Gram-negative bacteria and their complex cell envelope comprising an outer and inner membrane are an important and attractive system for studying the translocation of small molecules across biological membranes. In the outer membrane of Enterobacteriaceae, trimeric porins control the cellular penetration of small molecules, including nutrients and antibacterial agents. The synergistic action between relatively slow porin-mediated passive uptake across the outer membrane and active efflux transporters in the inner membrane creates a permeability barrier that reinforces the enzymatic modification barrier, which efficiently reduces the intracellular concentrations of small molecules and contributes to the emergence of antibiotic resistance. In this review, we discuss recent advances in our understanding of the molecular and functional roles of classic porins in small molecule translocation in Enterobacteriaceae and consider the crucial role of porins in antibiotic resistance. Commented [w1]: Is this specification necessary here?, in my opinion it deviates, better to put later… Commented [JP2]: Editor request... porins represent the preferred route for the entry of β-lactams, including cephalosporins, penicillins and carbapenems 14-16. The clinical relevance of membrane-associated mechanisms (MAMs) of resistance (i.e. porin defects and/or overexpression of multidrug efflux pumps) has been well established for these antibiotics. The Influx and Efflux rates control the internal concentration of antibiotics and represent the first lane (mechanical barrier) protecting the bacterial cells against therapeutic treatment 1-3,6. Consequently, studies on bacterial porins are receiving a renewed interest due to their key role in the bacterial susceptibility towards clinically used antibiotics. In combination with the expression of antibiotic-modifying enzymes expressed in the periplasm (e.g. β-lactamases), porins play a key role in β-lactam resistance 4,17. In this review, we discuss recent advances in our understanding of the molecular and functional roles of classic porins in antibiotic translocation in Enterobacteriaceae. We explore structural aspects and the insights gained into permeation and the pore translocation process, the regulation of porin expression as well as the role of porins in the emergence of antibiotic susceptibility. Enterobacterial general porins Structural aspects The crystal structures of a general porin from Rhodobacter capsulatus 18 , the OmpF and PhoE porins from E. coli 19 and other E. coli OmpF structures including mutants 20,21 were the first to be solved. Only a limited number of other enterobacterial porin structures have been reported, i.e. E. coli OmpC, K. pneumoniae OmpK36 and Salmonella typhi OmpF 22-24. The lack of data has hindered attempts to relate structure to function. Recently, the structures of two porins from P. stuartii as well as the structures of the OmpF and OmpC orthologs of K. pneumoniae, E. aerogenes and E. cloacae have been reported 12,25,26. Another recent study reported th...

The cyanobacterium, Microcystis aeruginosa, is able to proliferate in a wide range of freshwater ecosystems and to produce many secondary metabolites that are a threat to human and animal health. The dynamic of this production and more globally the metabolism of this species is still poorly known. A DNA microarray based on the genome of M. aeruginosa PCC 7806 was constructed and used to study the dynamics of gene expression in this cyanobacterium during the light/dark cycle, because light is a critical factor for this species, like for other photosynthetic microorganisms. This first application of transcriptomics to a Microcystis species has revealed that more than 25% of the genes displayed significant changes in their transcript abundance during the light/dark cycle and in particular during the dark/light transition. The metabolism of M. aeruginosa is compartmentalized between the light period, during which carbon uptake, photosynthesis and the reductive pentose phosphate pathway lead to the synthesis of glycogen, and the dark period, during which glycogen degradation, the oxidative pentose phosphate pathway, the TCA branched pathway and ammonium uptake promote amino acid biosynthesis. We also show that the biosynthesis of secondary metabolites, such as microcystins, aeruginosin and cyanopeptolin, occur essentially during the light period, suggesting that these metabolites may interact with the diurnal part of the central metabolism.

With the spreading of antibiotic resistance, the translocation of antibiotics through bacterial envelopes is crucial for their antibacterial activity. In Gram-negative bacteria, the interplay between membrane permeability and drug efflux pumps must be investigated as a whole. Here, we quantified the intracellular accumulation of a series of fluoroquinolones in population and in individual cells of Escherichia coli according to the expression of the AcrB efflux transporter. Computational results supported the accumulation levels measured experimentally and highlighted how fluoroquinolones side chains interact with specific residues of the distal pocket of the AcrB tight monomer during recognition and binding steps.

The efficacy of antibacterial molecules depends on their capacity to reach inhibitory concentrations in the vicinity of their target. This is particularly challenging for drugs directed against Gram-negative bacteria, which have a complex envelope comprising two membranes and efflux pumps. Precise determination of the bacterial drug content is an essential prerequisite for drug development. Here we describe three approaches that have been developed in our laboratories to quantify drugs accumulated in intact cells by spectrofluorimetry, microspectrofluorimetry, and kinetics microspectrofluorimetry (KMSF). These different procedures provide complementary results that highlight the contribution of membrane-associated mechanisms, including influx through the outer membrane (OM) and efflux, and the importance of the physicochemical properties of the transported drugs for the intracellular concentration of a given antibiotic in a given bacterial population. The three key stages of this protocol are preparation of the bacterial strains in the presence of the antibiotic; preparation of the whole-cell lysates (WCLs) and fluorescence readings; and data analysis, including normalization and quantitation of the intracellular antibiotic fluorescence relative to the internal standard and the antibiotic standard curve, respectively. Fluorimetry is limited to naturally fluorescent or labeled compounds, but in contrast to existing alternative methods such as mass spectrometry, it uniquely allows single-cell analysis. From culture growth to data analysis, the protocol described here takes 5 d.

Bacterial multidrug resistance is a worrying health issue. In Gram-negative antibacterial research, the challenge is to define the antibiotic permeation across the membranes. Passing through the membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibacterial molecules. A spectrofluorimetric methodology has been developed to detect fluoroquinolones in bacterial population and inside individual Gram-negative bacterial cells. In this work, we studied the antibiotic accumulation in cells expressing various levels of efflux pumps. The assays allow us to determine the intracellular concentration of the fluoroquinolones to study the relationships between the level of efflux activity and the antibiotic accumulation, and finally to evaluate the impact of fluoroquinolone structures in this process. This represents the first protocol to identify some structural parameters involved in antibiotic translocation and accumulation, and to illustrate the recently proposed “Structure Intracellular Concentration Activity Relationship” (SICAR) concept.

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A maltotriose-conjugate can deliver molecules into the cytoplasmic space of Gram-negative bacteria by parasitizing the maltose uptake pathway.

The transport of small molecules across membranes is a pivotal step for controlling the drug concentration into the bacterial cell and it efficiently contributes to the antibiotic susceptibility in Enterobacteriaceae. Two types of membrane transports, passive and active, usually represented by porins and efflux pumps, are involved in this process. Importantly, the expression of these transporters and channels are modulated by an armamentarium of tangled regulatory systems. Among them, Helix-turn-Helix (HTH) family regulators (including the AraC/XylS family) and the two-component systems (TCS) play a key role in bacterial adaptation to environmental stresses and can manage a decrease of porin expression associated with an increase of efflux transporters expression. In the present review, we highlight some recent genetic and functional studies that have substantially contributed to our better understanding of the sophisticated mechanisms controlling the transport of small solutes (antibiotics) across the membrane of Enterobacteriaceae. This information is discussed, taking into account the worrying context of clinical antibiotic resistance and fitness of bacterial pathogens. The localization and relevance of mutations identified in the respective regulation cascades in clinical resistant strains are discussed. The possible way to bypass the membrane/transport barriers is described in the perspective of developing new therapeutic targets to combat bacterial resistance.