Chrystala Constantinidou

Performance comparison of benchtop high-throughput sequencing platforms

Three benchtop high-throughput sequencing instruments are now available. The 454 GS Junior (Roche), MiSeq (Illumina) and Ion Torrent PGM (Life Technologies) are laser-printer sized and offer modest set-up and running costs. Each instrument can generate data required for a draft bacterial genome sequence in days, making them attractive for identifying and characterizing pathogens in the clinical setting. We compared the performance of these instruments by sequencing an isolate of Escherichia coli O104:H4, which caused an outbreak of food poisoning in Germany in 2011. The MiSeq had the highest throughput per run (1.6 Gb/run, 60 Mb/h) and lowest error rates. The 454 GS Junior generated the longest reads (up to 600 bases) and most contiguous assemblies but had the lowest throughput (70 Mb/run, 9 Mb/h). Run in 100-bp mode, the Ion Torrent PGM had the highest throughput (80–100 Mb/h). Unlike the MiSeq, the Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors (1.5 and 0.38 errors per 100 bases, respectively).

A Reassessment of the FNR Regulon and Transcriptomic Analysis of the Effects of Nitrate, Nitrite, NarXL, and NarQP as Escherichia coli K12 Adapts from Aerobic to Anaerobic Growth

The transcription factor FNR, the regulator of fumarate and nitrate reduction, regulates major changes as Escherichia coli adapts from aerobic to anaerobic growth. In an anaerobic glycerol/trimethylamine N-oxide/fumarate medium, the fnr mutant grew as well as the parental strain, E. coli K12 MG1655, enabling us to reveal the response to oxygen, nitrate, and nitrite in the absence of glucose repression or artifacts because of variations in growth rate. Hence, many of the discrepancies between previous microarray studies of the E. coli FNR regulon were resolved. The current microarray data confirmed 31 of the previously characterized FNR-regulated operons. Forty four operons not previously known to be included in the FNR regulon were activated by FNR, and a further 28 operons appeared to be repressed. For each of these operons, a match to the consensus FNR-binding site sequence was identified. The FNR regulon therefore minimally includes at least 103, and possibly as many as 115, operons. Comparison of transcripts in the parental strain and a narXL deletion mutant revealed that transcription of 51 operons is activated, directly or indirectly, by NarL, and a further 41 operons are repressed. The narP gene was also deleted from the narXL mutant to reveal the extent of regulation by phosphorylated NarP. Fourteen promoters were more active in the narP+ strain than in the mutant, and a further 37 were strongly repressed. This is the first report that NarP might function as a global repressor as well as a transcription activator. The data also revealed possible new defense mechanisms against reactive nitrogen species.

A Culture-Independent Sequence-Based Metagenomics Approach to the Investigation of an Outbreak of Shiga-Toxigenic Escherichia coli O104:H4

IMPORTANCE Identification of the bacterium responsible for an outbreak can aid in disease management. However, traditional culture-based diagnosis can be difficult, particularly if no specific diagnostic test is available for an outbreak strain. OBJECTIVE To explore the potential of metagenomics, which is the direct sequencing of DNA extracted from microbiologically complex samples, as an open-ended clinical discovery platform capable of identifying and characterizing bacterial strains from an outbreak without laboratory culture. DESIGN, SETTING, AND PATIENTS In a retrospective investigation, 45 samples were selected from fecal specimens obtained from patients with diarrhea during the 2011 outbreak of Shiga-toxigenic Escherichia coli (STEC) O104:H4 in Germany. Samples were subjected to high-throughput sequencing (August-September 2012), followed by a 3-phase analysis (November 2012-February 2013). In phase 1, a de novo assembly approach was developed to obtain a draft genome of the outbreak strain. In phase 2, the depth of coverage of the outbreak strain genome was determined in each sample. In phase 3, sequences from each sample were compared with sequences from known bacteria to identify pathogens other than the outbreak strain. MAIN OUTCOMES AND MEASURES The recovery of genome sequence data for the purposes of identification and characterization of the outbreak strain and other pathogens from fecal samples. RESULTS During phase 1, a draft genome of the STEC outbreak strain was obtained. During phase 2, the outbreak strain genome was recovered from 10 samples at greater than 10-fold coverage and from 26 samples at greater than 1-fold coverage. Sequences from the Shiga-toxin genes were detected in 27 of 40 STEC-positive samples (67%). In phase 3, sequences from Clostridium difficile, Campylobacter jejuni, Campylobacter concisus, and Salmonella enterica were recovered. CONCLUSIONS AND RELEVANCE These results suggest the potential of metagenomics as a culture-independent approach for the identification of bacterial pathogens during an outbreak of diarrheal disease. Challenges include improving diagnostic sensitivity, speeding up and simplifying workflows, and reducing costs.

The NsrR Regulon of Escherichia coli K-12 Includes Genes Encoding the Hybrid Cluster Protein and the Periplasmic, Respiratory Nitrite Reductase

Successful pathogens must be able to protect themselves against reactive nitrogen species generated either as part of host defense mechanisms or as products of their own metabolism. The regulatory protein NsrR (a member of the Rrf2 family of transcription factors) plays key roles in this stress response. Microarray analysis revealed that NsrR represses nine operons encoding 20 genes in Escherichia coli MG1655, including the hmpA, ytfE, and ygbA genes that were previously shown to be regulated by NsrR. Novel NsrR targets revealed by this study include hcp-hcr (which were predicted in a recent bioinformatic study to be NsrR regulated) and the well-studied nrfA promoter that directs the expression of the periplasmic respiratory nitrite reductase. Conversely, transcription from the ydbC promoter is strongly activated by NsrR. Regulation of the nrf operon by NsrR is consistent with the ability of the periplasmic nitrite reductase to reduce nitric oxide and hence protect against reactive nitrogen species. Gel retardation assays were used to show that both FNR and NarL bind to the hcp promoter. The expression of hcp and the contiguous gene hcr is not induced by hydroxylamine. As hmpA and ytfE encode a nitric oxide reductase and a mechanism to repair iron-sulfur centers damaged by nitric oxide, the demonstration that hcp-hcr, hmpA, and ytfE are the three transcripts most tightly regulated by NsrR highlights the possibility that the hybrid cluster protein, HCP, might also be part of a defense mechanism against reactive nitrogen stress.

Defining bacterial species in the genomic era : insights from the genus Acinetobacter

BackgroundMicrobial taxonomy remains a conservative discipline, relying on phenotypic information derived from growth in pure culture and techniques that are time-consuming and difficult to standardize, particularly when compared to the ease of modern high-throughput genome sequencing. Here, drawing on the genus Acinetobacter as a test case, we examine whether bacterial taxonomy could abandon phenotypic approaches and DNA-DNA hybridization and, instead, rely exclusively on analyses of genome sequence data.ResultsIn pursuit of this goal, we generated a set of thirteen new draft genome sequences, representing ten species, combined them with other publically available genome sequences and analyzed these 38 strains belonging to the genus. We found that analyses based on 16S rRNA gene sequences were not capable of delineating accepted species. However, a core genome phylogenetic tree proved consistent with the currently accepted taxonomy of the genus, while also identifying three misclassifications of strains in collections or databases. Among rapid distance-based methods, we found average-nucleotide identity (ANI) analyses delivered results consistent with traditional and phylogenetic classifications, whereas gene content based approaches appear to be too strongly influenced by the effects of horizontal gene transfer to agree with previously accepted species.ConclusionWe believe a combination of core genome phylogenetic analysis and ANI provides an appropriate method for bacterial species delineation, whereby bacterial species are defined as monophyletic groups of isolates with genomes that exhibit at least 95% pair-wise ANI. The proposed method is backwards compatible; it provides a scalable and uniform approach that works for both culturable and non-culturable species; is faster and cheaper than traditional taxonomic methods; is easily replicable and transferable among research institutions; and lastly, falls in line with Darwin’s vision of classification becoming, as far as is possible, genealogical.

Extensive Microbial and Functional Diversity within the Chicken Cecal Microbiome

Chickens are major source of food and protein worldwide. Feed conversion and the health of chickens relies on the largely unexplored complex microbial community that inhabits the chicken gut, including the ceca. We have carried out deep microbial community profiling of the microbiota in twenty cecal samples via 16S rRNA gene sequences and an in-depth metagenomics analysis of a single cecal microbiota. We recovered 699 phylotypes, over half of which appear to represent previously unknown species. We obtained 648,251 environmental gene tags (EGTs), the majority of which represent new species. These were binned into over two-dozen draft genomes, which included Campylobacter jejuni and Helicobacter pullorum. We found numerous polysaccharide- and oligosaccharide-degrading enzymes encoding within the metagenome, some of which appeared to be part of polysaccharide utilization systems with genetic evidence for the co-ordination of polysaccharide degradation with sugar transport and utilization. The cecal metagenome encodes several fermentation pathways leading to the production of short-chain fatty acids, including some with novel features. We found a dozen uptake hydrogenases encoded in the metagenome and speculate that these provide major hydrogen sinks within this microbial community and might explain the high abundance of several genera within this microbiome, including Campylobacter, Helicobacter and Megamonas.

Genomic Studies with Escherichia coli MelR Protein: Applications of Chromatin Immunoprecipitation and Microarrays

Escherichia coli MelR protein is a transcription activator that is essential for melibiose-dependent expression of the melAB genes. We have used chromatin immunoprecipitation to study the binding of MelR and RNA polymerase to the melAB promoter in vivo. Our results show that MelR is associated with promoter DNA, both in the absence and presence of the inducer melibiose. In contrast, RNA polymerase is recruited to the melAB promoter only in the presence of inducer. The MelR DK261 positive control mutant binds to the melAB promoter but cannot recruit RNA polymerase. Further analysis of immunoprecipitated DNA, by using an Affymetrix GeneChip array, showed that the melAB promoter is the major, if not the sole, target in E. coli for MelR. This was confirmed by a transcriptomics experiment to analyze RNA in cells either with or without melR.

Roles of rpoN, fliA, and flgR in expression of flagella in Campylobacter jejuni

Three potential regulators of flagellar expression present in the genome sequence of Campylobacter jejuni NCTC 11168, the genes rpoN, flgR, and fliA, which encode the alternative sigma factor sigma(54), the sigma(54)-associated transcriptional activator FlgR, and the flagellar sigma factor sigma(28), respectively, were investigated for their role in global regulation of flagellar expression. The three genes were insertionally inactivated in C. jejuni strains NCTC 11168 and NCTC 11828. Electron microscopic studies of the wild-type and mutant strains showed that the rpoN and flgR mutants were nonflagellate and that the fliA mutant had truncated flagella. Immunoblotting experiments with the three mutants confirmed the roles of rpoN, flgR, and fliA in the expression of flagellin.

Identification of Novel Imidazo[1,2-a]pyridine Inhibitors Targeting M. tuberculosis QcrB

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. Through the use of high throughput whole cell screening of an extensive compound library a number of imidazo[1,2-a]pyridine (IP) compounds were obtained as potent lead molecules active against M. tuberculosis and Mycobacterium bovis BCG. The IP inhibitors (1–4) demonstrated minimum inhibitory concentrations (MICs) in the range of 0.03 to 5 µM against a panel of M. tuberculosis strains. M. bovis BCG spontaneous resistant mutants were generated against IP 1, 3, and 4 at 5× MIC and subsequent whole genome sequencing identified a single nucleotide polymorphism 937ACC>937GCC (T313A) in qcrB, which encodes the b subunit of the electron transport ubiquinol cytochrome C reductase. This mutation also conferred cross-resistance against IP 1, 3 and 4 demonstrating a common target. Gene dosage experiments confirmed M. bovis BCG QcrB as the target where over-expression in M. bovis BCG led to an increase in MIC from 0.5 to >8 µM for IP 3. An acute murine model of TB infection established bacteriostatic activity of the IP series, which await further detailed characterization.

Regulators encoded in the Escherichia coli type III secretion system 2 gene cluster influence expression of genes within the locus for enterocyte effacement in enterohemorrhagic E. coli O157 : H7

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 subverts host cells through a type III secretion system encoded by the locus for enterocyte effacement (LEE). Genome sequencing of this pathotype revealed the existence of a gene cluster encoding components of a second cryptic type III secretion system, E. coli type III secretion system 2 (ETT2). Recently, we showed that the ETT2 gene cluster is present in whole or in part in the majority of E. coli strains but is unable to encode a functional secretion system in most strains, including EHEC O157:H7. However, here we show that mutational inhibition of two regulatory genes (ECs3720 or etrA and ECs3734 or eivF) from the ETT2 cluster in EHEC O157:H7 leads to greatly increased secretion of proteins encoded by the LEE and to increased adhesion to human intestinal cells. Studies in which transcriptional fusions and microarrays were used indicated that EtrA and EivF exert profound negative effects on gene transcription within the LEE. Consistent with these observations, expression of these regulators in an EHEC O26:H- strain led to suppression of protein secretion under LEE-inducing conditions. These findings provide fresh examples of the influence of mobile genetic elements on regulation of the LEE and of cross talk between type III secretion system gene clusters. In addition, they provide a cautionary tale because they show that the effects of regulatory genes can outlive widespread decay of other genes in a functionally coherent gene cluster, a phenomenon that we have named the “Cheshire cat effect.” It also seems likely that variations in the ETT2 regulator repertoire might account for strain-to-strain variation in secretion of LEE-encoded proteins.