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.

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.

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.

Pathoadaptive mutations in Salmonella enterica isolated after serial passage in mice.

How pathogenic bacteria adapt and evolve in the complex and variable environment of the host remains a largely unresolved question. Here we have used whole genome sequencing of Salmonella enterica serovar Typhimurium LT2 populations serially passaged in mice to identify mutations that adapt bacteria to systemic growth in mice. We found unique pathoadaptive mutations in two global regulators, phoQ and stpA, which increase the competitive indexes of the bacteria 3- to 5-fold. Also, all mouse-adapted lineages had changed the orientation of the hin invertable element, resulting in production of a FliC type of flagellum. Competition experiments in mice with locked flagellum mutants showed that strains expressing the FliC type of flagellum had a 5-fold increase in competitive index as compared to those expressing FljB type flagellum. Combination of the flagellum cassette inversion with the stpA mutation increased competitive indexes up to 20-fold. These experiments show that Salmonella can rapidly adapt to a mouse environment by acquiring a few mutations of moderate individual effect that when combined confer substantial increases in growth.

A strain of Yersinia pestis with a mutator phenotype from the Republic of Georgia.

We describe here a strain of Yersinia pestis, G1670A, which exhibits a baseline mutation rate elevated 250-fold over wild-type Y. pestis. The responsible mutation, a C to T substitution in the mutS gene, results in the transition of a highly conserved leucine at position 689 to arginine (mutS(L689R)). When the MutSL 689R protein of G1670A was expressed in a ΔmutS derivative of Y. pestis strain EV76, mutation rates observed were equivalent to those observed in G1670A, consistent with a causal association between the mutS mutation and the mutator phenotype. The observation of a mutator allele in Yersinia pestis has potential implications for the study of evolution of this and other especially dangerous pathogens.

Genetic barcodes for improved environmental tracking of an anthrax simulant.

The development of realistic risk models that predict the dissemination, dispersion and persistence of potential biothreat agents have utilized nonpathogenic surrogate organisms such as Bacillus atrophaeus subsp. globigii or commercial products such as Bacillus thuringiensis subsp. kurstaki. Comparison of results from outdoor tests under different conditions requires the use of genetically identical strains; however, the requirement for isogenic strains limits the ability to compare other desirable properties, such as the behavior in the environment of the same strain prepared using different methods. Finally, current methods do not allow long-term studies of persistence or reaerosolization in test sites where simulants are heavily used or in areas where B. thuringiensis subsp. kurstaki is applied as a biopesticide. To create a set of genetically heterogeneous yet phenotypically indistinguishable strains so that variables intrinsic to simulations (e.g., sample preparation) can be varied and the strains can be tested under otherwise identical conditions, we have developed a strategy of introducing small genetic signatures ("barcodes") into neutral regions of the genome. The barcodes are stable over 300 generations and do not impact in vitro growth or sporulation. Each barcode contains common and specific tags that allow differentiation of marked strains from wild-type strains and from each other. Each tag is paired with specific real-time PCR assays that facilitate discrimination of barcoded strains from wild-type strains and from each other. These uniquely barcoded strains will be valuable tools for research into the environmental fate of released organisms by providing specific artificial detection signatures.

Genomic signatures of strain selection and enhancement in Bacillus atrophaeus var. globigii, a historical biowarfare simulant.

BACKGROUND: Despite the decades-long use of Bacillus atrophaeus var. globigii (BG) as a simulant for biological warfare (BW) agents, knowledge of its genome composition is limited. Furthermore, the ability to differentiate signatures of deliberate adaptation and selection from natural variation is lacking for most bacterial agents. We characterized a lineage of BGwith a long history of use as a simulant for BW operations, focusing on classical bacteriological markers, metabolic profiling and whole-genome shotgun sequencing (WGS). RESULTS: Archival strains and two "present day" type strains were compared to simulant strains on different laboratory media. Several of the samples produced multiple colony morphotypes that differed from that of an archival isolate. To trace the microevolutionary history of these isolates, we obtained WGS data for several archival and present-day strains and morphotypes. Bacillus-wide phylogenetic analysis identified B. subtilis as the nearest neighbor to B. atrophaeus. The genome of B. atrophaeus is, on average, 86% identical to B. subtilis on the nucleotide level. WGS of variants revealed that several strains were mixed but highly related populations and uncovered a progressive accumulation of mutations among the "military" isolates. Metabolic profiling and microscopic examination of bacterial cultures revealed enhanced growth of "military" isolates on lactate-containing media, and showed that the "military" strains exhibited a hypersporulating phenotype. CONCLUSIONS: Our analysis revealed the genomic and phenotypic signatures of strain adaptation and deliberate selection for traits that were desirable in a simulant organism. Together, these results demonstrate the power of whole-genome and modern systems-level approaches to characterize microbial lineages to develop and validate forensic markers for strain discrimination and reveal signatures of deliberate adaptation.

Double-blind characterization of non-genome-sequenced bacteria by mass spectrometry-based proteomics.

Due to the possibility of a biothreat attack on civilian or military installations, a need exists for technologies that can detect and accurately identify pathogens in a near-real-time approach. One technology potentially capable of meeting these needs is a high-throughput mass spectrometry (MS)-based proteomic approach. This approach utilizes the knowledge of amino acid sequences of peptides derived from the proteolysis of proteins as a basis for reliable bacterial identification. To evaluate this approach, the tryptic digest peptides generated from double-blind biological samples containing either a single bacterium or a mixture of bacteria were analyzed using liquid chromatography-tandem mass spectrometry. Bioinformatic tools that provide bacterial classification were used to evaluate the proteomic approach. Results showed that bacteria in all of the double-blind samples were accurately identified with no false-positive assignment. The MS proteomic approach showed strain-level discrimination for the various bacteria employed. The approach also characterized double-blind bacterial samples to the respective genus, species, and strain levels when the experimental organism was not in the database due to its genome not having been sequenced. One experimental sample did not have its genome sequenced, and the peptide experimental record was added to the virtual bacterial proteome database. A replicate analysis identified the sample to the peptide experimental record stored in the database. The MS proteomic approach proved capable of identifying and classifying organisms within a microbial mixture.

Population-level variation of the preproricin gene contradicts expectation of neutral equilibrium for generalist plant defense toxins.

The preproricin gene encodes ricin, the highly toxic, type II ribosome-inactivating protein of castor bean (Ricinus communis L.). As a generalist plant defense gene, preproricin is expected to exhibit population-level variation consistent with the neutral equilibrium model and to comprise few functionally different alleles. We first test the hypothesis that the preproricin gene family should comprise six to eight members by searching the publicly available draft genome sequence of R. communis and analyzing its ricin-like loci. We then test the neutral equilibrium expectation for the preproricin gene by characterizing its allelic variation among 25 geographically diverse castor bean plants. We confirm the presence of six ricin-like loci that share with the preproricin gene 62.9-96.3% nucleotide identity and intact A-chains. DNA sequence variation among the preproricin haplotypes significantly rejects tests of the neutral equilibrium model. Replacement mutations preserve the 12 amino acids known to affect catalytic and electrostatic interactions of the native protein toxin, which suggests functional divergence among alleles has been minimal. Nucleotide polymorphism is maintained by purifying selection (omega < 0.3) yet includes an excess of rare silent mutations greater than predicted by the neutral equilibrium model. Development of robust detection methods for ricin contamination must account for the presence of these other ricin-like molecules and should leverage the specificity provided by the numerous single nucleotide polymorphisms in the preproricin gene.

Surface sampling of spores in dry-deposition aerosols.

The ability to reliably and reproducibly sample surfaces contaminated with a biological agent is a critical step in measuring the extent of contamination and determining if decontamination steps have been successful. The recovery operations following the 2001 attacks with Bacillus anthracis spores were complicated by the fact that no standard sample collection format or decontamination procedures were established. Recovery efficiencies traditionally have been calculated based upon biological agents which were applied to test surfaces in a liquid format and then allowed to dry prior to sampling tests, which may not be best suited for a real-world event with aerosolized biological agents. In order to ascertain if differences existed between air-dried liquid deposition and biological spores which were allowed to settle on a surface in a dried format, a study was undertaken to determine if differences existed in surface sampling recovery efficiencies for four representative surfaces. Studies were then undertaken to compare sampling efficiencies between liquid spore deposition and aerosolized spores which were allowed to gradually settle under gravity on four different test coupon types. Tests with both types of deposition compared efficiencies of four unique swabbing materials applied to four surfaces with various surface properties. Our studies demonstrate that recovery of liquid-deposited spores differs significantly from recovery of dry aerosol-deposited spores in most instances. Whether the recovery of liquid-deposited spores is overexaggerated or underrepresented with respect to that of aerosol-deposited spores depends upon the surface material being tested.

High-throughput real-time RT-PCR assay to detect the exotic Newcastle Disease Virus during the California 2002--2003 outbreak.

During the 2002--2003 Exotic Newcastle Disease (END) outbreak in Southern California, a high-throughput real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) system was developed to respond to the large diagnostic and surveillance sample workload. A 96-well RNA extraction method, using magnetic bead technology, combined with a 96-well RRT-PCR assay, allowed 1 technician to process and test more than 400 samples per day. A 3-technician team could complete testing on approximately 1,900 samples per day. The diagnostic sensitivity of the high-throughput RRT-PCR assay was 0.9967 (95% CI 0.9937-0.9997) based on 926 virus isolation confirmed positive samples. Diagnostic specificity using an initial 434 virus isolation confirmed negative samples was 100%. A diagnostic specificity of 0.9999 (95% CI 0.9999, >0.9999) was subsequently calculated on the basis of 2 false-positive results among 65,343 surveillance samples collected after the final END-positive case was confirmed in May 2003. Assay performance over 500 replicates, including reproducibility of the combined extraction and RRT-PCR amplification steps yielded a standard deviation of 0.70 RRT-PCR cycle thresholds (Ct) and a standard deviation of 0.59 Ct for the RRT-PCR steps alone. The high-throughput RRT-PCR developed for END contributed significantly to the 2002--2003 END control effort, reducing the predicted timeline for eradication from 3 years to just 11 months, primarily because of the large number of samples that could be rapidly tested. The 96-well approach described for high-throughput END RRT-PCR could be adapted to other rapid, high-volume testing needs, as required for potential foreign animal disease responses or intensive surveillance efforts.

A multiplexed PCR-coupled liquid bead array for the simultaneous detection of four biothreat agents.

We have developed a 10-plexed PCR assay coupled to a 12-plexed liquid bead array to rapidly screen environmental samples for B. anthracis, Y. pestis, F. tularensis, and B. melitensis. Highly validated species-specific primer sets were used to simultaneously amplify multiple diagnostic regions unique to each individual pathogen. Resolution of the mix of amplified products was achieved by PCR product hybridization to corresponding probe sequences, attached to unique sets of fluorescent beads. The hybridized beads were processed through a flow cytometer, which detected presence and quantity of each PCR product. The assay was optimized to allow for maximum sensitivity in a multiplexed format. A high-throughput demonstration was performed where 384 simulated environmental samples were spiked with different amounts of B. thuringensis spores and pathogen DNA. The samples were robotically processed to extract DNA and arrayed for multiplexed PCR-liquid bead detection. The assay correctly identified the presence or absence of each pathogen and collected over 3000 individual data points within a single 8-h shift for approximately $4.00 material costs per environmental sample in a 10-plexed assay.

Use of subtractive hybridization for comprehensive surveys of prokaryotic genome differences.

Comparative bacterial genomics shows that even different isolates of the same bacterial species can vary significantly in gene content. An effective means to survey differences across whole genomes would be highly advantageous for understanding this variation. Here we show that suppression subtractive hybridization (SSH) provides high, representative coverage of regions that differ between similar genomes. Using Helicobacter pylori strains 26695 and J99 as a model, SSH identified approximately 95% of the unique open reading frames in each strain, showing that the approach is effective. Furthermore, combining data from parallel SSH experiments using different restriction enzymes significantly increased coverage compared to using a single enzyme. These results suggest a powerful approach for assessing genome differences among closely related strains when one member of the group has been completely sequenced.