Platform approaches to mRNA analytical testing methods

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Site-specific methylation in Bacillus subtilis chemotaxis: effect of covalent modifications to the chemotaxis receptor McpB

The Bacillus subtilis chemotaxis pathway employs a receptor methylation system that functions differently from the one in the canonical Escherichia coli pathway. Previously, we hypothesized that B. subtilis employs a site-specific methylation system for adaptation where methyl groups are added and removed at different sites. This study investigated how covalent modifications to the adaptation region of the chemotaxis receptor McpB altered its apparent affinity for its cognate ligand, asparagine, and also its ability to activate the CheA kinase. This receptor has three closely spaced adaptation sites located at residues Gln371, Glu630 and Glu637. We found that amidation, a putative methylation mimic, of site 371 increased the receptor's apparent affinity for asparagine and its ability to activate the CheA kinase. Conversely, amidation of sites 630 and 637 reduced the receptor's ability to activate the kinase but did not affect the apparent affinity for asparagine, suggesting that activity and sensitivity are independently controlled in B. subtilis. We also examined how electrostatic interactions may underlie this behaviour, using homology models. These findings further our understanding of the site-specific methylation system in B. subtilis by demonstrating how the modification of specific sites can have varying effects on receptor function

Chemoperception of Specific Amino Acids Controls Phytopathogenicity in Pseudomonas syringae pv. tomato

IMPORTANCE There is substantive evidence that chemotaxis is a key requisite for efficient pathogenesis in plant pathogens. However, information regarding particular bacterial chemoreceptors and the specific plant signal that they sense is scarce. Our work shows that the phytopathogenic bacterium Pseudomonas syringae pv. tomato mediates not only chemotaxis but also the control of pathogenicity through the perception of the plant abundant amino acids Asp and Glu. We describe the specificity of the perception of L- and D-Asp and L-Glu by the PsPto-PscA chemoreceptor and the involvement of this perception in the regulation of pathogenicity-related traits. Moreover, a saturating concentration of D-Asp reduces bacterial virulence, and we therefore propose that ligand-mediated interference of key chemoreceptors may be an alternative strategy to control virulence.Supplemental material for this article may be found at https://doi.org/10.1128/mBio .01868-19.We acknowledge M. Trini Gallegos for kindly provide plasmid pCdrA::gfpS and S. Nebreda for technical assistance.Chemotaxis has been associated with the pathogenicity of bacteria in plants and was found to facilitate bacterial entry through stomata and wounds. However, knowledge regarding the plant signals involved in this process is scarce. We have addressed this issue using Pseudomonas syringae pv. tomato, which is a foliar pathogen that causes bacterial speck in tomato. We show that the chemoreceptor P. syringae pv. tomato PscA (PsPto-PscA) recognizes specifically and with high affinity L-Asp, L-Glu, and D-Asp. The mutation of the chemoreceptor gene largely reduced chemotaxis to these ligands but also altered cyclic di-GMP (c-di-GMP) levels, biofilm formation, and motility, pointing to cross talk between different chemosensory pathways. Furthermore, the PsPto-PscA mutant strain showed reduced virulence in tomato. Asp and Glu are the most abundant amino acids in plants and in particular in tomato apoplasts, and we hypothesize that this receptor may have evolved to specifically recognize these compounds to facilitate bacterial entry into the plant. Infection assays with the wild-type strain showed that the presence of saturating concentrations of D-Asp also reduced bacterial virulence.This work was supported by grants AGL2015-63851-R and RTI2018-095222-B100 (to E.L.-S.) and BIO2016-76779-P (to T.K.) from the Ministerio de Economía y Competitividad, Spain. J.P.C.-V. was supported by the FPI program (BES-2016-076452, MINECOSpain)

Logarithmic sensing in Bacillus subtilis aerotaxis

Aerotaxis, the directed migration along oxygen gradients, allows many microorganisms to locate favorable oxygen concentrations. Despite oxygen’s fundamental role for life, even key aspects of aerotaxis remain poorly understood. In Bacillus subtilis, for example, there is conflicting evidence of whether migration occurs to the maximal oxygen concentration available or to an optimal intermediate one, and how aerotaxis can be maintained over a broad range of conditions. Using precisely controlled oxygen gradients in a microfluidic device, spanning the full spectrum of conditions from quasi-anoxic to oxic (60 n mol/l–1 m mol/l), we resolved B. subtilis’ ‘oxygen preference conundrum’ by demonstrating consistent migration towards maximum oxygen concentrations (‘monotonic aerotaxis’). Surprisingly, the strength of aerotaxis was largely unchanged over three decades in oxygen concentration (131 n mol/l–196 μ mol/l). We discovered that in this range B. subtilis responds to the logarithm of the oxygen concentration gradient, a rescaling strategy called ‘log-sensing’ that affords organisms high sensitivity over a wide range of conditions. In these experiments, high-throughput single-cell imaging yielded the best signal-to-noise ratio of any microbial taxis study to date, enabling the robust identification of the first mathematical model for aerotaxis among a broad class of alternative models. The model passed the stringent test of predicting the transient aerotactic response despite being developed on steadystate data, and quantitatively captures both monotonic aerotaxis and log-sensing. Taken together, these results shed new light on the oxygen-seeking capabilities of B. subtilis and provide a blueprint for the quantitative investigation of the many other forms of microbial taxis

Second-step splicing factors and heavy metal stress management in Arabidopsis thaliana

Pre-mRNA splicing in plants is mechanistically the same as splicing in other eukaryotes, but its mode of intron recognition is unique. Whereas in animals a pyrimidine-tract contributes to the definition of exons, in plants, introns are defined by the boundaries of AU-rich intron and GC-rich exon. Some of these differences may depend on splicing factors mediating recognition of the unusual sequence-dependent transition points and/or factors mediating the first- and second-steps in intron definition and excision. The second transesterification step of splicing involves many protein factors (second-step splicing factors) that have not been previously characterized in Arabidopsis. Among these are numerous genes encoded by multicopy genes in this model plant: PRP16 (1 copy), PRP17 (2 copies), PRP18 (2 copies), PRP22 (3 copies) and SLU7 (3 copies). This work is aimed at defining the structural differences and expression patterns of these multiple second-step splicing factors and their involvements in heavy metal stress response. The first goal of this project was to determine unique characteristics of the multicopy second-step splicing factors and the tissue and developmental stages in which each of these genes is expressed. My studies have indicated that all of these second-step splicing factors genes are expressed in Arabidopsis. While most are constitutively expressed throughout each tissue type and developmental stage, a specialized subset including PRP17-2, PRP18B, PRP22-3 and SLU7-2 are primarily expressed in flower and silique tissue in four-week-old plants and throughout the entire seven-week-old plant. Each of these specialized genes has unique structural features when compared to their homologs, suggesting that they form unique spliceosomal networks. These include variations in sequence identity between each other and their homologs in other organisms. Additionally, homology modeling revealed specific sites in which changes in residues will likely contribute to their interactions with other proteins. For example, residues important for protein-protein interaction in ScPRP17 are unique on AtPRP17-2. Basic surface residues on ScPRP18 that contribute to interaction with ScSLU7 are in slightly different locations on both AtPRP18A and AtPRP18B and fewer in number on AtPRP18B. The second goal of this project was to determine the response of second-step splicing factors to heavy metal stress conditions (HgCl2, Hg(OAc)2, CdSO4, CuSO4 and ZnSO4). My studies have shown that plants subjected to increasing concentrations of Hg(OAc)2 and CdSO4 for three weeks from germination accumulate pre-mRNA transcripts of genes not subject to alternative splicing. This effect is observed for some of the second-step splicing factors and some genes that are involved in other cellular processes like plant defense and transcription regulation. In contrast, pre-mRNA transcripts of genes subject to alternative splicing, such as the Ser/Arg-rich (SR) proteins involved in intron recognition, accumulate varied proportions of alternatively spliced transcripts but not pre-mRNA transcripts. To determine whether translation-dependent nonsense-mediated decay (NMD) was involved in the accumulation of pre-mRNAs, the effects of chemically inactivating translation were examined. My studies have shown that the patterns of second-step splicing factor transcript accumulation observed when NMD is chemically knocked out most resemble the patterns of transcript accumulation when plants are treated with cadmium. The fact that these patterns do not match exactly suggests that while the metals may affect NMD, they do not abolish it in the same manner as chemically abolishing it by halting translation and other effects are independent of NMD. It was also determined that many of the second-step splicing factor expression levels are regulated by NMD under normal conditions and the loss of NMD causes an accumulation of these transcripts