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1.  Mutations in Global Regulators Lead to Metabolic Selection during Adaptation to Complex Environments 
PLoS Genetics  2014;10(12):e1004872.
Adaptation to ecologically complex environments can provide insights into the evolutionary dynamics and functional constraints encountered by organisms during natural selection. Adaptation to a new environment with abundant and varied resources can be difficult to achieve by small incremental changes if many mutations are required to achieve even modest gains in fitness. Since changing complex environments are quite common in nature, we investigated how such an epistatic bottleneck can be avoided to allow rapid adaptation. We show that adaptive mutations arise repeatedly in independently evolved populations in the context of greatly increased genetic and phenotypic diversity. We go on to show that weak selection requiring substantial metabolic reprogramming can be readily achieved by mutations in the global response regulator arcA and the stress response regulator rpoS. We identified 46 unique single-nucleotide variants of arcA and 18 mutations in rpoS, nine of which resulted in stop codons or large deletions, suggesting that subtle modulations of ArcA function and knockouts of rpoS are largely responsible for the metabolic shifts leading to adaptation. These mutations allow a higher order metabolic selection that eliminates epistatic bottlenecks, which could occur when many changes would be required. Proteomic and carbohydrate analysis of adapting E. coli populations revealed an up-regulation of enzymes associated with the TCA cycle and amino acid metabolism, and an increase in the secretion of putrescine. The overall effect of adaptation across populations is to redirect and efficiently utilize uptake and catabolism of abundant amino acids. Concomitantly, there is a pronounced spread of more ecologically limited strains that results from specialization through metabolic erosion. Remarkably, the global regulators arcA and rpoS can provide a “one-step” mechanism of adaptation to a novel environment, which highlights the importance of global resource management as a powerful strategy to adaptation.
Author Summary
Changing environmental conditions are the norm in biology. However, understanding adaptation to complex environments presents many challenges. For example, adaptation to resource-rich environments can potentially have many successful evolutionary trajectories to increased fitness. Even in conditions of plenty, the utilization of numerous but novel resources can require multiple mutations before a benefit is accrued. We evolved two bacterial species isolated from the gut of healthy humans in two different, resource-rich media commonly used in the laboratory. We anticipated that under weak selection the population would evolve tremendous genetic diversity. Despite such a complex genetic background we were able to identify a strong degree of parallel evolution and using a combination of population proteomic and population genomic approaches we show that two global regulators, arcA and rpoS, are the principle targets of selection. Up-regulation of the different metabolic pathways that are controlled by these global regulators in combination with up-regulation of transporters that transport nutrients into the cell revealed increased use of the novel resources. Thus global regulators can provide a one-step model to shift metabolism efficiently and provide rapid a one-step reprogramming of the cell metabolic profile.
PMCID: PMC4263409  PMID: 25501822
2.  Complete Genome Sequence of the Epidemic and Highly Virulent CTX-M-15-Producing H30-Rx Subclone of Escherichia coli ST131 
Genome Announcements  2013;1(6):e00988-13.
We report the complete genome sequence, including five complete plasmid sequences, of Escherichia coli ST131 isolate JJ1886. The isolate was obtained in 2007 in the United States from a patient with fatal urosepsis and belongs to the virulent, CTX-M-15-producing H30-Rx sublineage.
PMCID: PMC3853059  PMID: 24309736
3.  Protein Export by the Mycobacterial SecA2 System Is Determined by the Preprotein Mature Domain 
Journal of Bacteriology  2013;195(4):672-681.
At the core of the bacterial general secretion (Sec) pathway is the SecA ATPase, which powers translocation of unfolded preproteins containing Sec signal sequences through the SecYEG membrane channel. Mycobacteria have two nonredundant SecA homologs: SecA1 and SecA2. While the essential SecA1 handles “housekeeping” export, the nonessential SecA2 exports a subset of proteins and is required for Mycobacterium tuberculosis virulence. Currently, it is not understood how SecA2 contributes to Sec export in mycobacteria. In this study, we focused on identifying the features of two SecA2 substrates that target them to SecA2 for export, the Ms1704 and Ms1712 lipoproteins of the model organism Mycobacterium smegmatis. We found that the mature domains of Ms1704 and Ms1712, not the N-terminal signal sequences, confer SecA2-dependent export. We also demonstrated that the lipid modification and the extreme N terminus of the mature protein do not impart the requirement for SecA2 in export. We further showed that the Ms1704 mature domain can be efficiently exported by the twin-arginine translocation (Tat) pathway. Because the Tat system exports only folded proteins, this result implies that SecA2 substrates can fold in the cytoplasm and suggests a putative role of SecA2 in enabling export of such proteins. Thus, the mycobacterial SecA2 system may represent another way that bacteria solve the problem of exporting proteins that can fold in the cytoplasm.
PMCID: PMC3562099  PMID: 23204463
4.  Pathoadaptive Mutations in Salmonella enterica Isolated after Serial Passage in Mice 
PLoS ONE  2013;8(7):e70147.
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.
PMCID: PMC3723669  PMID: 23936152
5.  Detection and Tracking of a Novel Genetically Tagged Biological Simulant in the Environment 
Applied and Environmental Microbiology  2012;78(23):8281-8288.
A variant of Bacillus thuringiensis subsp. kurstaki containing a single, stable copy of a uniquely amplifiable DNA oligomer integrated into the genome for tracking the fate of biological agents in the environment was developed. The use of genetically tagged spores overcomes the ambiguity of discerning the test material from pre-existing environmental microflora or from previously released background material. In this study, we demonstrate the utility of the genetically “barcoded” simulant in a controlled indoor setting and in an outdoor release. In an ambient breeze tunnel test, spores deposited on tiles were reaerosolized and detected by real-time PCR at distances of 30 m from the point of deposition. Real-time PCR signals were inversely correlated with distance from the seeded tiles. An outdoor release of powdered spore simulant at Aberdeen Proving Ground, Edgewood, MD, was monitored from a distance by a light detection and ranging (LIDAR) laser. Over a 2-week period, an array of air sampling units collected samples were analyzed for the presence of viable spores and using barcode-specific real-time PCR assays. Barcoded B. thuringiensis subsp. kurstaki spores were unambiguously identified on the day of the release, and viable material was recovered in a pattern consistent with the cloud track predicted by prevailing winds and by data tracks provided by the LIDAR system. Finally, the real-time PCR assays successfully differentiated barcoded B. thuringiensis subsp. kurstaki spores from wild-type spores under field conditions.
PMCID: PMC3497391  PMID: 23001670
6.  Genetic Barcodes for Improved Environmental Tracking of an Anthrax Simulant 
Applied and Environmental Microbiology  2012;78(23):8272-8280.
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.
PMCID: PMC3497392  PMID: 23001658
7.  Effect of the Bacillus atrophaeus subsp. globigii Spo0F H101R Mutation on Strain Fitness 
Applied and Environmental Microbiology  2012;78(24):8601-8610.
Sporulation is a critical developmental process in Bacillus spp. that, once initiated, removes the possibility of further growth until germination. Therefore, the threshold conditions triggering sporulation are likely to be subject to evolutionary constraint. Our previous studies revealed two spontaneous hypersporulating mutants of Bacillus atrophaeus subsp. globigii, both containing point mutations in the spo0F gene. One of these strains (Detrick-2; contains the spo0F101 allele with a C:T [His101Arg] substitution) had been deliberately selected in the early 1940s as an anthrax surrogate. To determine whether the experimental conditions used during the selection of the “military” strains could have supported the emergence of hypersporulating variants, the relative fitness of strain Detrick-2 was measured in several experimental settings modeled on experimental conditions employed during its development in the 1940s as a simulant. The congenic strain Detrick-1 contained a wild-type spo0F gene and sporulated like the wild-type strain. The relative fitness of Detrick-1 and Detrick-2 was evaluated in competition experiments using quantitative single nucleotide polymorphism (SNP)-specific real-time PCR assays directed at the C:T substitution. The ancestral strain Detrick-1 had a fitness advantage under all conditions tested except when competing cultures were subjected to frequent heat shocks. The hypersporulating strain gained the maximum fitness advantage when cultures were grown at low oxygen tension and when heat shock was applied soon after the formation of the first heat-resistant spores. This is interpreted as gain of fitness by the hypersporulating strain in fast-changing fluctuating environments as a result of the increased rate of switching to the sporulating phenotype.
PMCID: PMC3502920  PMID: 23042165
8.  Detection of Burkholderia pseudomallei O-antigen serotypes in near-neighbor species 
BMC Microbiology  2012;12:250.
Burkholderia pseudomallei is the etiological agent of melioidosis and a CDC category B select agent with no available effective vaccine. Previous immunizations in mice have utilized the lipopolysaccharide (LPS) as a potential vaccine target because it is known as one of the most important antigenic epitopes in B. pseudomallei. Complicating this strategy are the four different B. pseudomallei LPS O-antigen types: A, B, B2, and rough. Sero-crossreactivity is common among O-antigens of Burkholderia species. Here, we identified the presence of multiple B. pseudomallei O-antigen types and sero-crossreactivity in its near-neighbor species.
PCR screening of O-antigen biosynthesis genes, phenotypic characterization using SDS-PAGE, and immunoblot analysis showed that majority of B. mallei and B. thailandensis strains contained the typical O-antigen type A. In contrast, most of B. ubonensis and B. thailandensis-like strains expressed the atypical O-antigen types B and B2, respectively. Most B. oklahomensis strains expressed a distinct and non-seroreactive O-antigen type, except strain E0147 which expressed O-antigen type A. O-antigen type B2 was also detected in B. thailandensis 82172, B. ubonensis MSMB108, and Burkholderia sp. MSMB175. Interestingly, B. thailandensis-like MSMB43 contained a novel serotype B positive O-antigen.
This study expands the number of species which express B. pseudomallei O-antigen types. Further work is required to elucidate the full structures and how closely these are to the B. pseudomallei O-antigens, which will ultimately determine the efficacy of the near-neighbor B serotypes for vaccine development.
PMCID: PMC3541218  PMID: 23126230
9.  Genomic Comparison of Escherichia coli O104:H4 Isolates from 2009 and 2011 Reveals Plasmid, and Prophage Heterogeneity, Including Shiga Toxin Encoding Phage stx2 
PLoS ONE  2012;7(11):e48228.
In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C–3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL–2050 and 2009EL–2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL–2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.
PMCID: PMC3486847  PMID: 23133618
10.  Comparative Genomics of 2009 Seasonal Plague (Yersinia pestis) in New Mexico 
PLoS ONE  2012;7(2):e31604.
Plague disease caused by the Gram-negative bacterium Yersinia pestis routinely affects animals and occasionally humans, in the western United States. The strains native to the North American continent are thought to be derived from a single introduction in the late 19th century. The degree to which these isolates have diverged genetically since their introduction is not clear, and new genomic markers to assay the diversity of North American plague are highly desired. To assay genetic diversity of plague isolates within confined geographic areas, draft genome sequences were generated by 454 pyrosequencing from nine environmental and clinical plague isolates. In silico assemblies of Variable Number Tandem Repeat (VNTR) loci were compared to laboratory-generated profiles for seven markers. High-confidence SNPs and small Insertion/Deletions (Indels) were compared to previously sequenced Y. pestis isolates. The resulting panel of mutations allowed clustering of the strains and tracing of the most likely evolutionary trajectory of the plague strains. The sequences also allowed the identification of new putative SNPs that differentiate the 2009 isolates from previously sequenced plague strains and from each other. In addition, new insertion points for the abundant insertion sequences (IS) of Y. pestis are present that allow additional discrimination of strains; several of these new insertions potentially inactivate genes implicated in virulence. These sequences enable whole-genome phylogenetic analysis and allow the unbiased comparison of closely related isolates of a genetically monomorphic pathogen.
PMCID: PMC3281092  PMID: 22359605
11.  Genomic Signatures of Strain Selection and Enhancement in Bacillus atrophaeus var. globigii, a Historical Biowarfare Simulant 
PLoS ONE  2011;6(3):e17836.
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).
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.
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.
PMCID: PMC3064580  PMID: 21464989
12.  Characterization of pPCP1 Plasmids in Yersinia pestis Strains Isolated from the Former Soviet Union 
Complete sequences of 9.5-kb pPCP1 plasmids in three Yersinia pestis strains from the former Soviet Union (FSU) were determined and compared with those of pPCP1 plasmids in three well-characterized, non-FSU Y. pestis strains (KIM, CO92, and 91001). Two of the FSU plasmids were from strains C2614 and C2944, isolated from plague foci in Russia, and one plasmid was from strain C790 from Kyrgyzstan. Sequence analyses identified four sequence types among the six plasmids. The pPCP1 plasmids in the FSU strains were most genetically related to the pPCP1 plasmid in the KIM strain and least related to the pPCP1 plasmid in Y. pestis 91001. The FSU strains generally had larger pPCP1 plasmid copy numbers compared to strain CO92. Expression of the plasmid's pla gene was significantly (P ≤ .05) higher in strain C2944 than in strain CO92. Given pla's role in Y. pestis virulence, this difference may have important implications for the strain's virulence.
PMCID: PMC3010648  PMID: 21197443
14.  An Inner Membrane Dioxygenase that Generates the 2-Hydroxymyristate Moiety of Salmonella Lipid A 
Biochemistry  2008;47(9):2814-2825.
The lipid A residues of certain Gram-negative bacteria, including most strains of Salmonella and Pseudomonas, are esterified with one or two secondary S-2-hydroxyacyl chains. The S-2 hydroxylation process is O2-dependent in vivo, but the relevant enzymatic pathways have not been fully characterized because in vitro assays have not been developed. We previously reported that expression of the Salmonella lpxO gene confers upon Escherichia coli K-12 the ability to synthesize 2-hydroxymyristate modified lipid A (Gibbons, H. S., Lin, S., Cotter, R. J., and Raetz, C. R. H. J. Biol. Chem. 275, 32940–49, 2000). We now demonstrate that inactivation of lpxO, which encodes a putative Fe2+/O2/α-ketoglutarate-dependent dioxygenase, abolishes S-2-hydroxymyristate formation in S. typhimurium. Membranes of E. coli strains expressing lpxO are able to hydroxylate Kdo2-[4′-32P]-lipid A in vitro in the presence of Fe2+, O2, α-ketoglutarate, ascorbate and Triton X-100. The Fe2+ chelator 2,2′-bipyridyl inhibits the reaction. The product generated in vitro is a mono-hydroxylated Kdo2-lipid A derivative. The [4′-32P]-lipid A released by mild acid hydrolysis from the in vitro product migrates with authentic S-2-hydroxlyated lipid A isolated from 32P-labeled S. typhimurium cells. Electrospray ionization mass spectrometry and gas chromatography/mass spectrometry of the in vitro product are consistent with the 2-hydroxylation of the 3′-secondary myristoyl chain of Kdo2-lipid A. LpxO contains two predicted trans-membrane helices (one at each end of the protein), and its active site likely faces the cytoplasm. LpxO is an unusual example of an integral membrane protein that is a member of the Fe2+/O2/α-ketoglutarate-dependent dioxygenase family.
PMCID: PMC2709818  PMID: 18254598
15.  ATPase Activity of Mycobacterium tuberculosis SecA1 and SecA2 Proteins and Its Importance for SecA2 Function in Macrophages▿  
Journal of Bacteriology  2008;190(14):4880-4887.
The Sec-dependent translocation pathway that involves the essential SecA protein and the membrane-bound SecYEG translocon is used to export many proteins across the cytoplasmic membrane. Recently, several pathogenic bacteria, including Mycobacterium tuberculosis, were shown to possess two SecA homologs, SecA1 and SecA2. SecA1 is essential for general protein export. SecA2 is specific for a subset of exported proteins and is important for M. tuberculosis virulence. The enzymatic activities of two SecA proteins from the same microorganism have not been defined for any bacteria. Here, M. tuberculosis SecA1 and SecA2 are shown to bind ATP with high affinity, though the affinity of SecA1 for ATP is weaker than that of SecA2 or Escherichia coli SecA. Amino acid substitution of arginine or alanine for the conserved lysine in the Walker A motif of SecA2 eliminated ATP binding. We used the SecA2(K115R) variant to show that ATP binding was necessary for the SecA2 function of promoting intracellular growth of M. tuberculosis in macrophages. These results are the first to show the importance of ATPase activity in the function of accessory SecA2 proteins.
PMCID: PMC2447007  PMID: 18487341
16.  Identification of Two Mycobacterium smegmatis Lipoproteins Exported by a SecA2-Dependent Pathway▿ †  
Journal of Bacteriology  2007;189(14):5090-5100.
The SecA2 protein is part of a specialized protein export system of mycobacteria. We set out to identify proteins exported to the bacterial cell envelope by the mycobacterial SecA2 system. By comparing the protein profiles of cell wall and membrane fractions from wild-type and ΔsecA2 mutant Mycobacterium smegmatis, we identified the Msmeg1712 and Msmeg1704 proteins as SecA2-dependent cell envelope proteins. These are the first endogenous M. smegmatis proteins identified as dependent on SecA2 for export. Both proteins are homologous to periplasmic sugar-binding proteins of other bacteria, and both contain functional amino-terminal signal sequences with lipobox motifs. These two proteins appeared to be genuine lipoproteins as shown by Triton X-114 fractionation and sensitivity to globomycin, an inhibitor of lipoprotein signal peptidase. The role of SecA2 in the export of these proteins was specific; not all mycobacterial lipoproteins required SecA2 for efficient localization or processing. Finally, Msmeg1704 was recognized by the SecA2 pathway of Mycobacterium tuberculosis, as indicated by the appearance of an export intermediate when the protein was expressed in a ΔsecA2 mutant of M. tuberculosis. Taken together, these results indicate that a select subset of envelope proteins containing amino-terminal signal sequences can be substrates of the mycobacterial SecA2 pathway and that some determinants for SecA2-dependent export are conserved between M. smegmatis and M. tuberculosis.
PMCID: PMC1951849  PMID: 17496088
California Medicine  1954;80(6):438-440.
The uses and actions of a few of the large number of new drugs that have been introduced to the medical profession in recent years are reviewed herein. The greatest number of drugs introduced are those affecting the autonomic nervous system. Their usefulness often depends upon the individual patient's own response. A knowledge of the type of drug he is administering and a comparison with others that are related better enables a physician to appraise the results of his treatment.
PMCID: PMC1532033  PMID: 13160813
22.  A Protest From Cooper Medical College 
PMCID: PMC1893325  PMID: 18735030
25.  Report on Medical Legislation* 
PMCID: PMC1649262  PMID: 18732843

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