Putative Phenotypically Neutral Genomic Insertion Points in Prokaryotes.

The barriers to effective genome editing in diverse prokaryotic organisms have been falling at an accelerated rate. As editing becomes easier in more organisms, quickly identifying genomic locations to insert new genetic functions without disrupting organism fitness becomes increasingly useful. When the insertion is noncoding DNA for applications such as information storage or barcoding, a neutral insertion point can be especially important. Here we describe an approach to identify putatively neutral insertion sites in prokaryotes. An algorithm (targetFinder) finds convergently transcribed genes with gap sizes within a specified range, and looks for annotations within the gaps. We report putative editing targets for 10 common synthetic biology chassis organisms, including coverage of available RNA-seq data, and provide software to apply to others. We further experimentally evaluate the neutrality of six identified targets in Escherichia coli through insertion of a DNA barcode. We anticipate this information and the accompanying tool will prove useful for synthetic biologists seeking neutral insertion points for genome editing.

barCoder: a tool to generate unique, orthogonal genetic tags for qPCR detection.

BACKGROUND: Tracking dispersal of microbial populations in the environment requires specific detection methods that discriminate between the target strain and all potential natural and artificial interferents, including previously utilized tester strains. Recent work has shown that genomic insertion of short identification tags, called "barcodes" here, allows detection of chromosomally tagged strains by real-time PCR. Manual design of these barcodes is feasible for small sets, but expansion of the technique to larger pools of distinct and well-functioning assays would be significantly aided by software-guided design. RESULTS: Here we introduce barCoder, a bioinformatics tool that facilitates the process of creating sets of uniquely identifiable barcoded strains. barCoder utilizes the genomic sequence of the target strain and a set of user-specified PCR parameters to generate a list of suggested barcode "modules" that consist of binding sites for primers and probes, and appropriate spacer sequences. Each module is designed to yield optimal PCR amplification and unique identification. Optimal amplification includes metrics such as ideal melting temperature and G+C content, appropriate spacing, and minimal stem-loop formation; unique identification includes low BLAST hits against the target organism, previously generated barcode modules, and databases (such as NCBI). We tested the ability of our algorithm to suggest appropriate barcodes by generating 12 modules for Bacillus thuringiensis serovar kurstaki-a simulant for the potential biowarfare agent Bacillus anthracis-and three each for other potential target organisms with variable G+C content. Real-time PCR detection assays directed at barcodes were specific and yielded minimal cross-reactivity with a panel of near-neighbor and potential contaminant materials. CONCLUSIONS: The barCoder algorithm facilitates the generation of synthetically barcoded biological simulants by (a) eliminating the task of creating modules by hand, (b) minimizing optimization of PCR assays, and (c) reducing effort wasted on non-unique barcode modules.

The ChpR transcriptional regulator of Sinorhizobium meliloti senses 3,5,6-trichloropyridinol, a degradation product of the organophosphate pesticide chlorpyrifos.

The global use of organophosphate insecticides (OPPs) and the growing concern of off-target side effects due to OPP exposure has prompted the need for sensitive and economical detection methods. Here we set out to engineer a previously identified OPP responsive transcription factor, ChpR, from Sinorhizobium melilotii to respond to alternative OPPs and generate a repertoire of whole-cell biosensors for OPPs. The ChpR transcription factor and cognate promoter P chpA, have been shown to activate transcription in the presence of the OPP chlorpyrifos (CPF). Utilizing a GFP reporter regulated by ChpR in a whole-cell biosensor we found that the system responds significantly better to 3,5,6-trichloro-2-pyridinol (TCP), the main degradation product of CPF, compared to CPF itself. This biosensor was able to respond to TCP at 390 nM within 4 h compared to 50 µM of CPF in 7 h. The ChpR-P chpA , and the activating ligand TCP, were able to regulate expression of a kanamycin resistance/sucrose sensitivity (kan/sacB) selection/counterselection module suitable for high throughput mutagenesis screening studies. The ability to control both GFP and the kan/sacB module demonstrates the utility of this reporter for the detection of CPF affected areas. The ChpR-P chpA system serves as an additional positive regulator switch to add to the growing repertoire of controllers available within synthetic biology.

The Essential Role of Hypermutation in Rapid Adaptation to Antibiotic Stress.

A common outcome of antibiotic exposure in patients and in vitro is the evolution of a hypermutator phenotype that enables rapid adaptation by pathogens. While hypermutation is a robust mechanism for rapid adaptation, it requires trade-offs between the adaptive mutations and the more common "hitchhiker" mutations that accumulate from the increased mutation rate. Using quantitative experimental evolution, we examined the role of hypermutation in driving the adaptation of Pseudomonas aeruginosa to colistin. Metagenomic deep sequencing revealed 2,657 mutations at ≥5% frequency in 1,197 genes and 761 mutations in 29 endpoint isolates. By combining genomic information, phylogenetic analyses, and statistical tests, we showed that evolutionary trajectories leading to resistance could be reliably discerned. In addition to known alleles such as pmrB, hypermutation allowed identification of additional adaptive alleles with epistatic relationships. Although hypermutation provided a short-term fitness benefit, it was detrimental to overall fitness. Alarmingly, a small fraction of the colistin-adapted population remained colistin susceptible and escaped hypermutation. In a clinical population, such cells could play a role in reestablishing infection upon withdrawal of colistin. We present here a framework for evaluating the complex evolutionary trajectories of hypermutators that applies to both current and emerging pathogen populations.

Publisher Correction: Shared strategies for ß-lactam catabolism in the soil microbiome.

In the version of the article originally published, the x axis of the graph in Fig. 4d was incorrectly labeled as "Retention time (min)". It should read "Reaction time (min)". The 'deceased' footnote was also formatted incorrectly when published. The footnote text itself should include the name of co-author Tara A. Gianoulis in addition to the previous link to her name in the author list through footnote number 10. The errors have been corrected in the HTML and PDF versions of the article.

Tracking a serial killer: Integrating phylogenetic relationships, epidemiology, and geography for two invasive meningococcal disease outbreaks.

BACKGROUND: While overall rates of meningococcal disease have been declining in the United States for the past several decades, New York City (NYC) has experienced two serogroup C meningococcal disease outbreaks in 2005-2006 and in 2010-2013. The outbreaks were centered within drug use and sexual networks, were difficult to control, and required vaccine campaigns. METHODS: Whole Genome Sequencing (WGS) was used to analyze preserved meningococcal isolates collected before and during the two outbreaks. We integrated and analyzed epidemiologic, geographic, and genomic data to better understand transmission networks among patients. Betweenness centrality was used as a metric to understand the most important geographic nodes in the transmission networks. Comparative genomics was used to identify genes associated with the outbreaks. RESULTS: Neisseria meningitidis serogroup C (ST11/ET-37) was responsible for both outbreaks with each outbreak having distinct phylogenetic clusters. WGS did identify some misclassifications of isolates that were more distant from the outbreak strains, as well as those that should have been included based on high genomic similarity. Genomes for the second outbreak were more similar than the first and no polymorphism was found to either be unique or specific to either outbreak lineage. Betweenness centrality as applied to transmission networks based on phylogenetic analysis demonstrated that the outbreaks were transmitted within focal communities in NYC with few transmission events to other locations. CONCLUSIONS: Neisseria meningitidis is an ever changing pathogen and comparative genomic analyses can help elucidate how it spreads geographically to facilitate targeted interventions to interrupt transmission.

Shared strategies for ß-lactam catabolism in the soil microbiome.

The soil microbiome can produce, resist, or degrade antibiotics and even catabolize them. While resistance genes are widely distributed in the soil, there is a dearth of knowledge concerning antibiotic catabolism. Here we describe a pathway for penicillin catabolism in four isolates. Genomic and transcriptomic sequencing revealed ß-lactamase, amidase, and phenylacetic acid catabolon upregulation. Knocking out part of the phenylacetic acid catabolon or an apparent penicillin utilization operon (put) resulted in loss of penicillin catabolism in one isolate. A hydrolase from the put operon was found to degrade in vitro benzylpenicilloic acid, the ß-lactamase penicillin product. To test the generality of this strategy, an Escherichia coli strain was engineered to co-express a ß-lactamase and a penicillin amidase or the put operon, enabling it to grow using penicillin or benzylpenicilloic acid, respectively. Elucidation of additional pathways may allow bioremediation of antibiotic-contaminated soils and discovery of antibiotic-remodeling enzymes with industrial utility.

A Standard Method To Inactivate Bacillus anthracis Spores to Sterility via Gamma Irradiation.

In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped preparations of gamma-irradiated spores of Bacillus anthracis that contained live spores. In response, a systematic evidence-based method for preparing, concentrating, irradiating, and verifying the inactivation of spore materials was developed. We demonstrate the consistency of spore preparations across multiple biological replicates and show that two different DoD institutions independently obtained comparable dose-inactivation curves for a monodisperse suspension of B. anthracis spores containing 3 × 1010 CFU. Spore preparations from three different institutions and three strain backgrounds yielded similar decimal reduction (D10) values and irradiation doses required to ensure sterility (DSAL) to the point at which the probability of detecting a viable spore is 10-6 Furthermore, spores of a genetically tagged strain of B. anthracis strain Sterne were used to show that high densities of dead spores suppress the recovery of viable spores. Together, we present an integrated method for preparing, irradiating, and verifying the inactivation of spores of B. anthracis for use as standard reagents for testing and evaluating detection and diagnostic devices and techniques.IMPORTANCE The inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of live Bacillus anthracis (anthrax) spores to U.S. and international destinations revealed the need to standardize inactivation methods for materials derived from biological select agents and toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of such an event. Following a retrospective analysis of the procedures previously employed to generate inactivated B. anthracis spores, a study was commissioned by the DoD to provide data required to support the production of inactivated spores for the biodefense community. The results of this work are presented in this publication, which details the method by which spores can be prepared, irradiated, and tested, such that the chance of finding residual living spores in any given preparation is 1/1,000,000. These irradiated spores are used to test equipment and methods for the detection of agents of biological warfare and bioterrorism.

Draft Genome Sequences of Three ß-Lactam-Catabolizing Soil Proteobacteria.

Most antibiotics are derived from the soil, but their catabolism there, which is necessary to close the antibiotic carbon cycle, remains uncharacterized. We report the first draft genome sequences of soil Proteobacteria identified for subsisting solely on ß-lactams as their carbon sources. The genomes encode multiple ß-lactamases, although their antibiotic catabolic pathways remain enigmatic.

Whole-Genome Sequencing in Microbial Forensic Analysis of Gamma-Irradiated Microbial Materials.

Effective microbial forensic analysis of materials used in a potential biological attack requires robust methods of morphological and genetic characterization of the attack materials in order to enable the attribution of the materials to potential sources and to exclude other potential sources. The genetic homogeneity and potential intersample variability of many of the category A to C bioterrorism agents offer a particular challenge to the generation of attributive signatures, potentially requiring whole-genome or proteomic approaches to be utilized. Currently, irradiation of mail is standard practice at several government facilities judged to be at particularly high risk. Thus, initial forensic signatures would need to be recovered from inactivated (nonviable) material. In the study described in this report, we determined the effects of high-dose gamma irradiation on forensic markers of bacterial biothreat agent surrogate organisms with a particular emphasis on the suitability of genomic DNA (gDNA) recovered from such sources as a template for whole-genome analysis. While irradiation of spores and vegetative cells affected the retention of Gram and spore stains and sheared gDNA into small fragments, we found that irradiated material could be utilized to generate accurate whole-genome sequence data on the Illumina and Roche 454 sequencing platforms.

Pathosphere.org: pathogen detection and characterization through a web-based, open source informatics platform.

BACKGROUND: The detection of pathogens in complex sample backgrounds has been revolutionized by wide access to next-generation sequencing (NGS) platforms. However, analytical methods to support NGS platforms are not as uniformly available. Pathosphere (found at Pathosphere.org) is a cloud - based open - sourced community tool that allows for communication, collaboration and sharing of NGS analytical tools and data amongst scientists working in academia, industry and government. The architecture allows for users to upload data and run available bioinformatics pipelines without the need for onsite processing hardware or technical support. RESULTS: The pathogen detection capabilities hosted on Pathosphere were tested by analyzing pathogen-containing samples sequenced by NGS with both spiked human samples as well as human and zoonotic host backgrounds. Pathosphere analytical pipelines developed by Edgewood Chemical Biological Center (ECBC) identified spiked pathogens within a common sample analyzed by 454, Ion Torrent, and Illumina sequencing platforms. ECBC pipelines also correctly identified pathogens in human samples containing arenavirus in addition to animal samples containing flavivirus and coronavirus. These analytical methods were limited in the detection of sequences with limited homology to previous annotations within NCBI databases, such as parvovirus. Utilizing the pipeline-hosting adaptability of Pathosphere, the analytical suite was supplemented by analytical pipelines designed by the United States Army Medical Research Insititute of Infectious Diseases and Walter Reed Army Institute of Research (USAMRIID-WRAIR). These pipelines were implemented and detected parvovirus sequence in the sample that the ECBC iterative analysis previously failed to identify. CONCLUSIONS: By accurately detecting pathogens in a variety of samples, this work demonstrates the utility of Pathosphere and provides a platform for utilizing, modifying and creating pipelines for a variety of NGS technologies developed to detect pathogens in complex sample backgrounds. These results serve as an exhibition for the existing pipelines and web-based interface of Pathosphere as well as the plug-in adaptability that allows for integration of newer NGS analytical software as it becomes available.

Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates.

The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is 'open', with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.

Genome sequencing of 18 francisella strains to aid in assay development and testing.

Francisella tularensis is a highly infectious bacterium with the potential to cause high fatality rates if infections are untreated. To aid in the development of rapid and accurate detection assays, we have sequenced and annotated the genomes of 18 F. tularensis and Francisella philomiragia strains.

Thirty-Two Complete Genome Assemblies of Nine Yersinia Species, Including Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica.

The genus Yersinia includes three human pathogens, of which Yersinia pestis is responsible for >2,000 illnesses each year. To aid in the development of detection assays and aid further phylogenetic elucidation, we sequenced and assembled the complete genomes of 32 strains (across 9 Yersinia species).

Complete genome sequences for 35 biothreat assay-relevant bacillus species.

In 2011, the Association of Analytical Communities (AOAC) International released a list of Bacillus strains relevant to biothreat molecular detection assays. We present the complete and annotated genome assemblies for the 15 strains listed on the inclusivity panel, as well as the 20 strains listed on the exclusivity panel.

Complete genome sequences for 59 burkholderia isolates, both pathogenic and near neighbor.

The genus Burkholderia encompasses both pathogenic (including Burkholderia mallei and Burkholderia pseudomallei, U.S. Centers for Disease Control and Prevention Category B listed), and nonpathogenic Gram-negative bacilli. Here we present full genome sequences for a panel of 59 Burkholderia strains, selected to aid in detection assay development.

Hybrid Vibrio cholerae El Tor lacking SXT identified as the cause of a cholera outbreak in the Philippines.

UNLABELLED: Cholera continues to be a global threat, with high rates of morbidity and mortality. In 2011, a cholera outbreak occurred in Palawan, Philippines, affecting more than 500 people, and 20 individuals died. Vibrio cholerae O1 was confirmed as the etiological agent. Source attribution is critical in cholera outbreaks for proper management of the disease, as well as to control spread. In this study, three V. cholerae O1 isolates from a Philippines cholera outbreak were sequenced and their genomes analyzed to determine phylogenetic relatedness to V. cholerae O1 isolates from recent outbreaks of cholera elsewhere. The Philippines V. cholerae O1 isolates were determined to be V. cholerae O1 hybrid El Tor belonging to the seventh-pandemic clade. They clustered tightly, forming a monophyletic clade closely related to V. cholerae O1 hybrid El Tor from Asia and Africa. The isolates possess a unique multilocus variable-number tandem repeat analysis (MLVA) genotype (12-7-9-18-25 and 12-7-10-14-21) and lack SXT. In addition, they possess a novel 15-kb genomic island (GI-119) containing a predicted type I restriction-modification system. The CTXΦ-RS1 array of the Philippines isolates was similar to that of V. cholerae O1 MG116926, a hybrid El Tor strain isolated in Bangladesh in 1991. Overall, the data indicate that the Philippines V. cholerae O1 isolates are unique, differing from recent V. cholerae O1 isolates from Asia, Africa, and Haiti. Furthermore, the results of this study support the hypothesis that the Philippines isolates of V. cholerae O1 are indigenous and exist locally in the aquatic ecosystem of the Philippines. IMPORTANCE: Genetic characterization and phylogenomics analysis of outbreak strains have proven to be critical for probing clonal relatedness to strains isolated in different geographical regions and over time. Recently, extensive genetic analyses of V. cholerae O1 strains isolated in different countries have been done. However, genome sequences of V. cholerae O1 isolates from the Philippines have not been available for epidemiological investigation. In this study, molecular typing and phylogenetic analysis of Vibrio cholerae isolated from both clinical and environmental samples in 2011 confirmed unique genetic features of the Philippines isolates, which are helpful to understand the global epidemiology of cholera.

Genomic sequences of six botulinum neurotoxin-producing strains representing three clostridial species illustrate the mobility and diversity of botulinum neurotoxin genes.

The whole genomes for six botulinum neurotoxin-producing clostridial strains were sequenced to provide references for under-represented toxin types, bivalent strains or unusual toxin complexes associated with a bont gene. The strains include three Clostridium botulinum Group I strains (CDC 297, CDC 1436, and Prevot 594), a Group II C. botulinum strain (Eklund 202F), a Group IV Clostridium argentinense strain (CDC 2741), and a Group V Clostridium baratii strain (Sullivan). Comparisons of the Group I genomic sequences revealed close relationships and conservation of toxin gene locations with previously published Group I C. botulinum genomes. The bont/F6 gene of strain Eklund 202F was determined to be a chimeric toxin gene composed of bont/F1 and bont/F2. The serotype G strain CDC 2741 remained unfinished in 20 contigs with the bont/G located within a 1.15Mb contig, indicating a possible chromosomal location for this toxin gene. Within the genome of C. baratii Sullivan strain, direct repeats of IS1182 insertion sequence (IS) elements were identified flanking the bont/F7 toxin complex that may be the mechanism of bont insertion into C. baratii. Highlights of the six strains are described and release of their genomic sequences will allow further study of unusual neurotoxin-producing clostridial strains.