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Total transcript amplification (TTA) from single eukaryotic cells for transcriptome analysis is established, but TTA from a single prokaryotic cell presents additional challenges with much less starting material, the lack of poly(A)-tails, and the fact that the messages can be polycistronic. Here, we describe a novel method for single-bacterium TTA using a model organism, Burkholderia thailandensis, exposed to a subinhibitory concentration of the antibacterial agent, glyphosate. Utilizing a B. thailandensis microarray to assess the TTA method showed low fold-change bias (less than twofold difference and Pearson correlation coefficient R ≈ 0.87–0.89) and drop-outs (4%–6% of 2842 detectable genes), compared with data obtained from the larger-scale nonamplified RNA samples. Further analysis of the microarray data suggests that B. thailandensis, when exposed to the aromatic amino acid biosynthesis inhibitor glyphosate, induces (or represses) genes to possibly recuperate and balance the intracellular amino acid pool. We validated our single-cell microarray data at the multi-cell and single-cell levels with lacZ and gfp reporter-gene fusions, respectively. Sanger sequencing of 192 clones generated from the TTA product of a single cell, with and without enrichment by elimination of rRNA and tRNA, detected only B. thailandensis sequences with no contamination. These data indicate that RNA-seq of TTA from a single cell is possible using this novel method.

The effects of several phosphonic additives on the formation of calcium sulfate scale on metal surface in a flow system were investigated thoroughly using multiple pipe flow system. Their chemical structure, concentration and run time are to be the main factors that influence the inhibitory capability of these anti-scalants. The relationships between these factors and the scale rate were established. The Langmuir adsorption isotherm is applied to predict the critical concentrations above which complete inhibition of scaling will occur. The inhibition efficiency order of the investigated organic additives is found as follows:EDTP > NTMP > NDPA > HEDP> NPDA, which corresponds to the number of phosphonate groups that the additives possess.

We propose a high-speed and energy-efficient two-cycle multiply-accumulate (MAC) architecture that supports two's complement numbers, and includes accumulation guard bits and saturation circuitry. The first MAC pipeline stage contains only partial-product generation circuitry and a reduction tree, while the second stage, thanks to a special sign-extension solution, implements all other functionality. Place-and-route evaluations using a 65-nm 1.1-V cell library show that the proposed architecture offers a 31% improvement in speed and a 32% reduction in energy per operation, averaged across operand sizes of 16, 32, 48, and 64 bits, over a reference two-cycle MAC architecture that employs a multiplier in the first stage and an accumulator in the second. When operating the proposed architecture at the lower frequency of the reference architecture the available timing slack can be used to downsize gates, resulting in a 52% reduction in energy compared to the reference. We extend the new architecture to create a versatile double-throughput MAC (DTMAC) unit that efficiently performs either multiply-accumulate or multiply operations for N-bit, 1 × N/2-bit, or 2 × N/2-bit operands. In comparison to a fixed-function 32-bit MAC unit, 16-bit multiply-accumulate operations can be executed with 67% higher energy efficiency on a 32-bit DTMAC unit.

Genetic manipulation of the category B select agents Burkholderia pseudomallei and Burkholderia mallei has been stifled due to the lack of compliant selectable markers. Hence, there is a need for additional select-agent-compliant selectable markers. We engineered a selectable marker based on the gat gene (encoding glyphosate acetyltransferase), which confers resistance to the common herbicide glyphosate (GS). To show the ability of GS to inhibit bacterial growth, we determined the effective concentrations of GS against Escherichia coli and several Burkholderia species. Plasmids based on gat , flanked by unique flip recombination target ( FRT ) sequences, were constructed for allelic-replacement. Both allelic-replacement approaches, one using the counterselectable marker pheS and the gat-FRT cassette and one using the DNA incubation method with the gat-FRT cassette, were successfully utilized to create deletions in the asd and dapB genes of wild-type B. pseudomallei strains. The asd and dapB genes encode an aspartate-semialdehyde dehydrogenase (BPSS1704, chromosome 2) and dihydrodipicolinate reductase (BPSL2941, chromosome 1), respectively. Mutants unable to grow on media without diaminopimelate (DAP) and other amino acids of this pathway were PCR verified. These mutants displayed cellular morphologies consistent with the inability to cross-link peptidoglycan in the absence of DAP. The B. pseudomallei 1026b Δ asd :: gat-FRT mutant was complemented with the B. pseudomallei asd gene on a site-specific transposon, mini-Tn 7 - bar , by selecting for the bar gene (encoding bialaphos/PPT resistance) with PPT. We conclude that the gat gene is one of very few appropriate, effective, and beneficial compliant markers available for Burkholderia select-agent species. Together with the bar gene, the gat cassette will facilitate various genetic manipulations of Burkholderia select-agent species.

Allelic replacement in the Burkholderia genus has been problematic due to the lack of appropriate counter-selectable and selectable markers. The counter-selectable marker sacB , commonly used in gram-negative bacteria, is nonselective on sucrose in many Burkholderia species. In addition, the use of antibiotic resistance markers of clinical importance for the selection of desirable genetic traits is prohibited in the United States for two potential bioterrorism agents, Burkholderia mallei and Burkholderia pseudomallei . Here, we engineered a mutated counter-selectable marker based on the B. pseudomallei PheS (the α-subunit of phenylalanyl tRNA synthase) protein and tested its effectiveness in three different Burkholderia species. The mutant PheS protein effectively killed 100% of the bacteria in the presence of 0.1% p -chlorophenylalanine. We assembled the mutant pheS on several allelic replacement vectors, in addition to constructing selectable markers based on tellurite (Tel r ) and trimethoprim (Tp r ) resistance that are excisable by flanking unique FLP recombination target (FRT) sequences. As a proof of concept, we utilized one of these gene replacement vectors (pBAKA) and the Tel r -FRT cassette to produce a chromosomal mutation in the Burkholderia thailandensis betBA operon, which codes for betaine aldehyde dehydrogenase and choline dehydrogenase. Chromosomal resistance markers could be excised by the introduction of pFLP-AB5 (Tp r ), which is one of two constructed flp -containing plasmids, pFLP-AB4 (Tel r ) and pFLP-AB5 (Tp r ). These flp -containing plasmids harbor the mutant pheS gene and allow self curing on media that contain p -chlorophenylalanine after Flp-FRT excision. The characterization of the Δ betBA ::Tel r -FRT and Δ betBA ::FRT mutants indicated a defect in growth with choline as a sole carbon source, while these mutants grew as well as the wild type with succinate and glucose as alternative carbon sources.

Beta-oxidative enzymes for fatty acid degradation (Fad) of long-chain fatty acids (LCFAs) are induced in vivo during lung infection in cystic fibrosis patients, and this may contribute to nutrient acquisition and pathogenesis of Pseudomonas aeruginosa. The promoter region of one P. aeruginosa beta-oxidation operon, fadBA5 (PA3014 and PA3013), was mapped. Focusing on the transposon mutagenesis of strain PAO1 carrying the P(fadBA5)-lacZ fusion, a regulator for the fadBA5 operon was identified to be PsrA (PA3006). Transcriptome analysis of the DeltapsrA mutant indicated its importance in regulating beta-oxidative enzymes. These microarray data were confirmed by real-time RT-PCR analyses of the fadB5 and lipA (encoding a lipase) genes. Induction of the fadBA5 operon was demonstrated to respond to novel LCFA signals, and this induction required the presence of PsrA, suggesting that LCFAs bind to PsrA to derepress fadBA5. Electrophoretic mobility shift assays indicate specific binding of PsrA to the fadBA5 promoter region. This binding is disrupted by specific LCFAs (C(18:1)(Delta9), C(16:0), C(14:0) and, to a lesser extent, C(12:0)), but not by other medium- or short-chain fatty acids or the first intermediate of beta-oxidation, acyl-CoA. It is shown here that PsrA is a fadBA5 regulator that binds and responds to LCFA signals in P. aeruginosa.

The quartz crystal microbalance (QCM) has been used extensively as a mass sensor due to its extremely high sensitivity to small mass loadings. Conventional measurement of the amount of scale deposited on a surface is restricted by the sensitivity limit of analytical balances. Thefirst attempt to investigate the deposition of gypsum scale on a surface using a rotating electrochemical QCMsystem was carried out to investigate the eflects of many factors at the early stages of scale formation. Results indicated there was almost no induction time for this system, and the long induction time observed in the conventional system was due to the limited sensitivity of the analytical balance. A slow increase in scale amount was observed at the beginning of the scaling process as shown by the plot offrequency or mass change against time. After this period the curve rises steeply and becomes almost linear. The supersaturation level of the solutions and the rotating speed have significant effects on the gypsum scaling. A QCM flow-cell system has also been developed to investigate the scaling of gypsum on the pipe wall. This system is similar to a conventional pipe flow system except that its size is much smaller and the deposition of scales can be monitored with the QCM electrode throughout the scaling process. The mass change is plotted against time and results are compared for the rotating QCM system and the conventional system. It is noticed that the formation of gypsum on the QCM electrode is greatly dependent on both the supersaturation of the solution and the flow rate of the fluid passing through the flow cell.

Without prior knowledge of the promoters of various genes in bacteria, it can be difficult to study gene regulation using reporter-gene fusions. Regulation studies of promoters are ideal at their native locus, which do not require prior knowledge of promoter regions. Based on a previous study with FRT–lacZ–KmR constructs, we constructed two novel FRT–lacZ–GmR plasmids. This allows easy engineering of Pseudomonas aeruginosa reporter-gene fusions, post-mutant construction, with the Flp–FRT system. We demonstrate the usefulness of one of these FRT–lacZ–GmR plasmids to study the regulation of the fadAB1 operon in P. aeruginosa at its native locus. The fadAB1 operon, involved in fatty acid (FA) degradation, was significantly induced in the presence of several medium chain-length fatty acids (MCFA) and, to a lesser degree, long chain-length fatty acids (LCFA). In addition to the previous work on the FRT–lacZ–KmR tools, these new constructs increase the repertoire of tools that can be applied to P. aeruginosa or other species and strains of bacteria where kanamycin resistance may not be appropriate.

Technology scaling and growing use of accelerators make optimization of data movement of increasing importance in all computing systems. Further, growing diversity in memory structures makes embedding such optimization in software non-portable. We propose a novel architectural solution called Data Layout Transformation (DLT) associated with a simple set of instructions that enable software to describe the required data movement compactly, and free the implementation to optimize the movement based on the knowledge of the memory hierarchy and system structure. The DLT architecture ideas can be applicable to both general-purpose and accelerator-based heterogeneous systems. Experiment results first show that the proposed DLT architecture can make use of the full bandwidth (>97%) of a wide range of memory systems (DDR3 and HMC) while its implementation cost is relatively low, occupying only 0.24 mm2 and consuming 75mW at 1GHz in 32nm CMOS technology. Our evaluation of using the DLT accelerator in accelerated-based heterogeneous system across DDR3 and HMC memory shows that the DLT can enhance system performance in range of 4.6x–99x (DDR3), 4.4x–115x (HMC) which turns out 2.8x–48x (DDR3), 1.4x–39x (HMC) improvement for energy efficiency.

Calcium sulphate scaling is a serious problem encountered in many industrial and domestic applications. Supersaturation has been proven to be the major driving force of scale formation, but the solubility of calcium sulphate changes with temperature. The main purpose of this work is to investigate the effects of temperature on the formation of calcium sulphate scales in pipes, using a pipe flow system. Various levels of supersaturation of the calcium sulphate solution have been employed at different temperatures. Results indicated that higher temperature produced a large increase of scale amounts and a significant decrease of induction periods. Many forms of hydrated calcium sulphate were created at high temperature. The relationship between deposited scale mass and temperature was deduced from experimental data. From the relationship between induction period and temperature activation energies of the surface nucleation were estimated to be in the range of 42 to 48 kJ mol − 1 .

The T7-expression system has been very useful for protein expression in Escherichia coli. However, it is often desirable to over-express proteins in species other than E. coli. Here, we constructed an inducible broad-host-range T7-expression transposon, which allows simple one-step construction of T7-expression strains in various species, providing the option to over-express proteins of interest in a broader host-range. This transposon contains the T7 RNA polymerase driven by the lacUV5 promoter, which is repressed by the lac-repressor. Leaky expression is prevented by the presence of T7-lysozyme on this construct. The complete T7-expression system is flanked by mariner transposon repeats of the suicidal R6Kgammaori plasmid, pBT20-Deltabla. Stable integration of the whole system is possible by a one-step selection for a Flp-excisable Gm(R)-marker. We showed the engineering of E. coli, Pseudomonas aeruginosa, Erwinia carotovora, Salmonella choleraesuis, Agrobacterium tumefaciens, and Chromobacterium violaceum strains with this construct and demonstrated the expression of the Burkholderia pseudomallei Asd protein in these hosts, by induction with isopropyl-beta-d-thiogalactopyranoside (IPTG).

We propose the Unified Automata Processor (UAP), a new architecture that provides general and efficient support for finite automata (FA). The UAP supports a wide range of existing finite automata models (DFAs, NFAs, A-DFAs, JFAs, counting-DFAs, and counting-NFAs), and future novel FA models. Evaluation on realistic workloads shows that UAP implements all models efficiently, achieving line rates 94.5% of ideal. A single UAP lane delivers line rates 50× greater than software approaches on CPUs and GPUs. Scaling UAP to 64 lanes achieves FA transition throughputs as high as 295 Gbps, more than 100× higher than CPUs and GPUs, and even exceeding ASIC approaches such as IBM's RegX by 6×. With efficient support for multiple input streams, UAP achieves throughputs that saturate even high speed stacked DRAM memory systems. The UAP design and implementation is efficient in instructions executed, memory references, and energy. UAP achieves a 1.2 Ghz clock rate (32nm TSMC CMOS), and a 64-lane UAP requires only 2.2 mm2 and 563 mW. At these levels of performance and energy-efficiency, UAP is a promising candidate for integration into general-purpose computing architectures.

In this paper, we perform a systematic comparison to study the energy cost of varying data formats and data types w.r.t. arithmetic logic and data movement for accelerator-based heterogeneous systems in which both compute-intensive (FFT accelerator) and data-intensive accelerators (DLT accelerator) are added. We explore evaluation for a wide range of design processes (e.g. 32nm bulk-CMOS and projected 7nm FinFET) and memory systems (e.g. DDR3 and HMC). First, our result shows that when varying data formats, the energy costs of using floating point over fixed point are 5.3% (DDR3), 6.2% (HMC) for core and 0.8% (DDR3), 1.5% (HMC) for system in 32nm process. These energy costs are negligible as 0.2% and 0.01% for core and system in 7nm FinFET process in DDR3 memory and slightly increasing in HMC. Second, we identify that the core and system energy of systems using fixed point, 16-bit, FFT accelerator is nearly half of using 32-bit if data movement is also accelerated. This evidence implies that system energy is highly proportional to the amount of moving data when varying data types.

Burkholderia cepacia strain LO6 is a betaproteobacterium that was isolated from a cystic fibrosis patient. Here we report the 6.4 Mb draft genome sequence assembled into 2 contigs. This genome sequence will aid the transcriptomic profiling of this bacterium and help us to better understand the mechanisms specific to pulmonary infections.

Scaling is a serious problem in many industries. Scale formation is promoted by the presence of dissolved salts in a supersaturated solution. The precipitation of a crystalline substance from a solution onto the site of scale formation requires three simultaneous factors: supersaturation, nucleation, and adequate contact time. There are many factors influencing the scaling rate: supersaturation, retention time, hydrodynamic conditions such as velocity, pipe diameter and Reynolds number, surface roughness and material, and temperature. The scale formation can be inhibited by many types of additives, which have different efficiency on different scalants. The effectiveness of the inhibitors depends on various factors: contact time, temperature, solution pH, additive concentration, and structure. The Langmuir isotherm can be used effectively to predict the efficiency of scale inhibitors.

Accelerators have long been used to improve the performance and energy efficiency of embedded signal processing systems relying on Fast Fourier Transforms (FFTs). We explore the benefits of processor-integrated FFT accelerators, characterizing their performance and energy efficiency for current and future memory architectures. First, we consider designs that deeply integrate an FFT accelerator into a simple 5-stage RISC pipeline and evaluate the performance and energy efficiency for a 32 nm process. Our results indicate that a 64-point processor-integrated FFT accelerator alone can increase performance for a 4K/32k-point 1D-FFT by 7/4-fold respectively. In term of energy efficiency, our 64-point FFT accelerator increases it at least 4-fold. Second, since memory performance is a critical constraint, we evaluate system configuration with 3D-stacked DRAM systems. Our results indicate that energy efficiency bottlenecks can be alleviated, as the 3D-stacked memory reduces energy by nearly 14-fold. When combined with our FFT accelerator, overall energy efficiency for 4k and 32k-point FFTs increases 86-fold and 70-fold respectively. Prospectively, with addition of a data layout transformation engine, cycle count and energy for the data transpose phase can be reduced 10x. Such a step would increase the accelerator benefit at least 10-fold in energy for DDR3 and more than 100-fold in 3D-stacked memory system.

Triclosan is an antiseptic frequently added to items as diverse as soaps, lotions, toothpaste, and many commonly used household fabrics and plastics. Although wild-type Pseudomonas aeruginosa expresses the triclosan target enoyl-acyl carrier protein reductase, it is triclosan resistant due to expression of the MexAB-OprM efflux system. Exposure of a susceptible Δ( mexAB-oprM ) strain to triclosan selected multidrug-resistant bacteria at high frequencies. These bacteria hyperexpressed the MexCD-OprJ efflux system due to mutations in its regulatory gene, nfxB . The MICs of several drugs for these mutants were increased up to 500-fold, including the MIC of ciprofloxacin, which was increased 94-fold. Whereas the MexEF-OprN efflux system also participated in triclosan efflux, this antimicrobial was not a substrate for MexXY-OprM.

Purification of proteins from Escherichia coli under native conditions is often hampered by inclusion-body formation after overexpression from T7 promoter-based expression vectors. This is probably due to the relatively high copy number of the ColE1-based expression vectors. To circumvent these problems, the low-copy-number pViet and pNam expression vectors were constructed. These vectors contain the pSC101 origin of replication and allow the expression of oligohistidine and intein chitin-binding domain fusion proteins, respectively. Since pViet and pNam do not replicate in E. coli B strains, an E. coli K-12 host strain [SA1503(DE3)] was constructed. This strain is defective in the Lon and OmpT proteases and allows IPTG-inducible expression of recombinant proteins from the T7 promoter. The new vectors were successfully tested by purification of three very insoluble proteins (RmlD, LasI and RhlI) under non-denaturing conditions, and all three proteins retained enzymatic activity. The purified hexahistidine (His6)-tagged Pseudomonas aeruginosa RhlI protein was subjected to more detailed analyses, which indicated that (1) only butyryl-acyl carrier protein (ACP) and S-adenosylmethionine (SAM) were required for synthesis of N-butyryl-L-homoserine lactone; (2) when present at physiological concentrations, butyryl-coenzyme A and NADPH were not substrates for RhlI; (3) RhlI was able to synthesize N-hexanoyl-L-homoserine lactone from hexanoyl-ACP and SAM; (4) RhlI was able to direct synthesis of N-butyryl-L-homoserine lactone from crotonyl-ACP in a reaction coupled to purified P. aeruginosa FabI (enoyl-ACP reductase).

The Pseudomonas aeruginosa fabI structural gene, encoding enoyl-acyl carrier protein (ACP) reductase, was cloned and sequenced. Nucleotide sequence analysis revealed that fabI is probably the last gene in a transcriptional unit that includes a gene encoding an ATP-binding protein of an ABC transporter of unknown function. The FabI protein was similar in size and primary sequence to other bacterial enoyl-ACP reductases, and it contained signature motifs for the FAD-dependent pyridine nucleotide reductase and glucose/ribitol dehydrogenase families, respectively. The chromosomal fabI gene was disrupted, and the resulting mutant was viable but possessed only 62% of the total enoyl-ACP reductase activity found in wild-type cell extracts. The fabI -encoded enoyl-ACP reductase activity was NADH dependent and inhibited by triclosan; the residual activity in the fabI mutant was also NADH dependent but not inhibited by triclosan. An polyhistidine-tagged FabI protein was purified and characterized. Purified FabI (i) could use NADH but not NADPH as a cofactor; (ii) used both crotonyl-coenzyme A and crotonyl-ACP as substrates, although it was sixfold more active with crotonyl-ACP; and (iii) was efficiently inhibited by low concentrations of triclosan. A FabI Gly 95 -to-Val active-site amino acid substitution was generated by site-directed mutagenesis, and the mutant protein was purified. The mutant FabI protein retained normal enoyl-ACP reductase activity but was highly triclosan resistant. When coupled to FabI, purified P. aeruginosa N -butyryl- l -homoserine lactone (C 4 -HSL) synthase, RhlI, could synthesize C 4 -HSL from crotonyl-ACP and S -adenosylmethionine. This reaction was NADH dependent and inhibited by triclosan. The levels of C 4 -HSL and N -(3-oxo)-dodecanoyl- l -homoserine lactones were reduced 50% in a fabI mutant, corroborating the role of FabI in acylated homoserine lactone synthesis in vivo.

An improved method for gene replacement in Pseudomonas aeruginosa was developed. The method employs several new gene replacement vectors that incorporate (1) the counterselectable sacB marker, (2) a lacZ alpha-allele for blue-white screening, (3) the pUC18/19 vectors multiple cloning site with 10 unique restriction sites, (4) an oriT for conjugation-mediated plasmid transfer and (5) carbenicillin, gentamicin (Gm) and tetracycline selectable markers. A cassette was constructed that contains a GmR selectable marker next to the green fluorescent protein structural gene, with both markers being flanked by Flp recombinase target (FRT) sites. The FRT cassette was used to insertionally inactivate the cloned P. aeruginosa pabC gene encoding aminodeoxychorismate lyase. After conjugal transfer into P. aeruginosa, plasmid integrants were selected, and deletion of unwanted DNA sequences was promoted by sucrose counterselection. The FRT cassette was excised with high frequencies (close to 100%) from the chromosome after conjugal transfer of a Flp recombinase-expressing plasmid; this sacB-containing plasmid was subsequently cured by sucrose counterselection, resulting in an unmarked P. aeruginosa delta pabC strain.

Pseudomonas aeruginosa can grow to very high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of P. aeruginosa in vivo. The phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs) are released by enzymatic cleavage of PC by bacterial phospholipase C and lipases. Three different bacterial pathways, the choline, glycerol, and fatty acid degradation pathways, are then involved in the degradation of these PC components. Here, we identified five potential FA degradation (Fad) related fadBA-operons (fadBA1-5, each encoding 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase). Through mutagenesis and growth analyses, we showed that three (fadBA145) of the five fadBA-operons are dominant in medium-chain and long-chain Fad. The triple fadBA145 mutant also showed reduced ability to degrade PC in vitro. We have previously shown that by partially blocking Fad, via mutagenesis of fadBA5 and fadDs, we could significantly reduce the ability of P. aeruginosa to replicate on FA and PC in vitro, as well as in the mouse lung. However, no studies have assessed the ability of mutants, defective in choline and/or glycerol degradation in conjunction with Fad, to grow on PC or in vivo. Hence, we constructed additional mutants (ΔfadBA145ΔglpD, ΔfadBA145ΔbetAB, and ΔfadBA145ΔbetABΔglpD) significantly defective in the ability to degrade FA, choline, and glycerol and, therefore, PC. The analysis of these mutants in the BALB/c mouse lung infection model showed significant inability to utilize PC in vitro, resulted in decreased replication fitness and competitiveness in vivo compared to the complement strain, although there was little to no variation in typical virulence factor production (e.g., hemolysin, lipase, and protease levels). This further supports the hypothesis that lung surfactant PC serves as an important nutrient for P. aeruginosa during CF lung infection.

Applicability of this method is demonstrated by total syntheses of Hirsutene (VII) and Illudol (VIII).

A tandem process provides high-value 1,6-enynes that are otherwise difficult to prepare. Two base-mediated reactions—fragmentation and olefination—are executed in a coordinated manner that is overall more efficient than either reaction on its own. The 1,6-enynes can be strategically employed in conjunction with carbocyclization to deliver important targets, as noted for reported syntheses of hirsutene and illudol.

A novel pUC19-based gene replacement vector has been developed. This vector incorporates (i) the counterselectable sacB marker, (ii) a lacZ alpha allele for blue-white screening, (iii) an oriT for conjugation-mediated plasmid transfer and (iv) unique cloning sites for SmaI and the rare-cutting meganuclease I-SceI. These rare restriction sites are also present on the helper plasmid pUC19Sce. The replacement vector is engineered to contain few restriction sites to gain greater access to restriction sites within cloned DNA fragments, thus facilitating their genetic manipulation. The usefulness of the system was demonstrated by chromosomal integration of a newly constructed xylE::GmR fusion cassette into the glpD gene of Pseudomonas aeruginosa.

2-Methyl-2-(1-oxo-5-heptynyl)-1,3-dithiane 2-Methyl-1,3-dithiane n-Butyllithium 2-Methyl-3-trifluoromethansulfonyloxy-cyclohex-2-en-1-one 2-Methyl-1,3-cyclohexanedione Trifluoromethanesulfonic anhydride Keywords: alkyne reactions; vinylogous acyl triflates (VATs); nucleophilic addition/fragmentation reactions; tethered keto-alkynes; 2-Methyl-2-(1-oxo-5-heptynyl)-1,3-dithiane

Burkholderia pseudomallei , the cause of serious and life-threatening diseases in humans, is of national biodefense concern because of its potential use as a bioterrorism agent. This microbe is listed as a select agent by the CDC; therefore, development of vaccines is of significant importance. Here, we further investigated the growth characteristics of a recently created B. pseudomallei 1026b Δ asd mutant in vitro , in a cell model, and in an animal model of infection. The mutant was typified by an inability to grow in the absence of exogenous diaminopimelate (DAP); upon single-copy complementation with a wild-type copy of the asd gene, growth was restored to wild-type levels. Further characterization of the B. pseudomallei Δ asd mutant revealed a marked decrease in RAW264.7 murine macrophage cytotoxicity compared to the wild type and the complemented Δ asd mutant. RAW264.7 cells infected by the Δ asd mutant did not exhibit signs of cytopathology or multinucleated giant cell (MNGC) formation, which were observed in wild-type B. pseudomallei cell infections. The Δ asd mutant was found to be avirulent in BALB/c mice, and mice vaccinated with the mutant were protected against acute inhalation melioidosis. Thus, the B. pseudomallei Δ asd mutant may be a promising live attenuated vaccine strain and a biosafe strain for consideration of exclusion from the select agent list.

Leakage power is an important concern in modern electronic designs. To efficiently employ power gating for leakage reduction in embedded processors, the architecture must provide a clear-cut software support for power gating and the power-gated unit must have significant idle times during the execution of the applications. We introduce power gating of individual datapath units for the embedded architecture of FlexCore, to evaluate if leakage reductions in temporarily idle units can reduce the overall power dissipation of compute-intensive applications. Post-layout multi-corner simulations for a 65-nm FlexCore datapath implementation demonstrate that power gating of the multiplier unit yields overall datapath energy savings, up to 14%, for two EEMBC benchmarks.

Designing a processor is a complex task that uses multiple and varied tools. The complete development cycle spans software as well as hardware design and verification. More often than not, in spite of the close dependencies between hardware and software, there is no common platform for quick and accurate testing of these dependencies. Though such systems are often employed in industry, it is not common for end-to-end frameworks to be deployed in educational and research settings.We present the FlexCore Design Exploration Framework (FlexDEF), an end-to-end tool-chain used to develop the FlexCore processor and its accompanying cache system. The tool-chain is a hierarchically linked system that spans the various development phases involved in design and verification. The processor system is intended to be a model, for use in research-oriented projects where both the software and hardware are in a constant state of flux. We discuss the complete framework and the advantages in each context. Finally, we summarize the developments and discuss the future of the FlexDEF tool-chain.

A proven approach to increase performance of general-purpose processors is to add hardware accelerators. In its basic configuration, the FlexCore processor has a limited set of datapath units. But thanks to a flexible datapath interconnect and a wide control word, the FlexCore datapath is explicitly designed to support integration of special units that, on demand, can accelerate certain data-intensive applications. We present the integration of a versatile accelerator for several Cyclic Redundancy Checking (CRC) keys. Furthermore, we investigate the accelerator’s impact on processor execution time and energy efficiency, using the Power Stone CRC benchmark. Our evaluation shows that the accelerated 65-nm 2.7-ns FlexCore datapath is, for example, 86% more energy and cycle efficient than a datapath lacking the CRC accelerator.

We propose a high-speed and energy-efficient 2-cycle Multiply-Accumulate (MAC) architecture. Our architecture is based on two's complement representation, it uses guarding bits to efficiently support longer MAC loops, and it includes output saturation. By performing carry propagation only in the second stage of the MAC pipeline, multiplication and accumulation have similar delays. But in contrast to previous MAC architectures that propose to only use one carry-propagation stage, our architecture requires no extra cycles to produce the final result. Instead it correctly produces the sum of the accumulated value and the product in each cycle. Our place-and-route evaluation shows that the proposed architecture, averaged across several operand sizes, offers a 33% improvement in speed and a 37% reduction of energy over a conventional 2-cycle MAC architecture.

The Pseudomonas aeruginosa PsrA autorepressor has dual roles as a repressor of the fadBA5beta-oxidation operon and an activator of the stationary-phase sigma factor rpoS and exsCEBA operon of the type III secretion system (TTSS). Previously, we demonstrated that the repression of the fadBA5 operon by PsrA is relieved by long-chain fatty acids (LCFAs). However, the signal affecting the activation of rpoS and exsC via PsrA is unknown. In this study, microarray and gene fusion data suggested that LCFA (e.g. oleate) affected the expression of rpoS and exsC. DNA binding studies confirmed that PsrA binds to the rpoS and exsC promoter regions. This binding was inhibited by LCFA, indicating that LCFA directly affects the activation of these two genes through PsrA. LCFA decreased rpoS and exsC expression, resulting in increased N-(butyryl)-l-homoserine-lactone quorum sensing signal and decreased ExoS/T production respectively. Based on the crystal structure of PsrA, site-directed mutagenesis of amino acid residues, within the hydrophobic channel thought to accommodate LCFA, created two LCFA-non-responsive PsrA mutants. The binding and activation of rpoS and exsC by these PsrA mutants was no longer inhibited by LCFA. These data support a mechanistic model where LCFAs influence PsrA regulation to control LCFA metabolism and some virulence genes in P. aeruginosa.

As a simple five-stage General-Purpose Processor (GPP), the baseline FlexCore processor has a limited set of datapath units. By utilizing a flexible datapath interconnect and a wide control word, a FlexCore processor is explicitly designed to support integration of special units that, on demand, can accelerate certain data-intensive applications. In this paper, we propose the integration of a novel Double Throughput Multiply-Accumulate (DTMAC) unit, whose different operating modes allow for on-thefly optimization of computational precision. For the two EEMBC benchmarks considered, the FlexCore processor performance is significantly enhanced when one DTMAC accelerator is included, translating into reduced execution time and energy dissipation. In comparison to the 32-bit GPP reference, the accelerated 32-bit FlexCore processor shows a 4.37× improvement in execution time and a 3.92× reduction in energy dissipation, for a benchmark with many consecutive 16-bit MAC operations.

There are few appropriate single-copy genetic tools for most Burkholderia species, and the high level of antibiotic resistance in this genus further complicates the development of genetic tools. In addition, the utilization of resistance genes for clinically important antibiotics is prohibited for the bioterrorism agents Burkholderia pseudomallei and Burkholderia mallei , necessitating the development of additional nonantibiotic-based genetic tools. Three single-copy systems devoid of antibiotic selection based on two nonantibiotic selectable markers, tellurite resistance (Tel r ) and Escherichia coli aspartate-semialdehyde dehydrogenase ( asd Ec ), were developed to facilitate genetic manipulation in Burkholderia species. These systems include one mariner transposon, a mini-Tn 7 -derived site-specific transposon, and six FRT reporter fusion vectors based on the lacZ , gfp , and luxCDABE reporter genes. Initially, we showed that the random mariner transposon pBT20-Δ bla -Tel r - FRT efficiently transposed within Burkholderia cenocepacia , Burkholderia thailandensis , B. pseudomallei , and B. mallei . We then utilized the mini-Tn 7 -Tel r -based transposon vector (mini-Tn7-Tel r - betBA ) and a transposase-containing helper plasmid (pTNS3- asd Ec ) to complement the B. thailandensis Δ betBA mutation. Next, one of the FRT - lacZ fusion vectors (pFRT1- lacZ -Tel r ) was integrated by Flp (encoded on a helper plasmid, pCD13SK-Flp- oriT - asd Ec ) to construct the B. thailandensis Δ betBA :: FRT - lacZ -Tel r reporter fusion strain. The betBA operon was shown to be induced in the presence of choline and under osmotic stress conditions by performing β-galactosidase assays on the B. thailandensis Δ betBA :: FRT - lacZ -Tel r fusion strain. Finally, we engineered B. thailandensis Δ betBA :: FRT - gfp -Tel r and Δ betBA :: FRT - lux -Tel r fusion strains by utilizing fusion vectors pFRT1- gfp -Tel r and pFRT1- lux -Tel r , respectively. The induction of the betBA operon by choline and osmotic stress was confirmed by performing fluorescent microscopy and bioluminescent imaging analyses.

One of the hallmarks of Pseudomonas aeruginosa infection in cystic fibrosis (CF) patients is very-high-cell-density (HCD) replication in the lung, allowing this bacterium to induce virulence controlled by the quorum-sensing systems. However, the nutrient sources sustaining HCD replication in this chronic infection are largely unknown. Here, we performed microarray studies of P. aeruginosa directly isolated from the lungs of CF patients to demonstrate its metabolic capability and virulence in vivo. In vivo microarray data, confirmed by real-time reverse transcription-PCR, indicated that the P. aeruginosa population expressed several genes for virulence, drug resistance, and utilization of multiple nutrient sources (lung surfactant lipids and amino acids) contributing to HCD replication. The most abundant lung surfactant lipid molecule, phosphatidylcholine (PC), induces key genes of P. aeruginosa pertinent to PC degradation in vitro as well as in vivo within the lungs of CF patients. The results support recent research indicating that P. aeruginosa exists in the lungs of CF patients as a diverse population with full virulence potential. The data also indicate that there is deregulation of several pathways, suggesting that there is in vivo evolution by deregulation of a large portion of the transcriptome during chronic infection in CF patients. To our knowledge, this is the first in vivo transcriptome analysis of P. aeruginosa in a natural infection in CF patients, and the results indicate several important aspects of P. aeruginosa pathogenesis, drug resistance, nutrient utilization, and general metabolism within the lungs of CF patients.

Calcium- and integrin-binding protein (CIB) is a novel member of the helix-loop-helix family of regulatory calcium-binding proteins which likely has a specific function in hemostasis through its interaction with platelet integrin alphaIIbbeta(3). The significant amino acid sequence homology between CIB and other regulatory calcium-binding proteins such as calmodulin, calcineurin B, and recoverin suggests that CIB may undergo a calcium-induced conformational change; however, the mechanism of calcium binding and the details of a structural change have not yet been investigated. Consequently, we have performed a variety of spectroscopic and microcalorimetric studies of CIB to determine its calcium binding characteristics, and the subsequent conformational changes that occur. Furthermore, we provide the first evidence for magnesium binding to CIB and determine the structural consequences of this interaction. Our results indicate that in the absence of any bound metal ions, apo-CIB adopts a folded yet highly flexible molten globule-like structure. Both calcium and magnesium binding induce conformational changes which stabilize both the secondary and tertiary structure of CIB, resulting in considerable increases in the thermal stability of the proteins. CIB was found to bind two Ca(2+) ions in a sequential manner with dissociation constants (K(d)) near 0.54 and 1.9 microM for sites EF-4 and EF-3, respectively. In contrast, CIB bound only one Mg(2+) ion to EF-3 with a K(d) near 120 microM. Together, our results suggest that CIB may exist in multiple structural and metal ion-bound states in vivo which may play a role in its regulation of target proteins such as platelet integrin.

The genomes of the two clonally derived Pseudomonas aeruginosa prototypic strains PAO1 and DSM-1707 differ by the presence of a 2.19-Mb inversion including oriC . Integration of two Flp recombinase target sites near the rrn operons containing the inversion endpoints in PAO1 led to Flp-catalyzed inversion of the intervening 1.59-Mb fragment, including oriC , at high frequencies (83%), favoring the chromosome configuration found in DSM-1707. The results indicate that the oriC -containing region of the P. aeruginosa chromosome can readily undergo and tolerate large inversions.

A DNA fragment containing the Pseudomonas aeruginosa fabD (encoding malonyl-coenzyme A [CoA]:acyl carrier protein [ACP] transacylase), fabG (encoding β-ketoacyl-ACP reductase), acpP (encoding ACP), and fabF (encoding β-ketoacyl-ACP synthase II) genes was cloned and sequenced. This fab gene cluster is delimited by the plsX (encoding a poorly understood enzyme of phospholipid metabolism) and pabC (encoding 4-amino-4-deoxychorismate lyase) genes; the fabF and pabC genes seem to be translationally coupled. The fabH gene (encoding β-ketoacyl-ACP synthase III), which in most gram-negative bacteria is located between plsX and fabD , is absent from this gene cluster. A chromosomal temperature-sensitive fabD mutant was obtained by site-directed mutagenesis that resulted in a W258Q change. A chromosomal fabF insertion mutant was generated, and the resulting mutant strain contained substantially reduced levels of cis -vaccenic acid. Multiple attempts aimed at disruption of the chromosomal fabG gene were unsuccessful. We purified FabD as a hexahistidine fusion protein (H 6 -FabD) and ACP in its native form via an ACP-intein-chitin binding domain fusion protein, using a novel expression and purification scheme that should be applicable to ACP from other bacteria. Matrix-assisted laser desorption–ionization spectroscopy, native polyacrylamide electrophoresis, and amino-terminal sequencing revealed that (i) most of the purified ACP was properly modified with its 4′-phosphopantetheine functional group, (ii) it was not acylated, and (iii) the amino-terminal methionine was removed. In an in vitro system, purified ACP functioned as acyl acceptor and H 6 -FabD exhibited malonyl-CoA:ACP transacylase activity.

The fatty acid (FA) degradation pathway of Pseudomonas aeruginosa, an opportunistic pathogen, was recently shown to be involved in nutrient acquisition during BALB/c mouse lung infection model. The source of FA in the lung is believed to be phosphatidylcholine, the major component of lung surfactant. Previous research indicated that P. aeruginosa has more than two fatty acyl-CoA synthetase genes (fadD; PA3299 and PA3300), which are responsible for activation of FAs using ATP and coenzyme A. Through a bioinformatics approach, 11 candidate genes were identified by their homology to the Escherichia coli FadD in the present study. Four new homologues of fadD (PA1617, PA2893, PA3860, and PA3924) were functionally confirmed by their ability to complement the E. coli fadD mutant on FA-containing media. Growth phenotypes of 17 combinatorial fadD mutants on different FAs, as sole carbon sources, indicated that the four new fadD homologues are involved in FA degradation, bringing the total number of P. aeruginosa fadD genes to six. Of the four new homologues, fadD4 (PA1617) contributed the most to the degradation of different chain length FAs. Growth patterns of various fadD mutants on plant-based perfumery substances, citronellic and geranic acids, as sole carbon and energy sources indicated that fadD4 is also involved in the degradation of these plant-derived compounds. A decrease in fitness of the sextuple fadD mutant, relative to the ΔfadD1D2 mutant, was only observed during BALB/c mouse lung infection at 24 h.