Proteins In Bacterial Cells Research Articles

Synergy and Mode of Action of Ceftazidime plus Quercetin or Luteolin on Streptococcus pyogenes.

Streptococcus pyogenes causes streptococcal toxic shock syndrome. The recommended therapy has been often failure through the interfering of beta-lactamase-producing bacteria (BLPB). The present study was to investigate antibacterial activity, synergy, and modes of action of luteolin and quercetin using alone and plus ceftazidime against S. pyogenes. The MICs of ceftazidime, luteolin, and quercetin against all S. pyogenes were 0.50, 128, and 128 µg mL−1, respectively. A synergistic effect was exhibited on luteolin and quercetin plus ceftazidime against these strains at fractional inhibitory concentration indices 0.37 and 0.27, respectively, and was confirmed by the viable count. These combinations increased cytoplasmic membrane (CM) permeability, caused irregular cell shape, peptidoglycan, and CM damage, and decreased nucleic acid but increased proteins in bacterial cells. Enzyme assay demonstrated that these flavonoids had an inhibitory activity against β-lactamase. In summary, this study provides evidence that the inhibitory mode of action of luteolin and quercetin may be mediated via three mechanisms: (1) inhibiting of peptidoglycan synthesis, (2) increasing CM permeability, and (3) decreasing nucleic acid but increasing the protein contents of bacterial cells. So, luteolin and quercetin propose the high potential to develop adjunct to ceftazidime for the treatment of coexistence of the BLPB and S. pyogenes infections.

Endophytic Bacillus subtilis strain E1R-J is a promising biocontrol agent for wheat powdery mildew.

In this study, the biocontrol efficacies of 14 endophytic bacterial strains were tested against Blumeria graminis f. sp. tritici (Bgt) in pot experiments under greenhouse conditions. Bacillus subtilis strain E1R-j significantly reduced disease index and exhibited the best control (90.97%). When different formulations of E1R-j were sprayed 24 h before Bgt inoculation, fermentation liquid without bacterial cell and crude protein suspension displayed the similar effects; and they reduced disease index more than bacterial cell suspension (109 cfu mL−1) and fermentation liquid without protein. The control effects were not significantly different between 1011 and 109 cfu mL−1 of bacterial cell suspension but were higher than 107 cfu mL−1. Further observations showed that conidial germination and appressorial formation of Bgt were retarded by spraying E1R-j 24 h before Bgt inoculation. Compared with the water check, conidial germination and appressorial formation were decreased by 43.3% and 42.7%, respectively. In the treatment with E1R-j, the number of houstoria significantly reduced and the speed of mycelial extension was slowed down in the wheat leaves. Scanning electron microscopy observation revealed that E1R-j significantly suppressed the conidial germination and caused rupture and deformation of germ tubes. On the surface of wheat leaves, mycelia and conidiophores became shrinking.

Abstract 668: Folate conjugated site-specifically to anti-human-CD3-Fab is efficacious in mouse models of ovarian cancer

Abstract Ovarian cancer affects ∼22,000 women in the US each year. Folate receptor (FOLR1) is overexpressed in a high proportion of ovarian tumors and is generally associated with advanced stage disease. We report a first-in-class bi-specific conjugate for ovarian cancer by using RECODE™ technology that site-specifically incorporates novel amino acids into proteins in bacterial cells. The Ambrx bi-specific conjugate (Fol-aCD3) is developed for epithelial ovarian cancer and consists of (i) Folate that can bind to FOLR1 receptor on ovarian cancer cells and (ii) Anti-human CD3ε Fab that can bind to the CD3ε receptor on cytotoxic T cells. The potential engagement of T cells by Fol-aCD3 provides for T cell recruitment especially in an immune evasive, suppressive and tolerogenic tumor microenvironment. Para-acetyl phenylalanine (pAcF) containing anti-CD3ε Fab is conjugated to hydroxyl-amine-folate drug-linker. The resulting compound was analyzed by LC-MS with >95% conjugation of a single folate per Fab and >95% main peak by size-exclusion chromatography. By an in vitro LDH cytotoxicity assay using activated human T-cells, Fol-aCD3 killed SKOV-3 and Ov-90 cells with an EC50 of 7.4 and 4.4 ng/mL, respectively. The pharmacokinetic half-life in CD1 mice is 20 and 60 minutes for IV and IP administration, respectively. Fol-aCD3 delivered IV at 5, 0.5 or 0.05 mg/kg daily for 5 days inhibited tumor growth in a dose-dependent manner for Ov-90 co-implanted subcutaneously with activated T-cells in NOD-SCID mice on a folate deficient diet. There was no body weight loss (BWL). Fol-aCD3 was tested in a peritoneal dissemination mouse model of ovarian cancer where the cancer cells, Fol-aCD3, and activated T-cells were injected IP. At a dose of 1 mg/kg of Fol-aCD3 delivered every 3rd day simultaneously with activated T-cells, the NOD-SCID mice exhibited transient BWL but recovered by the 4th day. The treated mice experienced 30% and 50% increase in life span (ILS) for SKOV-3 and OV-90, respectively. The increased survival was seen for mice treated immediately upon tumor cell inoculation (prevention mode) as well as for mice treated 17 days after inoculation (treatment mode). Thus, Fol-aCD3 is being considered for development as a therapeutic for ovarian cancer.. Citation Format: Sandra L. Biroc, Marco Gymnopoulos, Shailaja Srinagesh, Brad Hayes, Nick Knudsen, Anthony Manibusan, Jason Pinkstaff, Tim Buss, Kari Cox, Robin Marsden, Lillian Skidmore, Jinming Xia, Ying Sun, Ning Zou, Tsotne Javahishvili. Folate conjugated site-specifically to anti-human-CD3-Fab is efficacious in mouse models of ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 668. doi:10.1158/1538-7445.AM2014-668

Interaction of different Shiga toxin-producing Escherichia coli serotypes with Caco-2 cells.

Shiga toxin-producing Escherichia coli (STEC) is defined by the ability to produce one or more types of Shiga toxins. In an attempt to better understand the mechanisms that underlie pathogenicity among STEC foodborne infection, we compared different STEC serotypes recovered from food sources (O26:H11, O103:H2, and O157:H7) for their interaction with human intestinal epithelial cells using the Caco-2 cell line as an infection model. Bacterial uptake was determined using gentamicin protection assay and results were confirmed by fluorescent microscopy. Our results revealed no significant difference in adherence among tested serotypes. Nonetheless, E. coli O157:H7 exhibited a significant increase of internalization ability and survived significantly better over different time points for up to 24 h. To study cellular invasion mechanisms, multiple inhibitors with known effects on eukaryotic cell structures and processes were used. Inhibition of bacterial and host cell protein synthesis significantly diminished entry in all tested serotypes, suggesting that invasion is an active process that requires both bacterial and eukaryotic protein syntheses. Cytochalasin D (an actin microfilament inhibitor) and staurosporine (inhibitor of several protein kinases) significantly decreased internalization of all tested serotypes. Our results suggested that invasion by STEC varies between different serotypes, might correlate with clinical outcome, and is receptor mediated. This process is dependent on microfilament-dependent pathway and phosphorylation of cell proteins but not on formation of microtubules.

Avoiding acidic region streaking in two-dimensional gel electrophoresis: case study with two bacterial whole cell protein extracts.

Acidic region streaking (ARS) is one of the lacunae in two-dimensional gel electrophoresis (2DE) of bacterial proteome. This streaking is primarily caused by nucleic acid (NuA) contamination and poses major problem in the downstream processes like image analysis and protein identification. Although cleanup and nuclease digestion are practiced as remedial options, these strategies may incur loss in protein recovery and perform incomplete removal of NuA. As a result, ARS has remained a common observation across publications, including the recent ones. In this work, we demonstrate how ultrasound wave can be used to shear NuA in plain ice-cooled water, facilitating the elimination of ARS in the 2DE gels without the need for any additional sample cleanup tasks. In combination with a suitable buffer recipe, IEF program and frequent paper-wick changing approach, we are able to reproducibly demonstrate the production of clean 2DE gels with improved protein recovery and negligible or no ARS. We illustrate our procedure using whole cell protein extracts from two diverse organisms, Escherichia coli and Mycobacterium smegmatis. Our designed protocols are straightforward and expected to provide good 2DE gels without ARS, with comparable times and significantly lower cost.

Genetic Encoding of Caged Cysteine and Caged Homocysteine in Bacterial and Mammalian Cells

We report the genetic incorporation of caged cysteine and caged homocysteine into proteins in bacterial and mammalian cells. The genetic code of these cells was expanded with an engineered pyrrolysine tRNA/tRNA synthetase pair that accepts both light-activatable amino acids as substrates. Incorporation was validated by reporter assays, western blots, and mass spectrometry, and differences in incorporation efficiency were explained by molecular modeling of synthetase-amino acid interactions. As a proof-of-principle application, the genetic replacement of an active-site cysteine residue with a caged cysteine residue in Renilla luciferase led to a complete loss of enzyme activity; however, upon brief exposure to UV light, a >150-fold increase in enzymatic activity was observed, thus showcasing the applicability of the caged cysteine in live human cells. A simultaneously conducted genetic replacement with homocysteine yielded an enzyme with greatly reduced activity, thereby demonstrating the precise probing of a protein active site. These discoveries provide a new tool for the optochemical control of protein function in mammalian cells and expand the set of genetically encoded unnatural amino acids.

Hydrogen peroxide staining to visualize intracellular bacterial infections of seedling root cells.

Visualization of bacteria in living plant cells and tissues is often problematic due to lack of stains that pass through living plant cell membranes and selectively stain bacterial cells. In this article, we report the use of 3,3'-diaminobenzidine tetrachloride (DAB) to stain hydrogen peroxide associated with bacterial invasion of eukaryotic cells. Tissues were counterstained with aniline blue/lactophenol to stain protein in bacterial cells. Using this staining method to visualize intracellular bacterial (Burkholderia gladioli) colonization of seedling roots of switch grass (Panicum virgatum), we compared bacterial free seedling roots and those inoculated with the bacterium. To further assess application of the technique in multiple species of vascular plants, we examined vascular plants for seedling root colonization by naturally occurring seed-transmitted bacteria. Colonization by bacteria was only observed to occur within epidermal (including root hairs) and cortical cells of root tissues, suggesting that bacteria may not be penetrating deeply into root tissues. DAB/peroxidase with counter stain aniline blue/lactophenol was effective in penetration of root cells to selectively stain bacteria. Furthermore, this stain combination permitted the visualization of the bacterial lysis process. Before any evidence of H2 O2 staining, intracellular bacteria were seen to stain blue for protein content with aniline blue/lactophenol. After H2 O2 staining became evident, bacteria were often swollen, without internal staining by aniline blue/lactophenol; this suggests loss of protein content. This staining method was effective for seedling root tissues; however, it was not effective at staining bacteria in shoot tissues due to poor penetration.

A tailored galK counterselection system for efficient markerless gene deletion and chromosomal tagging in Magnetospirillum gryphiswaldense.

Magnetotactic bacteria have emerged as excellent model systems to study bacterial cell biology, biomineralization, vesicle formation, and protein targeting because of their ability to synthesize single-domain magnetite crystals within unique organelles (magnetosomes). However, only few species are amenable to genetic manipulation, and the limited methods for site-specific mutagenesis are tedious and time-consuming. Here, we report the adaptation and application of a fast and convenient technique for markerless chromosomal manipulation of Magnetospirillum gryphiswaldense using a single antibiotic resistance cassette and galK-based counterselection for marker recycling. We demonstrate the potential of this technique by genomic excision of the phbCAB operon, encoding enzymes for polyhydroxyalkanoate (PHA) synthesis, followed by chromosomal fusion of magnetosome-associated proteins to fluorescent proteins. Because of the absence of interfering PHA particles, these engineered strains are particularly suitable for microscopic analyses of cell biology and magnetosome biosynthesis.

Characterization of the activity and biological function of human protein arginine methyltransferase 9 (555.6)

Protein arginine methyltransferases (PRMTs) make up a family of enzymes that transfer methyl groups onto arginine residues and play important roles in DNA repair, splicing, transcriptional control and signaling. In mammals, a family of nine PRMTs are known. PRMT1,2,3,4,6, and 8 catalyze the formation of asymmetric dimethylarginine (ADMA) and ω‐monomethylarginine (MMA), PRMT5 catalyzes symmetric dimethylarginine (SDMA) and MMA formation, and PRMT7 catalyzes only MMA formation. The last member of this family, PRMT9 (also designated PRMT10), has not previously been characterized. In humans, it is encoded on a gene on chromosome 4 in position 4q31. We have now expressed human PRMT9 both as a GST‐fusion protein in bacterial cells and as a GFP‐fusion protein in human HEK293 cells. For both expression systems, we have also prepared catalytic mutants to serve as controls for possible contamination of other types of PRMTs. Using an in vitro methylation assay, we show that both forms of PRMT9 catalyze the methylation of myelin basic protein and a GST‐fusion protein of the N‐terminal domain of human fibrillarin. Studies are in progress to characterize the type of activity and physiological substrates for PRMT9.Grant Funding Source: Supported by Ruth L. Kirschstein National Research Service Award GM007185

Second Verse, Same as the First? Structures of Thioredoxin Proteins TrxA and TrxC from Mycobacterium tuberculosis (LB103)

Mycobacterium tuberculosis, the causative agent for tuberculosis (TB), infected 8.6 million and killed 1.3 million people in 2012 (WHO). TB is most prevalent in countries with a high incidence of infectious diseases, such as HIV, due to weakened immune systems. TB mainly affects the lungs, but can also affect other systems. When a host organism is infected wtih TB, the Mycobacteria in the lungs multiply, often resulting in pneumonia, chest pain, and prolonged coughing. In response, host macrophages, a part of the natural immune system, engulf the Mycobacteria and oxidize bacterial cell proteins in an attempt to destroy it. To protect itself against this attack, the bacterial thioreductase system, consisting of the redox protein thioredoxin reductase (TrxR) and the thioredoxin proteins TrxA, TrxB, and TrxC, gives electrons back to the oxidized proteins. As this system works to maintain cellular redox homeostasis, finding ways to stop it might provide a new method for treating people with TB. TrxA and TrxC have similar structures, thus it can be hypothesized that their functions are similar. Comparing binding sites between the proteins can provide insight if TrxA can react with TrxR similarly to TrxC. By modeling TrxA and TrxC with 3D printing technology, the Messmer SMART Team (Students Modeling A Research Topic) can compare the structures of the thioredoxins which may lead to new strategies for curing or preventing TB.Grant Funding Source: Supported by a grant from NIH‐CTSA.

On-Chip Analysis of Intermittent Molecular Encounters in Nuclease Digestion of Specific DNA Sequence

DNA-binding proteins such as transcription factors react with their targets at faster than estimation from diffusion-controlled rates. This is explained by the fact that the protein can slide along DNA molecule with appreciable affinity for nonspecific sequences. However, does this sliding function really play a key role in this type of reaction kinetics? Most of the previous methods are optimized for one-dimensional protein diffusion along DNA maintained in an extended configuration. Here we show that a site-specific nuclease digestion efficiently occurs in the course of an intermittent encounter by the same single DNA molecule to a number of nucleolytic enzyme molecules. We found in a limited protein's sliding-free condition that the intermittent molecular encounter and complex formation process gives an enhanced rate for the reaction. Our results demonstrate how the flowing DNA encounters the immobilized restriction enzyme ApaI by which the site-specific DNA-break occurs. Moving tags of both DNA ends transmitted the breakup of single DNA molecule into fragments. Quite a number of digestions occurred at the first encounter (62%), but the remaining 38% of digestion occurred in the course of the subsequent intermittent encounters. The complex formation was maintained after the DNA break. This is suggestive of an intrinsic behaviour in which the DNA and protein molecules are continuously held together by switching their binding positions with short-range interactions. It also provides a clue to understanding an efficient and effective reaction by the DNA-binding proteins in bacterial cells.

Identification of FkpA as a Key Quality Control Factor for the Biogenesis of Outer Membrane Proteins under Heat Shock Conditions

The outer membrane proteins (OMPs) of Gram-negative bacterial cells, as well as the mitochondrion and chloroplast organelles, possess unique and highly stable β-barrel structures. Biogenesis of OMPs in Escherichia coli involves such periplasmic chaperones as SurA and Skp. In this study, we found that the ΔsurA Δskp double-deletion strain of E. coli, although lethal and defective in the biogenesis of OMPs at the normal growth temperature, is viable and effective at the heat shock temperature. We identified FkpA as the multicopy suppressor for the lethal phenotype of the ΔsurA Δskp strain. We also demonstrated that the deletion of fkpA from the ΔsurA cells resulted in only a mild decrease in the levels of folded OMPs at the normal temperature but a severe decrease as well as lethality at the heat shock temperature, whereas the deletion of fkpA from the Δskp cells had no detectable effect on OMP biogenesis at either temperature. These results strongly suggest a functional redundancy between FkpA and SurA for OMP biogenesis under heat shock stress conditions. Mechanistically, we found that FkpA becomes a more efficient chaperone for OMPs under the heat shock condition, with increases in both binding rate and affinity. In light of these observations and earlier reports, we propose a temperature-responsive OMP biogenesis mechanism in which the degrees of functional importance of the three chaperones are such that SurA > Skp > FkpA at the normal temperature but FkpA ≥ SurA > Skp at the heat shock temperature.

Semi-quantitative colony immunoassay for determining and optimizing protein expression in Saccharomyces cerevisiae and Escherichia coli

This work describes a quick semi-quantitative colony immunoassay (QSCI) method for immunoblot detection of intracellularly expressed proteins in both yeast and bacterial cells. After induction of protein expression, only 4.5h is required for cell breakage, protein detection, and data analysis. This protocol was used to screen and unambiguously identify Saccharomyces cerevisiae cells efficiently overexpressing glutathione S-transferase (GST)-tagged Yih1 in addition to cells expressing the myc-tagged large 297-kDa Gcn1 protein. In addition, the method was used to identify Escherichia coli cells efficiently expressing His6-tagged Yih1 and a GST-tagged Gcn1 fragment, respectively. The protocol allows the use of both epitope-specific and protein-specific antibodies. The same colony immunoassay can also be used to determine the minimal concentration of inducing agent sufficient for induction of optimal protein expression (e.g., galactose for yeast, isopropyl β-d-1-thiogalactopyranoside [IPTG] for E. coli). To our knowledge, this is the first report on a rapid low-cost procedure that allows the calibration of inducing agent on solid medium.

Biochemical Characterization of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate ligase (MurE) from Verrucomicrobium spinosum DSM 4136T

Verrucomicrobium spinosum is a Gram-negative bacterium that is related to bacteria from the genus Chlamydia. The bacterium is pathogenic towards Drosophila melanogaster and Caenorhabditis elegans, using a type III secretion system to facilitate pathogenicity. V. spinosum employs the recently discovered l,l-diaminopimelate aminotransferase biosynthetic pathway to generate the bacterial cell wall and protein precursors diaminopimelate and lysine. A survey of the V. spinosum genome provides evidence that the bacterium should be able to synthesize peptidoglycan de novo, since all of the necessary genes are present. The enzyme UDP-N-acetylmuramoyl-l-alanyl-d-glutamate: meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.15) catalyzes a reaction in the cytoplasmic step of peptidoglycan biosynthesis by adding the third amino acid residue to the peptide stem. The murE ortholog from V. spinosum (murE Vs) was cloned and was shown to possess UDP-MurNAc-l-Ala-d-Glu:meso-2,6-diaminopimelate ligase activity in vivo using functional complementation. In vitro analysis using the purified recombinant enzyme demonstrated that MurEVs has a pH optimum of 9.6 and a magnesium optimum of 30 mM. meso-Diaminopimelate was the preferred substrate with a K m of 17 µM, when compared to other substrates that are structurally related. Sequence alignment and structural analysis using homology modeling suggest that key residues that make up the active site of the enzyme are conserved in MurEVs. Our kinetic analysis and structural model of MurEVs is consistent with other MurE enzymes from Gram-negative bacteria that have been characterized. To verify that V. spinosum incorporates diaminopimelate into its cell wall, we purified peptidoglycan from a V. spinosum culture; analysis revealed the presence of diaminopimelate, consistent with that of a bona fide peptidoglycan from Gram-negative bacteria.

Non-Equilibrium Polar Localization of Proteins in Bacterial Cells

Many proteins are observed to localize to the poles within bacterial cells. Some bacteria show unipolar localization, yet under different conditions bipolar patterns can emerge. One mechanism for spontaneous polar localization has been shown to involve the combination of protein aggregation and nucleoid occlusion. Whether the different observed patterns represent global energy minima for the cellular system remains to be determined. In this paper we show that for a model consisting only of protein aggregation along with an excluded volume effect due to the DNA polymer, that unipolar patterns are the global energy ground state regardless of protein concentration and DNA density. We extend the model to allow for proteins to be added to the cellular volume at a constant rate and show that bipolar (or multi-foci) patterns emerge as the result of the system being kinetically trapped in a local energy minimum. Lastly we also consider the situation of a growing cell that starts with a pre-existing aggregate at one of the poles and determine conditions under which either unipolar or bipolar patterns can exist at the point when it is ready to divide. This work sheds new interpretations on recently published experimental data and suggests experiments to test whether such a mechanism can drive patterning in bacteria.

Surface proteins of bacteria of the genus Bifidobacterium

Beneficial effects due to the presence of probiotic bacteria of the genus Bifidobacterium in the human intestinal tract are still an interesting object of study. So far activities have been confirmed of bifidobacteria in stimulation of the host immune system, stimulation of tumor cell apoptosis, improvement of bowel motility, alleviation of symptoms of lactose intolerance, cholesterol lowering capacity, prevention and treatment of diarrhea and irritable bowel syndrome, alleviation of allergy or atopic dermatitis, maintenance of homeostasis of the intestine, and stimulation of the development of normal intestinal microflora in infants. A multitude of therapeutic properties encourages researchers to investigate the possibility of using the potential of Bifidobacterium in the prevention and treatment of other conditions such as rheumatoid arthritis and depression. Although it is known that the beneficial effects are due to intestinal mucosal colonization by these bacteria, the cell components responsible for the colonization are still not determined. In addition to the beneficial effects of probiotic administration, there were also negative effects including sepsis. Therefore research has been directed to identify specific components of Bifidobacterium responsible for probiotic effects. Currently researchers are focused on identifying, isolating and evaluating the properties of surface proteins that are probably involved in the adhesion of bacterial cells to the intestinal epithelium, improving colonization. This paper is an overview of current knowledge on Bifidobacterium surface proteins. The ways of transport and anchoring proteins in Gram-positive bacterial cells, the assembly of cell wall, and a description of the genus Bifidobacterium are presented.

Modelling Protein Oscillations in Myxococcus xanthus

Spatio-temporal oscillations of proteins in bacterial cells play an importantrole in fundamental biological processes. Motility of the rod-shaped bacterium Myxococcus xanthus is due to two motility systems: an A-motilitysystem and a type-4 pili system [1]. Both motility systems depend on the correct localisation of regulatory proteins at the cell poles which set up apolarity (front-to-back) axis. The oscillatory motion of the individual cel lresults from dynamic inversion of the polarity axis due to a spatio-temporal oscillation of the regulatory proteins between the cell poles. A mathematical framework for a minimal macroscopic model is presented which produces self-sustained oscillations of the protein concentrations. The mathematicalmodel is based on a reaction-diusion system and is independent of external triggers. Necessary conditions on the reaction terms leading to oscillating solutions are derived theoretically. Several possible cases are studied based on dierent rates of interaction between the regulatory proteins. The interaction laws are then chosen according to mathematical analysis to produce dierent spatio-temporal oscillation patterns [2]. The dierent scenarios are numerically tested for robustness against parameter variation. Finally,possible extensions of the model will be addressed.This is joint work with B. A. Schmitt, S. Dahlke, P. Lenz, L. Sgaard-Andersen. This work has been supported by the Centre for Synthetic Microbiologyin Marburg, promoted by the LOEWE Excellence Program ofthe state of Hessen, Germany References [1] P. Lenz, L. Sgaard-Andersen, Temporal and spatial oscillations in bac-teria, Nat. Rev. Microbiol. 9 565{577, 2011. [2] P. Rashkov, B.A. Schmitt, L. Sgaard-Andersen, P. Lenz, S. Dahlke, Amodel of oscillatory protein dynamics in bacteria, Bull. Math. Biol. 742183{2203, 2012.

A Genetically Encoded AND Gate for Cell-Targeted Metabolic Labeling of Proteins

We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNA(Met). Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide-alkyne cycloaddition. Protein labeling is apparent within 5 min after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals.

Immunolabeling of Proteins in situ in Escherichia coli K12 Strains

This protocol was developed to label proteins in bacterial cells with antibodies conjugated to a fluorophore for fluorescence microscopy imaging. The procedure is optimized to minimize morphological changes and also to minimize the amount of antibodies needed for the staining. The protocol can also be used with primary antibodies conjugated to a fluorophore. The method has been verified extensively (van der Ploeg et al., 2013), but it should be noted that one case in Caulobacter crescentus (Hocking et al., 2012) has been reported in which the localization of a protein changed upon fixation by formaldehyde/glutaraldehyde. However, the localization of the same protein in E. coli did not change.

Strategies and molecular tools to fight antimicrobial resistance: resistome, transcriptome, and antimicrobial peptides.

The increasing number of antibiotic resistant bacteria motivates prospective research toward discovery of new antimicrobial active substances. There are, however, controversies concerning the cost-effectiveness of such research with regards to the description of new substances with novel cellular interactions, or description of new uses of existing substances to overcome resistance. Although examination of bacteria isolated from remote locations with limited exposure to humans has revealed an absence of antibiotic resistance genes, it is accepted that these genes were both abundant and diverse in ancient living organisms, as detected in DNA recovered from Pleistocene deposits (30,000 years ago). Indeed, even before the first clinical use of antibiotics more than 60 years ago, resistant organisms had been isolated. Bacteria can exhibit different strategies for resistance against antibiotics. New genetic information may lead to the modification of protein structure affecting the antibiotic carriage into the cell, enzymatic inactivation of drugs, or even modification of cellular structure interfering in the drug-bacteria interaction. There are still plenty of new genes out there in the environment that can be appropriated by putative pathogenic bacteria to resist antimicrobial agents. On the other hand, there are several natural compounds with antibiotic activity that may be used to oppose them. Antimicrobial peptides (AMPs) are molecules which are wide-spread in all forms of life, from multi-cellular organisms to bacterial cells used to interfere with microbial growth. Several AMPs have been shown to be effective against multi-drug resistant bacteria and have low propensity to resistance development, probably due to their unique mode of action, different from well-known antimicrobial drugs. These substances may interact in different ways with bacterial cell membrane, protein synthesis, protein modulation, and protein folding. The analysis of bacterial transcriptome may contribute to the understanding of microbial strategies under different environmental stresses and allows the understanding of their interaction with novel AMPs.