Quorum Sensing and Cyclic di-GMP Exert Control Over Motility of Vibrio fischeri KB2B1.

Bacterial motility is critical for symbiotic colonization by Vibrio fischeri of its host, the squid Euprymna scolopes, facilitating movement from surface biofilms to spaces deep inside the symbiotic organ. While colonization has been studied traditionally using strain ES114, others, including KB2B1, can outcompete ES114 for colonization for a variety of reasons, including superior biofilm formation. We report here that KB2B1 also exhibits an unusual pattern of migration through a soft agar medium: whereas ES114 migrates rapidly and steadily, KB2B1 migrates slowly and then ceases migration. To better understand this phenomenon, we isolated and sequenced five motile KB2B1 suppressor mutants. One harbored a mutation in the gene for the cAMP receptor protein (crp); because this strain also exhibited a growth defect, it was not characterized further. Two other suppressors contained mutations in the quorum sensing pathway that controls bacterial bioluminescence in response to cell density, and two had mutations in the diguanylate cyclase (DGC) gene VF_1200. Subsequent analysis indicated that (1) the quorum sensing mutations shifted KB2B1 to a perceived low cell density state and (2) the high cell density state inhibited migration via the downstream regulator LitR. Similar to the initial point mutations, deletion of the VF_1200 DGC gene increased migration. Consistent with the possibility that production of the second messenger c-di-GMP inhibited the motility of KB2B1, reporter-based measurements of c-di-GMP revealed that KB2B1 produced higher levels of c-di-GMP than ES114, and overproduction of a c-di-GMP phosphodiesterase promoted migration of KB2B1. Finally, we assessed the role of viscosity in controlling the quorum sensing pathway using polyvinylpyrrolidone and found that viscosity increased light production of KB2B1 but not ES114. Together, our data indicate that while the two strains share regulators in common, they differ in the specifics of the regulatory control over downstream phenotypes such as motility.

A High-Throughput Method for Identifying Novel Genes That Influence Metabolic Pathways Reveals New Iron and Heme Regulation in Pseudomonas aeruginosa.

Heme is an essential metabolite for most life on earth. Bacterial pathogens almost universally require iron to infect a host, often acquiring this nutrient in the form of heme. The Gram-negative pathogen Pseudomonas aeruginosa is no exception, where heme acquisition and metabolism are known to be crucial for both chronic and acute infections. To unveil unknown genes and pathways that could play a role with heme metabolic flux in this pathogen, we devised an omic-based approach we dubbed "Met-Seq," for metabolite-coupled transposon sequencing. Met-Seq couples a biosensor with fluorescence-activated cell sorting (FACS) and massively parallel sequencing, allowing for direct identification of genes associated with metabolic changes. In this work, we first construct and validate a heme biosensor for use with P. aeruginosa and exploit Met-Seq to identify 188 genes that potentially influence intracellular heme levels. Identified genes largely consisted of metabolic pathways not previously associated with heme, including many secreted virulence effectors, as well as 11 predicted small RNAs (sRNAs) and riboswitches whose functions are not currently understood. We verify that five Met-Seq hits affect intracellular heme levels; a predicted extracytoplasmic function (ECF) factor, a phospholipid acquisition system, heme biosynthesis regulator Dnr, and two predicted antibiotic monooxygenase (ABM) domains of unknown function (PA0709 and PA3390). Finally, we demonstrate that PA0709 and PA3390 are novel heme-binding proteins. Our data suggest that Met-Seq could be extrapolated to other biological systems and metabolites for which there is an available biosensor, and provides a new template for further exploration of iron/heme regulation and metabolism in P. aeruginosa and other pathogens.IMPORTANCE The ability to simultaneously and more directly correlate genes with metabolite levels on a global level would provide novel information for many biological platforms yet has thus far been challenging. Here, we describe a method to help address this problem, which we dub "Met-Seq" (metabolite-coupled Tn sequencing). Met-Seq uses the powerful combination of fluorescent biosensors, fluorescence-activated cell sorting (FACS), and next-generation sequencing (NGS) to rapidly identify genes that influence the levels of specific intracellular metabolites. For proof of concept, we create and test a heme biosensor and then exploit Met-Seq to identify novel genes involved in the regulation of heme in the pathogen Pseudomonas aeruginosa Met-Seq-generated data were largely comprised of genes which have not previously been reported to influence heme levels in this pathogen, two of which we verify as novel heme-binding proteins. As heme is a required metabolite for host infection in P. aeruginosa and most other pathogens, our studies provide a new list of targets for potential antimicrobial therapies and shed additional light on the balance between infection, heme uptake, and heme biosynthesis.

Involvement of condensin-directed gene associations in the organization and regulation of chromosome territories during the cell cycle.

Chromosomes are not randomly disposed in the nucleus but instead occupy discrete sub-nuclear domains, referred to as chromosome territories. The molecular mechanisms that underlie the formation of chromosome territories and how they are regulated during the cell cycle remain largely unknown. Here, we have developed two different chromosome-painting approaches to address how chromosome territories are organized in the fission yeast model organism. We show that condensin frequently associates RNA polymerase III-transcribed genes (tRNA and 5S rRNA) that are present on the same chromosomes, and that the disruption of these associations by condensin mutations significantly compromises the chromosome territory arrangement. We also find that condensin-dependent intra-chromosomal gene associations and chromosome territories are co-regulated during the cell cycle. For example, condensin-directed gene associations occur to the least degree during S phase, with the chromosomal overlap becoming largest. In clear contrast, condensin-directed gene associations become tighter in other cell-cycle phases, especially during mitosis, with the overlap between the different chromosomes being smaller. This study suggests that condensin-driven intra-chromosomal gene associations contribute to the organization and regulation of chromosome territories during the cell cycle.

Interaction between TBP and Condensin Drives the Organization and Faithful Segregation of Mitotic Chromosomes.

Genome/chromosome organization is highly ordered and controls various nuclear events, although the molecular mechanisms underlying the functional organization remain largely unknown. Here, we show that the TATA box-binding protein (TBP) interacts with the Cnd2 kleisin subunit of condensin to mediate interphase and mitotic chromosomal organization in fission yeast. TBP recruits condensin onto RNA polymerase III-transcribed (Pol III) genes and highly transcribed Pol II genes; condensin in turn associates these genes with centromeres. Inhibition of the Cnd2-TBP interaction disrupts condensin localization across the genome and the proper assembly of mitotic chromosomes, leading to severe defects in chromosome segregation and eventually causing cellular lethality. We propose that the Cnd2-TBP interaction coordinates transcription with chromosomal architecture by linking dispersed gene loci with centromeres. This chromosome arrangement can contribute to the efficient transmission of physical force at the kinetochore to chromosomal arms, thereby supporting the fidelity of chromosome segregation.

Printing-based assembly of quadruple-junction four-terminal microscale solar cells and their use in high-efficiency modules.

Expenses associated with shipping, installation, land, regulatory compliance and on-going maintenance and operations of utility-scale photovoltaics can be significantly reduced by increasing the power conversion efficiency of solar modules through improved materials, device designs and strategies for light management. Single-junction cells have performance constraints defined by their Shockley-Queisser limits. Multi-junction cells can achieve higher efficiencies, but epitaxial and current matching requirements between the single junctions in the devices hinder progress. Mechanical stacking of independent multi-junction cells circumvents these disadvantages. Here we present a fabrication approach for the realization of mechanically assembled multi-junction cells using materials and techniques compatible with large-scale manufacturing. The strategy involves printing-based stacking of microscale solar cells, sol-gel processes for interlayers with advanced optical, electrical and thermal properties, together with unusual packaging techniques, electrical matching networks, and compact ultrahigh-concentration optics. We demonstrate quadruple-junction, four-terminal solar cells with measured efficiencies of 43.9% at concentrations exceeding 1,000 suns, and modules with efficiencies of 36.5%.

Passive coherent combination of a diode laser array with 35 elements.

A monolithic diode laser array with 35 elements is operated as a coherent array through the use of a Self-Fourier cavity. By analyzing the far field interference pattern, the coherence was measured to be 0.57 with all 35 elements operating and was measured to be approximately constant for arrays with greater than 15 elements. These results are in rough agreement with previous analyses which predict a coherence equal to 0.65 for very large arrays of passively coupled laser elements and demonstrate how the use of regenerative feedback benefits the passive phasing of coherent laser arrays. These results demonstrate that it is possible to circumvent previous cold cavity theories that predict poor phasing properties for arrays with greater than ~10 elements.

Effect of Kerr and resonant nonlinearities on phase locking of a multistable fiber amplifier array.

An analysis is presented, based on a rigorous solution of the propagation equations, of an array of saturable fiber amplifiers with scatter in length that is subject to global feedback. Passively phase-locked states exhibiting multistability due to resonant or Kerr nonlinearity are predicted in respectively low and high regimes of optical feedback.

Enhanced ultraviolet responses in thin-film InGaP solar cells by down-shifting.

Layers of poly(methyl methacrylate) doped with the Eu complex Eu(DPEPO)(hfac)3 (EuDH) provide a means for down-shifting incident ultraviolet (UV) light into the visible range, with beneficial effects on the performance of solar cells, as demonstrated with thin-film InGaP devices formed by epitaxial liftoff. Experimental and computational results establish important aspects of gain and loss mechanisms in the UV range. Measurements show that InGaP cells with coatings of EuDH doped PMMA exhibit enhanced currents (8.68 mA cm(-2)) and power conversion efficiencies (9.48%), both due to increased responses at wavelengths between 300-360 nm.

Centromeric motion facilitates the mobility of interphase genomic regions in fission yeast.

Dispersed genetic elements, such as retrotransposons and Pol-III-transcribed genes, including tRNA and 5S rRNA, cluster and associate with centromeres in fission yeast through the function of condensin. However, the dynamics of these condensin-mediated genomic associations remains unknown. We have examined the 3D motions of genomic loci including the centromere, telomere, rDNA repeat locus, and the loci carrying Pol-III-transcribed genes or long-terminal repeat (LTR) retrotransposons in live cells at as short as 1.5-second intervals. Treatment with carbendazim (CBZ), a microtubule-destabilizing agent, not only prevents centromeric motion, but also reduces the mobility of the other genomic loci during interphase. Further analyses demonstrate that condensin-mediated associations between centromeres and the genomic loci are clonal, infrequent and transient. However, when associated, centromeres and the genomic loci migrate together in a coordinated fashion. In addition, a condensin mutation that disrupts associations between centromeres and the genomic loci results in a concomitant decrease in the mobility of the loci. Our study suggests that highly mobile centromeres pulled by microtubules in cytoplasm serve as 'genome mobility elements' by facilitating physical relocations of associating genomic regions.

Mechanisms of enhanced optical absorption for ultrathin silicon solar microcells with an integrated nanostructured backside reflector.

This paper investigates mechanisms of enhanced light absorption exhibited by ultrathin Si solar microcells integrated with a periodically nanostructured, semitransparent metallic reflector. This backside reflector comprises periodic nanoscale relief features formed by soft-imprint lithography with a thin (~35 nm) coating of Au. The work shows that microcells placed in direct contact above the nanostructured reflector's surface creates Fabry-Pérot cavities, which traps impinging light inside the Si slab via the excitation of cavity modes. Experimental measurements show that the short-circuit current and efficiency values for devices incorporating this thin, semitransparent backside reflector outperform similar Si microcells integrated with a planar thick (~300 nm) opaque mirror by ~10-15% because of enhanced absorption. Computational modeling that is supported by experimental measurements reveal that the dominant methods of enhancement stem from a complex interplay between backside diffraction/scattering and Fabry-Pérot resonances. These same data demonstrate that plasmonic interactions contribute minimally to the optical enhancements seen.

In-phase supermode selection in a multicore fiber laser array by means of a self-Fourier external cavity.

A procedure is developed to determine the transverse-mode structure of a cavity consisting of a dense, evanescently coupled, waveguide laser array, which, in addition, is externally coupled by feedback from an external cavity. The formalism is used to determine the loss and phasing properties of a multicore fiber array coupled to an external self-Fourier cavity. Best performance is predicted for linear arrays of up to five cores, or two-dimensional arrays of up to 25 cores. A low-loss, in-phase, fundamental array mode is predicted, which achieves better than 30 dB discrimination against higher-order modes at periodically spaced values of the array length. However, we show that a shift in operating wavelength of typically a few nanometers can bring about near-perfect phasing and loss operation over a continuum of fiber lengths. With increased fill factor, significantly more of the output power can be concentrated in the central lobe of the far field but at the penalty of increased loss in the fundamental eigenmode.