Proteome-wide measurement of non-canonical bacterial mistranslation by quantitative mass spectrometry of protein modifications.

The genetic code is virtually universal in biology and was likely established before the advent of cellular life. The extent to which mistranslation occurs is poorly understood and presents a fundamental question in basic research and production of recombinant proteins. Here we used shotgun proteomics combined with unbiased protein modification analysis to quantitatively analyze in vivo mistranslation in an E. coli strain with a defect in the editing mechanism of leucyl-tRNA synthetase. We detected the misincorporation of a non-proteinogenic amino acid norvaline on 10% of all measured leucine residues under microaerobic conditions and revealed preferential deployment of a tRNA(Leu)(CAG) isoacceptor during norvaline misincorporation. The strain with the norvalylated proteome demonstrated a substantial reduction in cell fitness under both prolonged aerobic and microaerobic cultivation. Unlike norvaline, isoleucine did not substitute for leucine even under harsh error-prone conditions. Our study introduces shotgun proteomics as a powerful tool in quantitative analysis of mistranslation

Characterizing the Syphilis-Causing Treponema pallidum ssp. pallidum Proteome Using Complementary Mass Spectrometry

YesBackground. The spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection. Methodology/Principal Findings. To better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) and electrospray ionization (ESI-LTQ-Orbitrap) tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26) was found between previous microarray signal data and protein abundance. Conclusions. This is the most comprehensive description of the global T. pallidum proteome to date. These data provide valuable insights into in vivo T. pallidum protein expression, paving the way for improved understanding of the pathogenicity of this enigmatic organism.This work was supported by the grants from the Flanders Research Foundation, SOFI-B Grant to CRK, http://www.fwo.be/, a Public Health Service Grant from the National Institutes of Health to CEC, (grant # AI-051334), https://www.nih.gov/ and a grant from the Grant Agency of the Czech Republic to DS and MS (P302/12/0574, GP14-29596P), https:// gacr.cz/

The kinases HipA and HipA7 phosphorylate different substrate pools in Escherichia coli to promote multidrug tolerance

The bacterial serine-threonine protein kinase HipA promotes multidrug tolerance by phosphorylating the glutamate-tRNA ligase (GltX), leading to a halt in translation, inhibition of growth, and induction of a physiologically dormant state (persistence). The HipA variant HipA7 substantially increases persistence despite being less efficient at inhibiting cell growth. We postulated that this phenotypic difference was caused by differences in the substrates targeted by both kinases. We overproduced HipA and HipA7 in Escherichia coli and identified their endogenous substrates by SILAC-based quantitative phosphoproteomics. We confirmed that GltX was the main substrate of both kinase variants and likely the primary determinant of persistence. When HipA and HipA7 were moderately overproduced from plasmids, HipA7 targeted only GltX, but HipA phosphorylated several additional substrates involved in translation, transcription, and replication, such as ribosomal protein L11 (RplK) and the negative modulator of replication initiation, SeqA. HipA7 showed reduced kinase activity compared to HipA and targeted a substrate pool similar to that of HipA only when produced from a high–copy number plasmid. The kinase variants also differed in autophosphorylation, which was substantially reduced for HipA7. When produced endogenously from the chromosome, HipA showed no activity because of inhibition by the antitoxin HipB, whereas HipA7 phosphorylated GltX and phage shock protein PspA. Initial testing did not reveal a connection between HipA-induced phosphorylation of RplK and persistence or growth inhibition, suggesting that other HipA-specific substrates were likely responsible for growth inhibition. Our results contribute to the understanding of HipA7 action and present a resource for elucidating HipA-related persistence

Performance analysis of max-pressure control system for traffic network using macroscopic fundamental diagram

Traffic signal control is known to improve performance and to reduce congestion of urban networks in traffic management. As a control system, the traffic signal control regulates vehicles through traffic lights to produce traffic conditions with maximum output, queue lengths dispersion and less travel time. Max pressure control is a widely known approach in the traffic management for controlling signalized intersections networks by only requiring information of queues adjacent to the intersection and does not call for knowledge of mean demands. Beside the simplicity of the approach, the formulation requires turning ratio information related to movement of vehicles entering and leaving links of the intersections and phase saturation flow. In practice, real time measurement of turning ratios is subjected to inaccuracies caused by sensor network limitations. Therefore, it is of interest to carry out performance analysis of max pressure control application due to inaccuracies of turning ratio information. The paper considers the max-pressure control performance analysis by means of macroscopic fundamental diagram (MFD) approach. The performance analysis is conducted on a traffic model of Bandung city, Indonesia consisting of five active signalized intersections. In which, four intersections are active signalized intersections that have two signal phases, and one intersection is an active signalized intersection that has three signal phases. The simulation results show the max-pressure control algorithm performs good performance based on the evaluation of the MFD diagram related to the density value and the maximum vehicle flow value produced. In addition, based on travel time graph evaluation, the max-pressure control is more adaptive in handling changes in the demand rate which increases drastically. The performance analysis of simulation results using MFD with the proposed critical velocity approach indicates the max pressure control is insensitive to turning ratio variations to some extent

Almost squares in almost squares: solving the final instance

The "almost-squares in almost-squares" (Asqas) problem is a rectangle packing problem in which a series of almost-squares (rectangles of dimensions n by (n+1)) needs to be placed inside an almost-square frame without open areas or overlaps. Asqas-34, consisting of almost-squares 1 by 2, 2 by 3, ..., 34 by 35, remains unsolved. This paper shows Asqas-34 is the only remaining unsolved instance of Asqas, and describes several solutions to Asqas-34, and the methods used to find them

On the Mechanism and Origin of Isoleucyl-tRNA Synthetase Editing against Norvaline.

Aminoacyl-tRNA synthetases (aaRSs), the enzymes responsible for coupling tRNAs to their cognate amino acids, minimize translational errors by intrinsic hydrolytic editing. Here, we compared norvaline (Nva), a linear amino acid not coded for protein synthesis, to the proteinogenic, branched valine (Val) in their propensity to mistranslate isoleucine (Ile) in proteins. We show that in the synthetic site of isoleucyl-tRNA synthetase (IleRS), Nva and Val are activated and transferred to tRNA at similar rates. The efficiency of the synthetic site in pre-transfer editing of Nva and Val also appears to be similar. Post-transfer editing was, however, more rapid with Nva and consequently IleRS misaminoacylates Nva-tRNAIle at slower rate than Val-tRNAIle. Accordingly, an Escherichia coli strain lacking IleRS post-transfer editing misincorporated Nva and Val in the proteome to a similar extent and at the same Ile positions. However, Nva mistranslation inflicted higher toxicity than Val, in agreement with IleRS editing being optimized for hydrolysis of Nva-tRNAIle. Furthermore, we found that the evolutionary-related IleRS, leucyl- and valyl-tRNA synthetases (I/L/VRSs), all efficiently hydrolyze Nva-tRNAs even when editing of Nva seems redundant. We thus hypothesize that editing of Nva-tRNAs had already existed in the last common ancestor of I/L/VRSs, and that the editing domain of I/L/VRSs had primarily evolved to prevent infiltration of Nva into modern proteins