Optomechanical deformation and strain in elastic dielectrics

Light forces induced by scattering and absorption in elastic dielectrics lead to local density modulations and deformations. These perturbations in turn modify light propagation in the medium and generate an intricate nonlinear response. We generalise an analytic approach where light propagation in one-dimensional media of inhomogeneous density is modelled as a result of multiple scattering between polarizable slices. Using the Maxwell stress tensor formalism we compute the local optical forces and iteratively approach self-consistent density distributions where the elastic back-action balances gradient- and scattering forces. For an optically trapped dielectric we derive the nonlinear dependence of trap position, stiffness and total deformation on the object's size and field configuration. Generally trapping is enhanced by deformation, which exhibits a periodic change between stretching and compression. This strongly deviates from qualitative expectations based on the change of photon momentum of light crossing the surface of a dielectric. We conclude that optical forces have to be treated as volumetric forces and that a description using the change of photon momentum at the surface of a medium is inappropriate

Quick change: post-transcriptional regulation in Pseudomonas

Pseudomonas species have evolved dynamic and intricate regulatory networks to fine-tune gene expression, with complex regulation occurring at every stage in the processing of genetic information. This approach enables Pseudomonas to generate precise individual responses to the environment in order to improve their fitness and resource economy. The weak correlations we observe between RNA and protein abundance highlight the significant regulatory contribution of a series of intersecting post-transcriptional pathways, influencing mRNA stability, translational activity and ribosome function, to Pseudomonas environmental responses. This review examines our current understanding of three major post-transcriptional regulatory systems in Pseudomonas spp.; Gac/Rsm, Hfq and RimK, and presents an overview of new research frontiers, emerging genome-wide methodologies, and their potential for the study of global regulatory responses in Pseudomonas

The Pseudomonas aeruginosa Transcriptome in Planktonic Cultures and Static Biofilms Using RNA Sequencing

In this study, we evaluated how gene expression differs in mature Pseudomonas aeruginosa biofilms as opposed to planktonic cells by the use of RNA sequencing technology that gives rise to both quantitative and qualitative information on the transcriptome. Although a large proportion of genes were consistently regulated in both the stationary phase and biofilm cultures as opposed to the late exponential growth phase cultures, the global biofilm gene expression pattern was clearly distinct indicating that biofilms are not just surface attached cells in stationary phase. A large amount of the genes found to be biofilm specific were involved in adaptation to microaerophilic growth conditions, repression of type three secretion and production of extracellular matrix components. Additionally, we found many small RNAs to be differentially regulated most of them similarly in stationary phase cultures and biofilms. A qualitative analysis of the RNA-seq data revealed more than 3000 putative transcriptional start sites (TSS). By the use of rapid amplification of cDNA ends (5′-RACE) we confirmed the presence of three different TSS associated with the pqsABCDE operon, two in the promoter of pqsA and one upstream of the second gene, pqsB. Taken together, this study reports the first transcriptome study on P. aeruginosa that employs RNA sequencing technology and provides insights into the quantitative and qualitative transcriptome including the expression of small RNAs in P. aeruginosa biofilms

RsmW, Pseudomonas aeruginosa small non-coding RsmA-binding RNA upregulated in biofilm versus planktonic growth conditions

BACKGROUND: Biofilm development, specifically the fundamentally adaptive switch from acute to chronic infection phenotypes, requires global regulators and small non-coding regulatory RNAs (sRNAs). This work utilized RNA-sequencing (RNA-seq) to detect sRNAs differentially expressed in Pseudomonas aeruginosa biofilm versus planktonic state. RESULTS: A computational algorithm was devised to detect and categorize sRNAs into 5 types: intergenic, intragenic, 5′-UTR, 3′-UTR, and antisense. Here we report a novel RsmY/RsmZ-type sRNA, termed RsmW, in P. aeruginosa up-transcribed in biofilm versus planktonic growth. RNA-Seq, 5’-RACE and Mfold predictions suggest RsmW has a secondary structure with 3 of 7 GGA motifs located on outer stem loops. Northern blot revealed two RsmW binding bands of 400 and 120 bases, suggesting RsmW is derived from the 3’-UTR of the upstream hypothetical gene, PA4570. RsmW expression is elevated in late stationary versus logarithmic growth phase in PB minimal media, at higher temperatures (37°C versus 28°C), and in both gacA and rhlR transposon mutants versus wild-type. RsmW specifically binds to RsmA protein in vitro and restores biofilm production and reduces swarming in an rsmY/rsmZ double mutant. PA4570 weakly resembles an RsmA/RsmN homolog having 49% and 51% similarity, and 16% and 17% identity to RsmA and RsmN amino acid sequences, respectively. PA4570 was unable to restore biofilm and swarming phenotypes in ΔrsmA deficient strains. CONCLUSION: Collectively, our study reveals an interesting theme regarding another sRNA regulator of the Rsm system and further unravels the complexities regulating adaptive responses for Pseudomonas species

Identification and functional characterization of small non-coding RNAs in Xanthomonas oryzae pathovar oryzae

<p>Abstract</p> <p>Background</p> <p>Small non-coding RNAs (sRNAs) are regarded as important regulators in prokaryotes and play essential roles in diverse cellular processes. <it>Xanthomonas oryzae </it>pathovar <it>oryzae </it>(<it>Xoo</it>) is an important plant pathogenic bacterium which causes serious bacterial blight of rice. However, little is known about the number, genomic distribution and biological functions of sRNAs in <it>Xoo</it>.</p> <p>Results</p> <p>Here, we performed a systematic screen to identify sRNAs in the <it>Xoo </it>strain PXO99. A total of 850 putative non-coding RNA sequences originated from intergenic and gene antisense regions were identified by cloning, of which 63 were also identified as sRNA candidates by computational prediction, thus were considered as <it>Xoo </it>sRNA candidates. Northern blot hybridization confirmed the size and expression of 6 sRNA candidates and other 2 cloned small RNA sequences, which were then added to the sRNA candidate list. We further examined the expression profiles of the eight sRNAs in an <it>hfq </it>deletion mutant and found that two of them showed drastically decreased expression levels, and another exhibited an Hfq-dependent transcript processing pattern. Deletion mutants were obtained for seven of the Northern confirmed sRNAs, but none of them exhibited obvious phenotypes. Comparison of the proteomic differences between three of the ΔsRNA mutants and the wild-type strain by two-dimensional gel electrophoresis (2-DE) analysis showed that these sRNAs are involved in multiple physiological and biochemical processes.</p> <p>Conclusions</p> <p>We experimentally verified eight sRNAs in a genome-wide screen and uncovered three Hfq-dependent sRNAs in <it>Xoo</it>. Proteomics analysis revealed <it>Xoo </it>sRNAs may take part in various metabolic processes. Taken together, this work represents the first comprehensive screen and functional analysis of sRNAs in rice pathogenic bacteria and facilitates future studies on sRNA-mediated regulatory networks in this important phytopathogen.</p

The RNA Chaperone Hfq Is Important for Growth and Stress Tolerance in Francisella novicida

The RNA-binding protein Hfq is recognized as an important regulatory factor in a variety of cellular processes, including stress resistance and pathogenesis. Hfq has been shown in several bacteria to interact with small regulatory RNAs and act as a post-transcriptional regulator of mRNA stability and translation. Here we examined the impact of Hfq on growth, stress tolerance, and gene expression in the intracellular pathogen Francisella novicida. We present evidence of Hfq involvement in the ability of F. novicida to tolerate several cellular stresses, including heat-shock and oxidative stresses, and alterations in hfq gene expression under these conditions. Furthermore, expression of numerous genes, including several associated with virulence, is altered in a hfq mutant strain suggesting they are regulated directly or indirectly by Hfq. Strikingly, we observed a delayed entry into stationary phase and increased biofilm formation in the hfq mutant. Together, these data demonstrate a critical role for Hfq in F. novicida growth and survival

A Small RNA Controls Expression of the Chitinase ChiA in Listeria monocytogenes

In recent years, more than 60 small RNAs (sRNAs) have been identified in the gram-positive human pathogen Listeria monocytogenes, but their putative roles and mechanisms of action remain largely unknown. The sRNA LhrA was recently shown to be a post-transcriptional regulator of a single gene, lmo0850, which encodes a small protein of unknown function. LhrA controls the translation and degradation of the lmo0850 mRNA by an antisense mechanism, and it depends on the RNA chaperone Hfq for efficient binding to its target. In the present study, we sought to gain more insight into the functional role of LhrA in L. monocytogenes. To this end, we determined the effects of LhrA on global-wide gene expression. We observed that nearly 300 genes in L. monocytogenes are either positively or negatively affected by LhrA. Among these genes, we identified lmo0302 and chiA as direct targets of LhrA, thus establishing LhrA as a multiple target regulator. Lmo0302 encodes a hypothetical protein with no known function, whereas chiA encodes one of two chitinases present in L. monocytogenes. We show here that LhrA acts as a post-transcriptional regulator of lmo0302 and chiA by interfering with ribosome recruitment, and we provide evidence that both LhrA and Hfq act to down-regulate the expression of lmo0302 and chiA. Furthermore, in vitro binding experiments show that Hfq stimulates the base pairing of LhrA to chiA mRNA. Finally, we demonstrate that LhrA has a negative effect on the chitinolytic activity of L. monocytogenes. In marked contrast to this, we found that Hfq has a stimulating effect on the chitinolytic activity, suggesting that Hfq plays multiple roles in the complex regulatory pathways controlling the chitinases of L. monocytogenes

Identification of Genes Contributing to the Virulence of Francisella tularensis SCHU S4 in a Mouse Intradermal Infection Model

Background: Francisella tularensis is a highly virulent human pathogen. The most virulent strains belong to subspecies tularensis and these strains cause a sometimes fatal disease. Despite an intense recent research effort, there is very limited information available that explains the unique features of subspecies tularensis strains that distinguish them from other F. tularensis strains and that explain their high virulence. Here we report the use of targeted mutagenesis to investigate the roles of various genes or pathways for the virulence of strain SCHU S4, the type strain of subspecies tularensis. Methodology/Principal Findings: The virulence of SCHU S4 mutants was assessed by following the outcome of infection after intradermal administration of graded doses of bacteria. By this route, the LD\u2085\u2080 of the SCHU S4 strain is one CFU. The virulence of 20 in-frame deletion mutants and 37 transposon mutants was assessed. A majority of the mutants did not show increased prolonged time to death, among them notably \u394pyrB and \u394recA. Of the remaining, mutations in six unique targets, tolC, rep, FTT0609, FTT1149c, ahpC, and hfq resulted in significantly prolonged time to death and mutations in nine targets, rplA, wbtI, iglB, iglD, purL, purF, ggt, kdtA, and glpX, led to marked attenuation with an LD\u2085\u2080 of >10\ub3 CFU. In fact, the latter seven mutants showed very marked attenuation with an LD\u2085\u2080 of 6510\u2077 CFU. Conclusions/Significance: The results demonstrate that the characterization of targeted mutants yielded important information about essential virulence determinants that will help to identify the so far little understood extreme virulence of F. tularensis subspecies tularensis.Peer reviewed: YesNRC publication: Ye

Label-Free Optical Detection of Biomolecular Translocation through Nanopore Arrays

In recent years, nanopores have emerged as exceptionally promising single-molecule sensors due to their ability to detect biomolecules at subfemtomole levels in a label-free manner. Development of a high-throughput nanopore-based biosensor requires multiplexing of nanopore measurements. Electrical detection, however, poses a challenge, as each nanopore circuit must be electrically independent, which requires complex nanofluidics and embedded electrodes. Here, we present an optical method for simultaneous measurements of the ionic current across an array of solid-state nanopores, requiring no additional fabrication steps. Proof-of-principle experiments are conducted that show simultaneous optical detection and characterization of ssDNA and dsDNA using an array of pores. Through a comparison with electrical measurements, we show that optical measurements are capable of accessing equivalent transmembrane current information