parA). The origin of replication of pTAV400 contains a short A+T-rich region and several imperfect palindromic sequences. Curing experiments demonstrated that the megaplasmid bears genes required for growth in minimal media and can therefore be referred to as a mini-chromosome. Megaplasmids pTAV3 of P. versutus UW1 and pKLW2 of Paracoccus pantotrophus DSM 11073 were found to carry closely related, incompatible replicons. It has been shown that plasmid pORI6 (containing oriV of pTAV3 cloned into plasmid pABW1, which does not replicate in Paracoccus spp.) can be trans activated not only by pTAV3, but also by pKLW2. Using pORI6, it was demonstrated that replication systems related to pTAV3 are also present in the replicons of Paracoccus alcaliphilus JCM 7364, Paracoccus thiocyanatus IAM 12816 and Paracoccus methylutens DM 12.

Analysis of the insulin-sensitive phosphodiesterase 3B gene in type 2 diabetes

We screened for mutations in the gene of insulin–sensitive phosphodiesterase 3B (PDE3B), which regulates antilipolytic actions of insulin via reduction of intracellular cyclic AMP levels, in Japanese patients with type 2 diabetes mellitus and lipoatrophic diabetes mellitus using single-stranded conformation polymorphism analysis and Southern analysis and investigated frequencies of variable number of tandem repeats. A silent polymorphism at the Arg 463 codon (AGG→AGA) in exon 4 was identified after examining all 16 exons and exon–intron splicing junctions of the gene. This polymorphism was found in 53 of 100 subjects with type 2 diabetes mellitus, 2 of 5 lipoatrophic diabetic patients and 24 of 50 control subjects, without any significant difference in allele frequency between groups. An EcoRI restriction fragment length polymorphism was identified in patients with type 2 diabetes mellitus and control subjects, again with no differences in occurrence. The allelic distribution of two polymorphic tandem repeats sequences in introns 5 and 12 of the gene did not differ significantly between patients with type 2 diabetes mellitus and control subjects. In conclusion, alterations in the PDE3B gene are unlikely to contribute importantly to the pathogenesis of type 2 diabetes mellitus or lipoatrophic diabetes mellitus in Japan.

Genetic diversity of Bacillus thuringiensis serovars revealed by RFLP using random DNA probes.

EcoRI and HindIII restriction fragment length polymorphism (RFLP) profiles using 2 random DNA probes, named 104 and 106, were generated for 85 B. thuringiensis strains. These include 80 serovars, 4 intra-serovar strains: kurstaki HD-1, dendrolimus, tenebrionis and sandiego, and a non-serotypeable strain B. thuringiensis var. wuhanensis. A total of 47 EcoRI and 65 HindIII restriction patterns were generated when hybridization results from both probes were combined. Seventy-seven B. thuringiensis strains showed distinctive hybridization profiles. The dendrogram resulting from the numerical analysis of the distance matrix revealed fourteen distinct phylogenetic groups at the 96% banding patterns similarity. The intra-serovar strains showed higher similarity with their respective type serovars. However, different serovars from a common H-serotype did not always cluster in the same phylogenetic group. Alternatively, several mosquitocidal serovars clustered in a single phylogenetic group. The correlation between serotyping and banding pattern similarity is discussed.

Isolation and characterization of five Erwinia amylovora bacteriophages and assessment of phage resistance in strains of Erwinia amylovora.

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages phiEa1 and phiEa7 and 3 novel phages named phiEa100, phiEa125, and phiEa116C, were identified based on differences in genome size and restriction fragment pattern. phiEa1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages phiEa100, phiEa7, and phiEa125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. phiEa116C contained an approximately 75-kb genome. phiEa1, phiEa7, phiEa100, phiEa125, and phiEa116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. phiEa116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10(5) CFU.

Identification of sat, an autotransporter toxin produced by uropathogenic Escherichia coli.

Urinary tract infection (UTI) is a very common extraintestinal infection, and Escherichia coli is by far the most common causative organism. Uropathogenic E. coli possess traits that distinguish them from commensal strains of E. coli, such as secretion systems that allow virulence factors to be targeted to extracytoplasmic compartments. One of at least five characterized secretion mechanisms is the autotransporter system, which involves translocation of a protein across the inner membrane, presumably via the sec system, and across the outer membrane through a beta-barrel porin structure formed by the carboxy-terminus autotransporter domain. We identified a 107 kDa protein that was expressed significantly more often by E. coli strains associated with the clinical syndrome of acute pyelonephritis than by faecal strains (P = 0.029). We isolated the protein from E. coli CFT073, a strain cultured from the blood and urine of a patient with acute pyelonephritis. The N-terminal amino acid sequence showed highest similarity to two known SPATE (serine protease autotransporters of Enterobacteriaceae) proteins, Pet and EspC. Using a 509 bp probe from the 5' region of pet, 10 cosmid clones of an E. coli CFT073 gene library were positive for hybridization. From one cosmid clone, a 7.5 kb EcoRI restriction fragment, which reacted strongly with the probe, was shown to include the entire 3885 bp gene. The predicted 142 kDa protein product possesses the three domains that are typical of SPATE autotransporters: an unusually long signal sequence of 49 amino acids; a 107 kDa passenger domain containing a consensus serine protease active site (GDSGSG); and a C-terminal autotransporter domain of 30 kDa. The protein exhibited serine protease activity and displayed cytopathic activity on VERO primary kidney, HK-2 bladder and HEp-2 cell lines; the name Sat (secreted autotransporter toxin) was derived from these properties. In addition, Sat antibodies were present in the serum of mice infected with E. coli CFT073. Based upon its association with pathogenic isolates, its cytopathic phenotype and its ability to elicit a strong antibody response after infection, we postulate that Sat represents a novel virulence determinant of uropathogenic E. coli.