The MerR-like regulator BrlR confers biofilm tolerance by activating multidrug-efflux pumps in Pseudomonas aeruginosa biofilms

A defining characteristic of biofilms is antibiotic tolerance that can be up to 1,000-fold greater than that of planktonic cells. In Pseudomonas aeruginosa, biofilm tolerance to antimicrobial agents requires the biofilm-specific MerR-type transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm tolerance has not been elucidated. Genome-wide transcriptional profiling indicated that brlR was required for maximal expression of genes associated with antibiotic resistance, in particular those encoding the multidrug efflux pumps MexAB-OprM and MexEF-OprN. Chromatin immunoprecipitation (ChIP) analysis revealed a direct regulation of these genes by BrlR, with DNA binding assays confirming BrlR binding to the promoter regions of the mexAB-oprM and mexEF-oprN operons. Quantitative reverse transcriptase PCR (qRT-PCR) analysis further indicated BrlR to be an activator of mexAB-oprM and mexEF-oprN gene expression. Moreover, immunoblot analysis confirmed increased MexA abundance in cells overexpressing brlR. Inactivation of both efflux pumps rendered biofilms significantly more susceptible to five different classes of antibiotics by affecting MIC but not the recalcitrance of biofilms to killing by bactericidal agents. Overexpression of either efflux pump in a ⌬brlR strain partly restored tolerance of ⌬brlR biofilms to antibiotics. Expression of brlR in mutant biofilms lacking both efflux pumps partly restored antimicrobial tolerance of biofilms to wild-type levels. Our results indicate that BrlR acts as an activator of multidrug efflux pumps to confer tolerance to P. aeruginosa biofilms and to resist the action of antimicrobial agents

The Anti-Sigma Factor MucA of Pseudomonas aeruginosa: Dramatic Differences of a mucA22 vs. a ΔmucA Mutant in Anaerobic Acidified Nitrite Sensitivity of Planktonic and Biofilm Bacteria in vitro and During Chronic Murine Lung Infection

Mucoid mucA22 Pseudomonas aeruginosa (PA) is an opportunistic lung pathogen of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) patients that is highly sensitive to acidified nitrite (A-NO2-). In this study, we first screened PA mutant strains for sensitivity or resistance to 20 mM A-NO2- under anaerobic conditions that represent the chronic stages of the aforementioned diseases. Mutants found to be sensitive to A-NO2- included PA0964 (pmpR, PQS biosynthesis), PA4455 (probable ABC transporter permease), katA (major catalase, KatA) and rhlR (quorum sensing regulator). In contrast, mutants lacking PA0450 (a putative phosphate transporter) and PA1505 (moaA2) were A-NO2- resistant. However, we were puzzled when we discovered that mucA22 mutant bacteria, a frequently isolated mucA allele in CF and to a lesser extent COPD, were more sensitive to A-NO2- than a truncated ΔmucA deletion (Δ157–194) mutant in planktonic and biofilm culture, as well as during a chronic murine lung infection. Subsequent transcriptional profiling of anaerobic, A-NO2--treated bacteria revealed restoration of near wild-type transcript levels of protective NO2- and nitric oxide (NO) reductase (nirS and norCB, respectively) in the ΔmucA mutant in contrast to extremely low levels in the A-NO2--sensitive mucA22 mutant. Proteins that were S-nitrosylated by NO derived from A-NO2- reduction in the sensitive mucA22 strain were those involved in anaerobic respiration (NirQ, NirS), pyruvate fermentation (UspK), global gene regulation (Vfr), the TCA cycle (succinate dehydrogenase, SdhB) and several double mutants were even more sensitive to A-NO2-. Bioinformatic-based data point to future studies designed to elucidate potential cellular binding partners for MucA and MucA22. Given that A-NO2- is a potentially viable treatment strategy to combat PA and other infections, this study offers novel developments as to how clinicians might better treat problematic PA infections in COPD and CF airway diseases

Expression of mucoid induction factor MucE is dependent upon the alternate sigma factor AlgU in Pseudomonas aeruginosa

Background Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T (σ22) to initiate transcription of the alginate biosynthetic operon. Results In the current study, we mapped the mucE transcriptional start site, and determined that PmucE activity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in PmucE activity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ΔalgU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE. Conclusions Our data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa

Methods and Compositions Based on Culturing Microorganisms in Low Sedimental Fluid Shear Conditions

The benefits of applying a low sedimental fluid shear environment to manipulate microorganisms were examined. Microorganisms obtained from a low sedimental fluid shear culture, which exhibit modified phenotypic and molecular genetic characteristics, are useful for the development of novel and improved diagnostics, therapeutics, vaccines, and bio-industrial products. Furthermore, application of low sedimental fluid conditions to microorganisms permits identification of molecules uniquely expressed under these conditions, providing a basis for the design of new therapeutic targets

The AllWISE Motion Survey, Part 2

We use the AllWISE Data Release to continue our search for WISE-detected motions. In this paper, we publish another 27,846 motion objects, bringing the total number to 48,000 when objects found during our original AllWISE motion survey are included. We use this list, along with the lists of confirmed WISE-based motion objects from the recent papers by Luhman and by Schneider et al. and candidate motion objects from the recent paper by Gagne et al. to search for widely separated, common-proper-motion systems. We identify 1,039 such candidate systems. All 48,000 objects are further analyzed using color-color and color-mag plots to provide possible characterizations prior to spectroscopic follow-up. We present spectra of 172 of these, supplemented with new spectra of 23 comparison objects from the literature, and provide classifications and physical interpretations of interesting sources. Highlights include: (1) the identification of three G/K dwarfs that can be used as standard candles to study clumpiness and grain size in nearby molecular clouds because these objects are currently moving behind the clouds, (2) the confirmation/discovery of several M, L, and T dwarfs and one white dwarf whose spectrophotometric distance estimates place them 5-20 pc from the Sun, (3) the suggestion that the Na 'D' line be used as a diagnostic tool for interpreting and classifying metal-poor late-M and L dwarfs, (4) the recognition of a triple system including a carbon dwarf and late-M subdwarf, for which model fits of the late-M subdwarf (giving [Fe/H] ~ -1.0) provide a measured metallicity for the carbon star, and (5) a possible 24-pc-distant K5 dwarf + peculiar red L5 system with an apparent physical separation of 0.1 pc.Comment: 62 pages with 80 figures, accepted for publication in The Astrophysical Journal Supplement Series, 23 Mar 2016; second version fixes a few small typos and corrects the footnotes for Table

Discovery of Two Nearby, Peculiar L Dwarfs from the 2MASS Proper Motion Survey: Young or Metal-Rich?

We present the discovery of two nearby L dwarfs from our 2MASS proper motion search, which uses multi-epoch 2MASS observations covering ~4700 square degrees of sky. 2MASS J18212815+1414010 and 2MASS J21481628+4003593 were overlooked by earlier surveys due to their faint optical magnitudes and their proximity to the Galactic Plane (10 degrees < |b| < 15 degrees). Assuming that both dwarfs are single, we derive spectrophotometric distances of ~10 pc, thus increasing the number of known L dwarfs within 10 pc to 10. In the near-infrared, 2MASS J21481628+4003593 shows a triangular-shaped H-band spectrum, strong CO absorption, and a markedly red J-Ks color (2.38+/-0.06) for its L6 optical spectral type. 2MASS J18212815+1414010 also shows a triangular-shaped H-band spectrum and a slightly red J-Ks color (1.78+/-0.05) for its L4.5 optical spectral type. Both objects show strong silicate absorption at 9-11 microns. Cumulatively, these features imply an unusually dusty photosphere for both of these objects. We examine several scenarios to explain the underlying cause for their enhanced dust content and find that a metal-rich atmosphere or a low-surface gravity are consistent with these results. 2MASS J18212815+1414010 may be young (and therefore have a low-surface gravity) based on its low tangential velocity of 10 km/s. On the other hand, 2MASS J21481628+4003593 has a high tangential velocity of 62 km/s and is therefore likely old. Hence, high metallicity and low-surface gravity may lead to similar effects.Comment: 9 pages, 4 tables, 13 figures. Accepted to Ap

Mitochondrial genome sequence analysis: A custom bioinformatics pipeline substantially improves Affymetrix MitoChip v2.0 call rate and accuracy

BACKGROUND: Mitochondrial genome sequence analysis is critical to the diagnostic evaluation of mitochondrial disease. Existing methodologies differ widely in throughput, complexity, cost efficiency, and sensitivity of heteroplasmy detection. Affymetrix MitoChip v2.0, which uses a sequencing-by-genotyping technology, allows potentially accurate and high-throughput sequencing of the entire human mitochondrial genome to be completed in a cost-effective fashion. However, the relatively low call rate achieved using existing software tools has limited the wide adoption of this platform for either clinical or research applications. Here, we report the design and development of a custom bioinformatics software pipeline that achieves a much improved call rate and accuracy for the Affymetrix MitoChip v2.0 platform. We used this custom pipeline to analyze MitoChip v2.0 data from 24 DNA samples representing a broad range of tissue types (18 whole blood, 3 skeletal muscle, 3 cell lines), mutations (a 5.8 kilobase pair deletion and 6 known heteroplasmic mutations), and haplogroup origins. All results were compared to those obtained by at least one other mitochondrial DNA sequence analysis method, including Sanger sequencing, denaturing HPLC-based heteroduplex analysis, and/or the Illumina Genome Analyzer II next generation sequencing platform. RESULTS: An average call rate of 99.75% was achieved across all samples with our custom pipeline. Comparison of calls for 15 samples characterized previously by Sanger sequencing revealed a total of 29 discordant calls, which translates to an estimated 0.012% for the base call error rate. We successfully identified 4 known heteroplasmic mutations and 24 other potential heteroplasmic mutations across 20 samples that passed quality control. CONCLUSIONS: Affymetrix MitoChip v2.0 analysis using our optimized MitoChip Filtering Protocol (MFP) bioinformatics pipeline now offers the high sensitivity and accuracy needed for reliable, high-throughput and cost-efficient whole mitochondrial genome sequencing. This approach provides a viable alternative of potential utility for both clinical diagnostic and research applications to traditional Sanger and other emerging sequencing technologies for whole mitochondrial genome analysis

Discoveries from a Near-infrared Proper Motion Survey using Multi-epoch 2MASS Data

We have conducted a 4030-square-deg near-infrared proper motion survey using multi-epoch data from the Two Micron All-Sky Survey (2MASS). We find 2778 proper motion candidates, 647 of which are not listed in SIMBAD. After comparison to DSS images, we find that 107 of our proper motion candidates lack counterparts at B-, R-, and I-bands and are thus 2MASS-only detections. We present results of spectroscopic follow-up of 188 targets that include the infrared-only sources along with selected optical-counterpart sources with faint reduced proper motions or interesting colors. We also establish a set of near-infrared spectroscopic standards with which to anchor near-infrared classifications for our objects. Among the discoveries are six young field brown dwarfs, five "red L" dwarfs, three L-type subdwarfs, twelve M-type subdwarfs, eight "blue L" dwarfs, and several T dwarfs. We further refine the definitions of these exotic classes to aid future identification of similar objects. We examine their kinematics and find that both the "blue L" and "red L" dwarfs appear to be drawn from a relatively old population. This survey provides a glimpse of the kinds of research that will be possible through time-domain infrared projects such as the UKIDSS Large Area Survey, various VISTA surveys, and WISE, and also through z- or y-band enabled, multi-epoch surveys such as Pan-STARRS and LSST.Comment: To appear in the September 2010 issue of The Astrophysical Journal, Supplement Serie

The Phylogeny of the Four Pan-American MtDNA Haplogroups: Implications for Evolutionary and Disease Studies

Only a limited number of complete mitochondrial genome sequences belonging to Native American haplogroups were available until recently, which left America as the continent with the least amount of information about sequence variation of entire mitochondrial DNAs. In this study, a comprehensive overview of all available complete mitochondrial DNA (mtDNA) genomes of the four pan-American haplogroups A2, B2, C1, and D1 is provided by revising the information scattered throughout GenBank and the literature, and adding 14 novel mtDNA sequences. The phylogenies of haplogroups A2, B2, C1, and D1 reveal a large number of sub-haplogroups but suggest that the ancestral Beringian population(s) contributed only six (successful) founder haplotypes to these haplogroups. The derived clades are overall starlike with coalescence times ranging from 18,000 to 21,000 years (with one exception) using the conventional calibration. The average of about 19,000 years somewhat contrasts with the corresponding lower age of about 13,500 years that was recently proposed by employing a different calibration and estimation approach. Our estimate indicates a human entry and spread of the pan-American haplogroups into the Americas right after the peak of the Last Glacial Maximum and comfortably agrees with the undisputed ages of the earliest Paleoindians in South America. In addition, the phylogenetic approach also indicates that the pathogenic status proposed for various mtDNA mutations, which actually define branches of Native American haplogroups, was based on insufficient grounds

Reconstructing Roma History from Genome-Wide Data

The Roma people, living throughout Europe and West Asia, are a diverse population linked by the Romani language and culture. Previous linguistic and genetic studies have suggested that the Roma migrated into Europe from South Asia about 1,000–1,500 years ago. Genetic inferences about Roma history have mostly focused on the Y chromosome and mitochondrial DNA. To explore what additional information can be learned from genome-wide data, we analyzed data from six Roma groups that we genotyped at hundreds of thousands of single nucleotide polymorphisms (SNPs). We estimate that the Roma harbor about 80% West Eurasian ancestry–derived from a combination of European and South Asian sources–and that the date of admixture of South Asian and European ancestry was about 850 years before present. We provide evidence for Eastern Europe being a major source of European ancestry, and North-west India being a major source of the South Asian ancestry in the Roma. By computing allele sharing as a measure of linkage disequilibrium, we estimate that the migration of Roma out of the Indian subcontinent was accompanied by a severe founder event, which appears to have been followed by a major demographic expansion after the arrival in Europe.Országos Tudományos Kutatási Alapprogramok (OTKA K 103983)Országos Tudományos Kutatási Alapprogramok (OTKA 73430)National Science Foundation (U.S.) (HOMINID grant 1032255)National Institutes of Health (U.S.) (grant GM100233