Search

Lineage-specific expansion (LSE) of protein families is a widespread phenomenon in many eukaryotic genomes, but is generally more limited in bacterial genomes. Here, we report the presence of 434 genes encoding solute-binding proteins (SBPs) from the tripartite tricarboxylate transporter (TTT) family, within the 8.2 Mb genome of the α-proteobacterium Rhodoplanes sp. Z2-YC6860, a gene family over-representation of unprecedented abundance in prokaryotes. Representing over 6 % of the total number of coding sequences, the SBP genes are distributed across the whole genome but are found rarely in low-GC islands, where the gene density for this family is much lower. This observation, and the much higher sequence identity between the 434 Rhodoplanes TTT SBPs compared with the average identity between homologues from different species, is indicative of a key role for LSE in the expansion. The TTT SBP genes were found in the vicinity of genes encoding membrane components of transport systems from different families, as well as regulatory proteins such as histidine-kinases and transcription factors, indicating a broad range of functions around the sensing, response and transport of organic compounds. A smaller expansion of TTT SBPs is known in some species of the β-proteobacteria Bordetella and we observed similar expansions in other β-proteobacterial lineages, including members of the genus Comamonas and the industrial biotechnology organism Cupriavidus necator, indicating that strong environmental selection can drive SBP duplication and specialisation from multiple evolutionary starting points.

The pathogen Vibrio cholerae is the causative agent of cholera. Emergence of antibiotic-resistant V. cholerae strains is increasing, but the underlying mechanisms remain unclear. Herein, we report that the stringent response regulator and stress alarmone guanosine tetra- and pentaphosphate ((p)ppGpp) significantly contributes to antibiotic tolerance in V. cholerae We found that N16961, a pandemic V. cholerae strain, and its isogenic (p)ppGpp-overexpressing mutant Δ relA Δ spoT are both more antibiotic-resistant than (p)ppGpp 0 (Δ relA Δ relV Δ spoT ) and Δ dksA mutants, which cannot produce or utilize (p)ppGpp, respectively. We also found that additional disruption of the aconitase B-encoding and tricarboxylic acid (TCA) cycle gene acnB in the (p)ppGpp 0 mutant increases its antibiotic tolerance. Moreover, expression of TCA cycle genes, including acnB , was increased in (p)ppGpp 0 , but not in the antibiotic-resistant Δ relA Δ spoT mutant, suggesting that (p)ppGpp suppresses TCA cycle activity, thereby entailing antibiotic resistance. Importantly, when grown anaerobically or incubated with an iron chelator, the (p)ppGpp 0 mutant became antibiotic-tolerant, suggesting that reactive oxygen species (ROS) are involved in antibiotic-mediated bacterial killing. Consistent with that hypothesis, tetracycline treatment markedly increased ROS production in the antibiotic-susceptible mutants. Interestingly, expression of the Fe(III) ABC transporter substrate-binding protein FbpA was increased 10-fold in (p)ppGpp 0 , and fbpA gene deletion restored viability of tetracycline-exposed (p)ppGpp 0 cells. Of note, FbpA expression was repressed in the (p)ppGpp-accumulating mutant, resulting in a reduction of intracellular free iron, required for the ROS-generating Fenton reaction. Our results indicate that (p)ppGpp-mediated suppression of central metabolism and iron uptake reduces antibiotic-induced oxidative stress in V. cholerae ., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Genome engineering has become a powerful tool for creating useful strains in research and industry. In this study, we applied singleplex and multiplex genome engineering approaches to construct an E. coli strain for the production of L-DOPA from glucose. We first used the singleplex genome engineering approach to create an L-DOPA-producing strain, E. coli DOPA-1, by deleting transcriptional regulators (tyrosine repressor tyrR and carbon storage regulator A csrA), altering glucose transport from the phosphotransferase system (PTS) to ATP-dependent uptake and the phosphorylation system overexpressing galactose permease gene (galP) and glucokinase gene (glk), knocking out glucose-6-phosphate dehydrogenase gene (zwf) and prephenate dehydratase and its leader peptide genes (pheLA) and integrating the fusion protein chimera of the downstream pathway of chorismate. Then, multiplex automated genome engineering (MAGE) based on 23 targets was used to further improve L-DOPA production. The resulting strain, E. coli DOPA-30N, produced 8.67 g/L of L-DOPA in 60 h in a 5 L fed-batch fermentation. This titer is the highest achieved in metabolically engineered E. coli having PHAH activity from glucose.

GluB is a substrate-binding protein (SBP) which participates in the uptake of glutamic acid in Corynebacterium glutamicum, a Gram-positive bacterium. It is part of an ATP-binding cassette (ABC) transporter system. Together with the transmembrane proteins GluC and GluD and the cytoplasmic protein GluA, which couples the hydrolysis of ATP to the translocation of glutamate, they form a highly active glutamate-uptake system. As part of efforts to study the amino-acid metabolism, especially the metabolism of glutamic acid by C. glutamicum, a bacterium that is widely used in the industrial production of glutamic acid, the GluB protein was expressed, purified and crystallized, an X-ray diffraction data set was collected to a resolution of 1.9 Å and preliminary crystallographic analysis was performed. The crystal belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 82.50, c = 72.69 Å.

The protein-gene pairs ArgP-argO of Escherichia coli and LysG-lysE of Corynebacterium glutamicum are orthologous, with the first member of each pair being a LysR-type transcriptional regulator and the second its target gene encoding a basic amino acid exporter. Whereas LysE is an exporter of arginine (Arg) and lysine (Lys) whose expression is induced by Arg, Lys, or histidine (His), ArgO exports Arg alone, and its expression is activated by Arg but not Lys or His. We have now reconstituted in E. coli the activation of lysE by LysG in the presence of its coeffectors and have shown that neither ArgP nor LysG can regulate expression of the noncognate orthologous target. Of several ArgP-dominant (ArgP(d)) variants that confer elevated Arg-independent argO expression, some (ArgP(d)-P274S, -S94L, and, to a lesser extent, -P108S) activated lysE expression in E. coli. However, the individual activating effects of LysG and ArgP(d) on lysE were mutually extinguished when both proteins were coexpressed in Arg- or His-supplemented cultures. In comparison with native ArgP, the active ArgP(d) variants exhibited higher affinity of binding to the lysE regulatory region and less DNA bending at both argO and lysE. We conclude that the transcription factor LysG from a Gram-positive bacterium, C. glutamicum, is able to engage appropriately with the RNA polymerase from a Gram-negative bacterium, E. coli, for activation of its cognate target lysE in vivo and that single-amino-acid-substitution variants of ArgP can also activate the distantly orthologous target lysE, but by a subtly different mechanism that renders them noninterchangeable with LysG.

Protein production through dedicated secretion systems might offer an potential alternative to the conventional cytoplasmical expression. The application of Type 1 secretion systems of Gram-negative bacteria, however, where often not successful in the past for a wide range of proteins. Recently, two studies using the E. coli maltose binding protein (MalE) and the rat intestinal fatty acid binding protein (IFABP) revealed a rational to circumvent these limitations. Here, wild-type passenger proteins were not secreted, while folding mutants with decreased folding kinetics were efficiently exported to the extracellular space. Subsequently, an one-step purification protocol yielded homogeneous and active protein. Taken together, theses two studies suggest that the introduction of slow-folding mutations into a protein sequence might be the key to use Type 1 secretion systems for the biotechnological production of proteins.

p19 RNA binding protein from the Carnation Italian ringspot virus (CIRV) is an RNA-silencing suppressor that binds small interfering RNA (siRNA) with high affinity. We created a bifunctional p19 fusion protein with an N-terminal maltose binding protein (MBP), for protein purification, and a C-terminal chitin binding domain (CBD) to bind p19 to chitin magnetic beads. The fusion protein binds dsRNAs in the size range of 20-23 nucleotides, but does not bind ssRNA or dsDNA. Relative affinities of the p19 fusion protein for different-length RNA and DNA substrates were determined. Binding specificity of the p19 fusion protein for small dsRNA allows detection of miRNA:RNA probe duplexes. Using radioactive RNA probes, we were able to detect low levels of miRNAs in the sub-femtomole range and in the presence of a million-fold excess of total RNA. Detection is linear over three logs. Unlike most nucleic acid detection methods, p19 selects for RNA hybrids of correct length and structure. Rules for designing optimal RNA probes for p19 detection of miRNAs were determined by in vitro binding of 18 different dsRNA oligos to p19. These studies demonstrate the potential of p19 fusion protein to detect miRNAs and isolate endogenous siRNAs.

Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier-driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail.

The use of Lactococcus lactis to deliver a chosen antigen to the mucosal surface has been shown to elicit an immune response in mice and is a possible method of vaccination in humans. The recent discovery on Gram-positive bacteria of pili that are covalently attached to the bacterial surface and the elucidation of the residues linking the major and minor subunits of such pili suggests that the presentation of an antigen on the tip of pili external to the surface of L. lactis might constitute a successful vaccine strategy. As a proof of principle, we have fused a foreign protein (the Escherichia coli maltose-binding protein) to the C-terminal region of the native tip protein (Cpa) of the T3 pilus derived from Streptococcus pyogenes and expressed this fusion protein (MBP*) in L. lactis. We find that MBP* is incorporated into pili in this foreign host, as shown by Western blot analyses of cell wall proteins and by immunogold electron microscopy. Furthermore, since the MBP* on these pili retains its native biological activity, it appears to retain its native structure. Mucosal immunization of mice with this L. lactis strain expressing pilus-linked MBP* results in production of both a systemic and a mucosal response (IgG and IgA antibodies) against the MBP antigen. We suggest that this type of mucosal vaccine delivery system, which we term UPTOP (for unhindered presentation on tips of pili), may provide an inexpensive and stable alternative to current mechanisms of immunization for many serious human pathogens.

The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed in E. coli, due to problems related to disulfide bond formation, post-translational modifications, and folding. In such cases, other expression systems are required for producing proteins for biomolecular NMR analyses. In this paper, we review the recent advances in expression systems for isotopically labeled heterologous proteins, utilizing non-E. coli prokaryotic and eukaryotic cells.

Potassium deficiency enhanced the synthesis of fifteen proteins in the nitrogen-fixing cyanobacterium Anabaena torulosa and of nine proteins in Escherichia coli. These were termed potassium deficiency-induced proteins or PDPs and constitute hitherto unknown potassium deficiency-induced stimulons. Potassium deficiency also enhanced the synthesis of certain osmotic stress-induced proteins. Addition of K+ repressed the synthesis of a majority of the osmotic stress-induced proteins and of PDPs in these bacteria. These proteins contrast with the dinitrogenase reductase of A. torulosa and the glycine betaine-binding protein of E. coli, both of which were osmo-induced to a higher level in potassium-supplemented conditions. The data demonstrate the occurrence of novel potassium deficiency-induced stimulons and a wider role of K+ in regulation of gene expression and stress responses in bacteria