Coli DNA Research Articles

Regulation of the Activity of the Dual-Function DnaA Protein in Caulobacter crescentus

DnaA is a conserved essential bacterial protein that acts as the initiator of chromosomal replication as well as a master transcriptional regulator in Caulobacter crescentus. Thus, the intracellular levels of active DnaA need to be tightly regulated during the cell cycle. Our previous work suggested that DnaA may be regulated at the level of its activity by the replisome-associated protein HdaA. Here, we describe the construction of a mutant DnaA protein [DnaA(R357A)]. The R357 residue in the AAA+ domain of the C. crescentus DnaA protein is equivalent to the R334 residue of the E. coli DnaA protein, which is required for the Regulatory Inactivation of DnaA (RIDA). We found that the expression of the DnaA(R357A) mutant protein in C. crescentus, but not the expression of the wild-type DnaA protein at similar levels, causes a severe phenotype of over-initiation of chromosomal replication and that it blocks cell division. Thus, the mutant DnaA(R357A) protein is hyper-active to promote the initiation of DNA replication, compared to the wild-type DnaA protein. DnaA(R357A) could not replace DnaA in vivo, indicating that the switch in DnaA activity once chromosomal replication has started may be an essential process in C. crescentus. We propose that the inactivation of DnaA is the main mechanism ensuring that chromosomal replication starts only once per cell cycle. We further observed that the R357A substitution in DnaA does not promote the activity of DnaA as a direct transcriptional activator of four important genes, encoding HdaA, the GcrA master cell cycle regulator, the FtsZ cell division protein and the MipZ spatial regulator of cell division. Thus, the AAA+ domain of DnaA may play a role in temporally regulating the bifunctionality of DnaA by reallocating DnaA molecules from initiating DNA replication to transcribing genes within the unique DnaA regulon of C. crescentus.

DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.

Cathelicidin Signaling via the Toll-like Receptor Protects Against Colitis in Mice

Cathelicidin (encoded by Camp) is an antimicrobial peptide in the innate immune system. We examined whether macrophages express cathelicidin in colons of mice with experimental colitis and patients with inflammatory bowel disease, and we investigated its signaling mechanisms. Quantitative, real-time, reverse-transcription polymerase chain reaction (PCR), bacterial 16S PCR, immunofluorescence, and small interfering RNA (siRNA) analyses were performed. Colitis was induced in mice using dextran sulfate sodium (DSS); levels of cathelicidin were measured in human primary monocytes. Expression of cathelicidin increased in the inflamed colonic mucosa of mice with DSS-induced colitis compared with controls. Cathelicidin expression localized to mucosal macrophages in inflamed colon tissues of patients and mice. Exposure of human primary monocytes to Escherichia coli DNA induced expression of Camp messenger RNA, which required signaling by extracellular signal-regulated kinase (ERK); expression was reduced by siRNAs against Toll-like receptor (TLR)9 and MyD88. Intracolonic administration of bacterial DNA to wild-type mice induced expression of cathelicidin in colons of control mice and mice with DSS-induced colitis. Colon expression of cathelicidin was significantly reduced in TLR9(-/-) mice with DSS-induced colitis. Compared with wild-type mice, Camp(-/-) mice developed a more severe form of DSS-induced colitis, particularly after intracolonic administration of E coli DNA. Expression of cathelicidin from bone marrow-derived immune cells regulated DSS induction of colitis in transplantation studies in mice. Cathelicidin protects against induction of colitis in mice. Increased expression of cathelicidin in monocytes and experimental models of colitis involves activation of TLR9-ERK signaling by bacterial DNA. This pathway might be involved in the pathogenesis of ulcerative colitis.

Tales of conjugation and sex pheromones: A plasmid and enterococcal odyssey.

This review covers highlights of the author's experience becoming and working as a plasmid biologist. The account chronicles a progression from studies of ColE1 DNA in Escherichia coli to Gram-positive bacteria with an emphasis on conjugation in enterococci. It deals with gene amplification, conjugative transposons and sex pheromones in the context of bacterial antibiotic resistance.

Helicobacter pylori DNA decreases pro-inflammatory cytokine production by dendritic cells and attenuates dextran sodium sulphate-induced colitis

Background and aimsEpidemiological data have recently emerged to suggest Helicobacter pylori may protect against certain chronic inflammatory diseases such as inflammatory bowel disease (IBD). However, the mechanism for the observed...

Molecular Beacon Based Bioassay for Highly Sensitive and Selective Detection of Nicotinamide Adenine Dinucleotide and the Activity of Alanine Aminotransferase

We have developed a new approach to detect nicotinamide adenine dinucleotide (NAD(+)) with high specificity and sensitivity using molecular beacons (MBs) and employed it in the investigation of NAD(+) related biological processes, such as calorie restriction and alanine aminotransferase (ALT) activation. The E. coli DNA ligase would catalyze the ligation of two short oligonucleotides that complement with an MB only in the presence of NAD(+), resulting in the opening of the MB and the restoration of fluorescent signal. Thanks to the high sensitivity of the MB probe and the fidelity of E. coli DNA ligase toward its substrates, this approach can detect 0.3 nM NAD(+) with high selectivity against other NAD(+) analogs. This novel assay can also provide a convenient and robust way to analyze NAD(+) in biological samples such as cell lysate. As NAD(+) plays an essential role in many biochemical processes, this method can be used to investigate NAD(+) related life processes. For instance, the effect of calorie restriction on the intracellular NAD(+) level in MCF7 cells has been studied using this new assay. Moreover, this approach was also successfully used to analyze the activity of ALT. Therefore, this novel NAD(+) assay holds wide applicability as an analytical tool in biochemical and biomedical research.

Assessment of clustering algorithms for unsupervised transcription factor binding site discovery

Identification of transcription factor binding sites is a key task to understand gene regulation mechanism to discover gene networks and functions. Clustering approach is proved to be useful when finding such patterns residing in promoter regions of co-regulated genes. Four clustering algorithms, Self-Organizing Map, K-Means, Fuzzy C-Means and Expectation-Maximization are studied in this paper to discover motifs in datasets extracted from Saccharomyces cerevisiae, Escherichia coli, Droshophila melanogaster and Homo sapiens DNA sequences. Required modifications to clustering algorithms in order to adapt them to motif finding task are presented through the paper. Then, their motif-finding performances are discussed carefully and evaluated against a popular motif-finding method, MEME.

Binding Interactions of Toluidine Blue O with Escherichia Coli DNA: Formation of Bridged Structure

The interaction(s) of Toluidine Blue O (TBO) with DNA was investigated using absorption and emission spectral methods. The binding of TBO with DNA was understood from the observed hypochromism in the absorption spectra and decrease in the emission intensity of TBO. From the absorption and emission spectral data, two binding constants were estimated for the binding of TBO with DNA. Based on the binding constant values both intercalative and electrostatic interactions of TBO with DNA were suggested. The TBO-DNA binding constant values reveal that the electrostatic interaction of TBO with DNA is weaker than the intercalative interaction. The emission quenching and salt effect studies showed that the TBO was partially intercalated with DNA. The two modes of binding between TBO and DNA may lead to the formation of bridging of a pair of DNA duplexes by TBO molecule. The electrostatic interaction is more important for the formation of the bridged structure of TBO with DNA. This was verified by studying the interaction of an anionic dye, Eosin Y (EY).

A rapid procedure to purify Escherichia coli DNA topoisomerase I

On the basis of the asymmetrical charge distribution of Escherichia coli DNA topoisomerase I, we developed a new procedure to purify E. coli DNA topoisomerase I in the milligram range. The new procedure includes using both cation- and anion-exchange columns, i.e., SP-Sepharose FF and Q-Sepharose FF columns. The E. coli DNA topoisomerase I purified here is free of DNase contamination. The kinetic constants of the DNA relaxation reaction of E. coli DNA topoisomerase I were also determined.

Origins for the Thermostability of Taq DNA Polymerase: Entropy Versus Heat Capacity

The thermal stability of Taq DNA polymerase is well known, and is the basis for its use in PCR. The free energy versus temperature stability curves of the large fragment domains of Taq (Klentaq) and E. coli (Klenow) DNA polymerases have been compared, and by utilizing the ΔG and ΔCp information obtained from the stability curves, the ΔH and TΔS of folding of each protein as a function of temperature can be calculated directly from the Gibbs-Helmholtz equation. This analysis reveals that Klentaq's extreme stability originates from a significantly decreased entropic folding penalty (TΔS of folding). In contrast, Klentaq's native state enthalpic stabilization (ΔH of folding) is actually considerably less favorable than Klenow's at all temperatures. We have also examined published data on 17 other mesophilic-thermophilic protein pairs using the same approach to calculate ΔH and TΔS as a function of temperature from the stability curves. There are 6 other data sets (7 total including Klenow/Klentaq) with the same thermodynamic pattern, where a reduced entropic penalty is the primary basis for thermostabilization. In contrast, 8 of the 18 total data sets exhibit a highly popularized thermodynamic pattern where a significantly reduced ΔCp of folding is correlated with the enhanced stability of the thermophile. ΔCp has been correlated with changes in accessible surface area during protein folding, so a reduced ΔCp of folding for a thermophilic protein has been suggested to reflect a more compact denatured state of the thermophilic protein, although direct empirical evidence of this is still lacking for any mesophilic-thermophilic protein pair. It is notable that there is minimal overlap between the 8 systems exhibiting a lowered ΔCp of folding and the 7 systems showing a lowered entropic folding penalty.

Strand Invasion Structures in the Inverted Repeat of Candida albicans Mitochondrial DNA Reveal a Role for Homologous Recombination in Replication

Molecular recombination and transcription are proposed mechanisms to initiate mitochondrial DNA (mtDNA) replication in yeast. We conducted a comprehensive analysis of mtDNA from the yeast Candida albicans. Two-dimensional agarose gel electrophoresis of mtDNA intermediates reveals no bubble structures diagnostic of specific replication origins, but rather supports recombination-driven replication initiation of mtDNA in yeast. Specific species of Y structures together with DNA copy number analyses of a C. albicans mutant strain provide evidence that a region in a mainly noncoding inverted repeat is predominantly involved in replication initiation via homologous recombination. Our further findings show that the C. albicans mtDNA forms a complex branched network that does not contain detectable amounts of circular molecules. We provide topological evidence for recombination-driven mtDNA replication initiation and introduce C. albicans as a suitable model organism to study wild-type mtDNA maintenance in yeast.

Abstract 1700: An improved synthesis and mutagenicity studies of O2-[4-(3-pyridyl-4-oxobut-1-yl]thymidine, the major adduct formed by tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in vivo

Abstract Among the four pyridyloxobutyl (POB)-DNA adducts formed in the lung and liver of rats treated with tobacco specific nitrosamines [N′-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)], the O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd) has been found to be the most persistent. It is an important biomarker to assess the human exposure to these carcinogens and also serves as a useful precursor for the synthesis of site-specifically modified oligomers required for mutagenicity studies. The only synthetic method reported for O2-POB-dThd requires repeated HPLC purifications and could only be used to prepare an analytical standard due to very low yield (0.4%). Our continued efforts have now led to the development of an efficient method for the total synthesis of O2-POB-dThd and its site-specifically adducted oligonucleotides. The synthesis of O2-POB-dThd was achieved by optimizing reaction between O2-5′-anhydrothymidine and sodium salt of 4-(1,3-dithian-2-yl)-4-(3-pyridyl)butan-1-ol, followed by deprotection of the dithiane group. The product was characterized by NMR (1H, 13C, and COSY spectra), MS, and HPLC analysis. This synthetic strategy provided for the first time a reliable method for a gram scale total synthesis of O2-POB-dThd that allowed for solid state site-specifically adducted oligomer synthesis. O2-POB-dThd was converted to its phosphoramidite and subsequently used for the synthesis of the oligomer by standard methods. The oligomer was characterized by MS and HPLC analysis. We examined the in vitro kinetics of the synthesized oligomer with the Klenow fragment of E. Coli DNA polymerase I with the exonuclease activity inactivated (Kf(exo-)) and human DNA polymerasesl eta, kappa, and iota). O2-POB-dThd was replicated in a mutagenic manner with with Kf(exo-)and human pol eta and was unreactive with pol kappa and iota. In summary, highly facile syntheses of O2-POB-dThd and its site-specifically adducted oligonucleotide are developed. The study will facilitate elucidation of mechanistic aspects and mutagenic potential of O2-POB-dThd adduct, and will provide further insight into the role it plays in inducing cancers in smokers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1700.

Binding Sites of the E. Coli DNA Recombinase Protein to the ssDNA: A Computational Study

Homologous recombination (HR) is a major pathway for the repair of double strand breaks, which is of primary importance for genomic stability and survival of all organisms. The crucial step in HR is the formation of the nucleoprotein filament composed by a single stranded DNA chain surrounded by the recombinases protein. The filament leads the search for an undamaged homologue, a sister-chromatid or homologous chromosome, as a template for the repair process. The theoretical study presented in this work is aimed to increase the understanding of the mechanism of interaction between a trimer of the recombinase enzyme in the Escherichia coli, EcRecA, with a model system of single stranded DNA, dT9. The molecular dynamics (MD) calculations were performed using the Amber 10 computer code. The binding free energies are calculated with the Molecular Mechanics (MM) Poisson-Boltzmann (PB) and Generalized Born (GB) solvent accessible surface area (SA), MM-PB(GB) SA model. The study is extended by the use of the Principal Component Analysis (PCA) to reduce the dimensionality of the conformational space. Several mutants are considered and the corresponding calculated binding free energies are compared. An experimental correlation between the binding free energy and the enzyme activity is absent in the literature, however our results confirm how the wild type RecA has a higher binding affinity compared to the mutants examined. Moreover, for all cases considered, a significant contribution of the binding free energy is due to one amino-acid, R196, located in the binding loop 2 of the enzyme. This is consistent with the complete loss of the enzyme activity for any mutation on that point.

Coordination of genomic structure and transcription by the main bacterial nucleoid‐associated protein HU

The histone-like protein HU is a highly abundant DNA architectural protein that is involved in compacting the DNA of the bacterial nucleoid and in regulating the main DNA transactions, including gene transcription. However, the coordination of the genomic structure and function by HU is poorly understood. Here, we address this question by comparing transcript patterns and spatial distributions of RNA polymerase in Escherichia coli wild-type and hupA/B mutant cells. We demonstrate that, in mutant cells, upregulated genes are preferentially clustered in a large chromosomal domain comprising the ribosomal RNA operons organized on both sides of OriC. Furthermore, we show that, in parallel to this transcription asymmetry, mutant cells are also impaired in forming the transcription foci-spatially confined aggregations of RNA polymerase molecules transcribing strong ribosomal RNA operons. Our data thus implicate HU in coordinating the global genomic structure and function by regulating the spatial distribution of RNA polymerase in the nucleoid.

Importance of Proteins Controlling Initiation of DNA Replication in the Growth of the High-Pressure-Loving Bacterium Photobacterium profundum SS9

The molecular mechanism(s) by which deep-sea bacteria grow optimally under high hydrostatic pressure at low temperatures is poorly understood. To gain further insight into the mechanism(s), a previous study screened transposon mutant libraries of the deep-sea bacterium Photobacterium profundum SS9 and identified mutants which exhibited alterations in growth at high pressure relative to that of the parent strain. Two of these mutants, FL23 (PBPRA3229::mini-Tn10) and FL28 (PBPRA1039::mini-Tn10), were found to have high-pressure sensitivity and enhanced-growth phenotypes, respectively. The PBPRA3229 and PBPRA1039 genes encode proteins which are highly similar to Escherichia coli DiaA, a positive regulator, and SeqA, a negative regulator, respectively, of the initiation of DNA replication. In this study, we investigated the hypothesis that PBPRA3229 and PBPRA1039 encode DiaA and SeqA homologs, respectively. Consistent with this, we determined that the plasmid-carried PBPRA3229 and PBPRA1039 genes restored synchrony to the initiation of DNA replication in E. coli mutants lacking DiaA and SeqA, respectively. Additionally, PBPRA3229 restored the cold sensitivity phenotype of an E. coli dnaA(Cs) diaA double mutant whereas PBPRA1039 suppressed the cold sensitivity phenotype of an E. coli dnaA(Cs) single mutant. Taken together, these findings show that the genes disrupted in FL23 and FL28 encode DiaA and SeqA homologs, respectively. Consequently, our findings add support to a model whereby high pressure affects the initiation of DNA replication in P. profundum SS9 and either the presence of a positive regulator (DiaA) or the removal of a negative regulator (SeqA) promotes growth under these conditions.

A slow ATP‐induced conformational change limits the rate of DNA binding but not the rate of β clamp binding by the Escherichia coli γ‐complex clamp loader

In the E. coli DNA replication system, the five‐subunit clamp loader, γ‐complex, loads the βsliding clamp onto DNA. The βclamp, a ring shaped dimer of identical subunits, tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, γ‐complex binds to βand DNA to form a ternary complex. Binding to primed template DNA triggers the clamp loader to hydrolyze ATP. Upon ATP hydrolysis, βis released onto DNA and γ‐complex dissociates from the complex. We have developed a βbinding fluorescence assay by covalently attaching a fluorophore, pyrene, onto a Cys residue (Q299C) ofβ. The fluorescence increases two‐fold when γ‐ complex binds β‐pyrene. Using this assay, the rates of association, kon of 3.5 x 107 M−1s−1 and dissociation, koff of 0.005 s−1 of γ‐complex binding to βwere determined in real time. In addition, the kinetics of γ‐ complex binding to DNA were measured in real time to compare to those for βbinding. When γ‐complex is pre‐incubated with ATP, the clamp loader can bind βor DNA with no preferred order. When there is no ATP pre‐incubation, γ‐complex must undergo a slow ATP‐induced conformational change before binding to DNA but does not have to undergo this change when binding toβ. Thus, there may be a kinetic preference for γ‐complex to bind βbefore DNA during the clamp loading reaction cycle.This work was supported by NIH grant GM055596.

Charge Redistribution in Oxidized and Semiquinone E. coli DNA Photolyase upon Photoexcitation: Stark Spectroscopy Reveals a Rationale for the Position of Trp382

The electronic structure of the two lowest excited electronic states of FAD and FADH(*) in folate-depleted E. coli DNA photolyase (PL(OX) and PL(SQ), respectively) was measured using absorption Stark spectroscopy. The experimental analysis was supported by TDDFT calculations of both the charge redistribution and the difference dipole moments for the transitions of both oxidation states using lumiflavin as a model. The difference dipole moments and polarizabilities for PL(OX) are similar to those obtained in our previous work for flavins in simple solvents and in an FMN-containing flavoprotein. No such comparison can be made for PL(SQ), as we believe this to be the first experimental report of the direction and magnitude of excited-state charge redistribution in any flavosemiquinone. The picture that emerges from these studies is discussed in the context of electron transfer in photolyase, particularly for the semiquinone photoreduction process, which involves nearby tryptophan residues as electron donors. The direction of charge displacement derived from an analysis of the Stark spectra rationalizes the positioning of the critical Trp382 residue relative to the flavin for efficient vectorial electron transfer leading to photoreduction. The ramifications of vectorial charge redistribution are discussed in the context of the wider class of flavoprotein blue light photoreceptors.

High-Density Gene Expression Analysis of Tumor-Associated Macrophages from Mouse Mammary Tumors

Clinical and experimental evidence indicates that tumor-associated macrophages (TAMs) promote malignant progression. In breast cancer, TAMs enhance tumor angiogenesis, tumor cell invasion, matrix remodeling, and immune suppression against the tumor. In this study, we examined late-stage mammary tumors from a transgenic mouse model of breast cancer. We used flow cytometry under conditions that minimized gene expression changes to isolate a rigorously defined TAM population previously shown to be associated with invasive carcinoma cells. The gene expression signature of this population was compared with a similar population derived from spleens of non-tumor-bearing mice using high-density oligonucleotide arrays. Using stringent selection criteria, transcript abundance of 460 genes was shown to be differentially regulated between the two populations. Bioinformatic analyses of known functions of these genes indicated that formerly ascribed TAM functions, including suppression of immune activation and matrix remodeling, as well as multiple mediators of tumor angiogenesis, were elevated in TAMs. Further bioinformatic analyses confirmed that a pure and valid TAM gene expression signature in mouse tumors could be used to assess expression of TAMs in human breast cancer. The data derived from these more physiologically relevant autochthonous tumors compared with previous studies in tumor xenografts suggest tactics by which TAMs may regulate tumor angiogenesis and thus provide a basis for exploring other transcriptional mediators of TAM trophic functions within the tumor microenvironment.

Modulation of Escherichia coli DNA Methyltransferase Activity by Biologically Derived GATC-flanking Sequences

Escherichia coli DNA adenine methyltransferase (EcoDam) methylates the N-6 position of the adenine in the sequence 5'-GATC-3' and plays vital roles in gene regulation, mismatch repair, and DNA replication. It remains unclear how the small number of critical GATC sites involved in the regulation of replication and gene expression are differentially methylated, whereas the approximately 20,000 GATCs important for mismatch repair and dispersed throughout the genome are extensively methylated. Our prior work, limited to the pap regulon, showed that methylation efficiency is controlled by sequences immediately flanking the GATC sites. We extend these studies to include GATC sites involved in diverse gene regulatory and DNA replication pathways as well as sites previously shown to undergo differential in vivo methylation but whose function remains to be assigned. EcoDam shows no change in affinity with variations in flanking sequences derived from these sources, but methylation kinetics varied 12-fold. A-tracts immediately adjacent to the GATC site contribute significantly to these differences in methylation kinetics. Interestingly, only when the poly(A) is located 5' of the GATC are the changes in methylation kinetics revealed. Preferential methylation is obscured when two GATC sites are positioned on the same DNA molecule, unless both sites are surrounded by large amounts of nonspecific DNA. Thus, facilitated diffusion and sequences immediately flanking target sites contribute to higher order specificity for EcoDam; we suggest that the diverse biological roles of the enzyme are in part regulated by these two factors, which may be important for other enzymes that sequence-specifically modify DNA.

PCR-Based Assay Using Occult Blood Detection Cards for Detection of Diarrheagenic Escherichia coli in Specimens from U.S. Travelers to Mexico with Acute Diarrhea

Large field studies of travelers' diarrhea for multiple destinations are limited by the need to perform stool cultures on site in a timely manner. A method for the collection, transport, and storage of fecal specimens that does not require immediate processing and refrigeration and that is stable for months would be advantageous. This study was designed to determine if enterotoxigenic Escherichia coli (ETEC) and enteroaggregative E. coli (EAEC) DNA could be identified from cards that were processed for the evaluation of fecal occult blood. U.S. students traveling to Mexico during 2005 to 2007 were monitored for the occurrence of diarrheal illness. When ill, students provided a stool specimen for culture and occult blood by the standard methods. Cards then were stored at room temperature prior to DNA extraction. Fecal PCR was performed to identify ETEC and EAEC in DNA extracted from stools and from occult blood cards. Significantly more EAEC cases were identified by PCR that was performed on DNA that was extracted from cards (49%) or from frozen feces (40%) than from culture methods that used HEp-2 adherence assays (13%) (P < 0.001). Similarly, more ETEC cases were detected from card DNA (38%) than from fecal DNA (30%) or by culture that was followed by hybridization (10%) (P < 0.001). The sensitivity and specificity of the card test were 75 and 62%, respectively, compared to those for EAEC by culture and were 50 and 63%, respectively, compared to those for ETEC. DNA extracted from fecal cards that was used for the detection of occult blood is of use in identifying diarrheagenic E. coli.