Comparative genetic, biochemical, and biophysical analyses of the four E. coli ABCF paralogs support distinct functions related to mRNA translation.

Multiple paralogous ABCF ATPases are encoded in most genomes, but the physiological functions remain unknown for most of them. We herein compare the four Escherichia coli K12 ABCFs - EttA, Uup, YbiT, and YheS - using assays previously employed to demonstrate EttA gates the first step of polypeptide elongation on the ribosome dependent on ATP/ADP ratio. A Δ uup knockout, like Δ ettA , exhibits strongly reduced fitness when growth is restarted from long-term stationary phase, but neither Δ ybiT nor Δ yheS exhibits this phenotype. All four proteins nonetheless functionally interact with ribosomes based on in vitro translation and single-molecule fluorescence resonance energy transfer experiments employing variants harboring glutamate-to-glutamine active-site mutations (EQ 2 ) that trap them in the ATP-bound conformation. These variants all strongly stabilize the same global conformational state of a ribosomal elongation complex harboring deacylated tRNA Val in the P site. However, EQ 2 -Uup uniquely exchanges on/off the ribosome on a second timescale, while EQ 2 -YheS-bound ribosomes uniquely sample alternative global conformations. At sub-micromolar concentrations, EQ 2 -EttA and EQ 2 -YbiT fully inhibit in vitro translation of an mRNA encoding luciferase, while EQ 2 -Uup and EQ 2 -YheS only partially inhibit it at ~10-fold higher concentrations. Moreover, tripeptide synthesis reactions are not inhibited by EQ 2 -Uup or EQ 2 -YheS, while EQ 2 -YbiT inhibits synthesis of both peptide bonds and EQ 2 -EttA specifically traps ribosomes after synthesis of the first peptide bond. These results support the four E. coli ABCF paralogs all having different activities on translating ribosomes, and they suggest that there remains a substantial amount of functionally uncharacterized "dark matter" involved in mRNA translation.

Regulation of the macrolide resistance ABC-F translation factor MsrD.

Antibiotic resistance ABC-Fs (ARE ABC-Fs) are translation factors that provide resistance against clinically important ribosome-targeting antibiotics which are proliferating among pathogens. Here, we combine genetic and structural approaches to determine the regulation of streptococcal ARE ABC-F gene msrD in response to macrolide exposure. We show that binding of cladinose-containing macrolides to the ribosome prompts insertion of the leader peptide MsrDL into a crevice of the ribosomal exit tunnel, which is conserved throughout bacteria and eukaryotes. This leads to a local rearrangement of the 23 S rRNA that prevents peptide bond formation and accommodation of release factors. The stalled ribosome obstructs the formation of a Rho-independent terminator structure that prevents msrD transcriptional attenuation. Erythromycin induction of msrD expression via MsrDL, is suppressed by ectopic expression of mrsD, but not by mutants which do not provide antibiotic resistance, showing correlation between MsrD function in antibiotic resistance and its action on this stalled complex.

ABC-F translation factors: from antibiotic resistance to immune response.

Energy-dependent translational throttle A (EttA) from Escherichia coli is a paradigmatic ABC-F protein that controls the first step in polypeptide elongation on the ribosome according to the cellular energy status. Biochemical and structural studies have established that ABC-F proteins generally function as translation factors that modulate the conformation of the peptidyl transferase center upon binding to the ribosomal tRNA exit site. These factors, present in both prokaryotes and eukaryotes but not in archaea, use related molecular mechanisms to modulate protein synthesis for heterogenous purposes, ranging from antibiotic resistance and rescue of stalled ribosomes to modulation of the mammalian immune response. Here, we review the canonical studies characterizing the phylogeny, regulation, ribosome interactions, and mechanisms of action of the bacterial ABC-F proteins, and discuss the implications of these studies for the molecular function of eukaryotic ABC-F proteins, including the three human family members.

ABC-F proteins in mRNA translation and antibiotic resistance.

The ATP binding cassette protein superfamily comprises ATPase enzymes which are, for the most part, involved in transmembrane transport. Within this superfamily however, some protein families have other functions unrelated to transport. One example is the ABC-F family, which comprises an extremely diverse set of cytoplasmic proteins. All of the proteins in the ABC-F family characterized to date act on the ribosome and are translation factors. Their common function is ATP-dependent modulation of the stereochemistry of the peptidyl transferase center (PTC) in the ribosome coupled to changes in its global conformation and P-site tRNA binding geometry. In this review, we give an overview of the function, structure, and theories for the mechanisms-of-action of microbial proteins in the ABC-F family, including those involved in mediating resistance to ribosome-binding antibiotics.

OXA-48-producing Enterobacterales in different ecological niches in Algeria: clonal expansion, plasmid characteristics and virulence traits.

OBJECTIVES: To investigate the prevalence and molecular characteristics of OXA-48-carbapenemase-producing Enterobacterales strains recovered from various ecological niches in Algeria. METHODS: In total, 3309 samples were collected from different ecological niches (human carriage, animal farms, wild animals, pets, food products, aquatic environment and wastewater treatment plants) distributed among six provinces in Algeria between December 2015 and April 2017. The potential presence of OXA-48-producing Enterobacterales isolates was screened on selective medium. Resistance and virulence profiles were characterized by PCR and sequencing. The clonal relatedness of the different isolates was studied using Rep-PCR and MLST. Conjugation was performed for all OXA-48-producing isolates. The plasmids were analysed by PCR-based replicon typing and WGS. RESULTS: A total of 78 OXA-48-producing Enterobacterales isolates were detected from 3309 samples (2.4%). OXA producers were observed in all the screened sources. The blaCTX-M-15 gene was only observed in two isolates. Clonality analysis revealed distinct lineages of the isolates and a clonal expansion of Klebsiella pneumoniae ST13. K. pneumoniae and Escherichia coli had few virulence factors. Plasmid analysis confirmed that all the isolates harboured a very similar transferable plasmid (belonging to IncL) with a similar structure to the pOXA-48a plasmid carried by K. pneumoniae strain Kp11978. CONCLUSIONS: This study suggests a global dissemination of OXA-48-producing Enterobacterales in different niches due mainly to the spread of an epidemic plasmid. Furthermore, it clearly shows that K. pneumoniae and commensal E. coli can be reservoirs of the blaOXA-48 gene, contributing to the dissemination and transfer of this gene to diverse bacteria among different sources.

The new strategies to overcome challenges in protein production in bacteria.

Recombinant proteins are essential for biotechnology. Here we review some of the key points for improving the production of heterologous proteins, and what can be the future of the field.

Lactococcus lactis is an Efficient Expression System for Mammalian Membrane Proteins Involved in Liver Detoxification, CYP3A4, and MGST1.

Despite the great importance of human membrane proteins involved in detoxification mechanisms, their wide use for biochemical approaches is still hampered by several technical difficulties considering eukaryotic protein expression in order to obtain the large amounts of protein required for functional and/or structural studies. Lactococcus lactis has emerged recently as an alternative heterologous expression system to Escherichia coli for proteins that are difficult to express. The aim of this work was to check its ability to express mammalian membrane proteins involved in liver detoxification, i.e., CYP3A4 and two isoforms of MGST1 (rat and human). Genes were cloned using two different strategies, i.e., classical or Gateway-compatible cloning, and we checked the possible influence of two affinity tags (6×-His-tag and Strep-tag II). Interestingly, all proteins could be successfully expressed in L. lactis at higher yields than those previously obtained for these proteins with classical expression systems (E. coli, Saccharomyces cerevisiae) or those of other eukaryotic membrane proteins expressed in L. lactis. In addition, rMGST1 was fairly active after expression in L. lactis. This study highlights L. lactis as an attractive system for efficient expression of mammalian detoxification membrane proteins at levels compatible with further functional and structural studies.