BC4707 Is a Major Facilitator Superfamily Multidrug Resistance Transport Protein from Bacillus cereus Implicated in Fluoroquinolone Tolerance

Transcriptional profiling highlighted a subset of genes encoding putative multidrug transporters in the pathogen Bacillus cereus that were up-regulated during stress produced by bile salts. One of these multidrug transporters (BC4707) was selected for investigation. Functional characterization of the BC4707 protein in Escherichia coli revealed a role in the energized efflux of xenobiotics. Phenotypic analyses after inactivation of the gene bc4707 in Bacillus cereus ATCC14579 suggested a more specific, but modest role in the efflux of norfloxacin. In addition to this, transcriptional analyses showed that BC4707 is also expressed during growth of B. cereus under non-stressful conditions where it may have a role in the normal physiology of the bacteria. Altogether, the results indicate that bc4707, which is part of the core genome of the B. cereus group of bacteria, encodes a multidrug resistance efflux protein that is likely involved in maintaining intracellular homeostasis during growth of the bacteria.Peer reviewe

Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions

This article reviews current methods for the reliable heterologous overexpression in Escherichia coli and purification of milligram quantities of bacterial membrane sensor kinase (MSK) proteins belonging to the two-component signal transduction family of integral membrane proteins. Many of these methods were developedatLeedsalongsideProfessor SteveBaldwintowhomthisreviewisdedicated.Italsoreviewstwo biophysical methods that we have adapted successfully for studies of purified MSKs and other membrane proteins – synchrotron radiation circular dichroism (SRCD) spectroscopy and analytical ultracentrifugation (AUC), both of which are non-immobilization and matrix-free methods that require no labelling strategies. Other techniques such as isothermal titration calorimetry (ITC) also share these features but generally require high concentrations of material. In common with many other biophysical techniques, both of these biophysical methods provide information regarding membrane protein conformation, oligomerization state and ligand binding, but they possess the additional advantage of providing direct assessments of whether ligand binding interactions are accompanied by conformational changes. Therefore, both methods provide a powerful means by which to identify and characterize inhibitor binding and any associated protein conformational changes, thereby contributing valuable information for future drug intervention strategies directed towards bacterial MSKs

The role of relatedness in structuring the social network of a wild guppy population

The role of relatedness in structuring animal societies has attracted considerable interest. Whilst a significant number of studies have documented kin recognition in shoaling fish under laboratory conditions, there is little evidence that relatedness plays a significant role in structuring social interactions in wild populations that are characterised by fission–fusion dynamics. Previous work has tended to compare relatedness within and among entire shoals. Such an approach however, does not have the ability to detect social sub-structuring within groups, which appears to be a major factor driving the social organisation of fission–fusion animal societies. Here, we use social network analysis combined with DNA microsatellite genotyping to examine the role of relatedness in structuring social relationships in a wild population of guppies (Poecilia reticulata). Consistent with previous findings, female–female dyads formed the strongest social relationships, which were stable over time. Interestingly, we also observed significant co-occurrence of male–male interactions, which is in contrast to previous work. Although we observed social sub-structuring in the population, we found no evidence for relatedness playing a significant role in underpinning this structure. Indeed, only seven first-degree relative dyads were identified among the 180 fish genotyped, indicating that the majority of individuals do not have a first-degree relative in the population. The high genetic diversity observed in this population is indicative of a large effective population size typical of lowland guppy populations. We discuss our findings in the context of the evolution of social organisation and the mechanisms and constraints that may drive the observed patterns in wild populations

Active membrane transport and receptor proteins from bacteria

A general strategy for the expression of bacterial membrane transport and receptor genes in Escherichia coli is described. Expression is amplified so that the encoded proteins comprise 5-35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGSH6 or a Strep tag at the C-terminus. These enable purification in mg quantities for crystallization and NMR studies. Examples of one nutrient uptake and one multidrug extrusion protein from Helicobacter pylori are described. This strategy is successful for membrane proteins from H. pylori, E. coli, Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Microbacterium liquefaciens, Brucella abortus, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides, Streptomyces coelicolor and Rhodobacter sphaeroides

Accumulation of Norfloxacin in <i>E. coli</i> DH5α <i>ΔacrAB</i> (pBC4707) and DH5α <i>ΔacrAB</i> (pTTQ18).

<p>The fluorescence of intracellularly accumulated norfloxacin was measured at 280 nm excitation and 445 nm emission in the supernatant of bacterial extracts. Bacteria expressing BC4707-RGSHis₆ are indicated by circles whereas bacteria carrying the empty vector are shown as diamonds. The assay was started by the addition of 100 µM norfloxacin at time 0 min. At 15 min, the protonophore CCCP (100 µM) was added to disrupt the proton motive force. Shown are averages and standard deviations from two independent experiments.</p

Transcriptional profile of <i>B. cereus</i> ATCC14579 <i>Δbc4707</i> (AH1598) compared to wild type (AH1709) grown to logarithmic stage (OD₆₀₀∼0.5) in rich medium followed by bile salt stress for 15 min.

<p>Transcriptional profile of <i>B. cereus</i> ATCC14579 <i>Δbc4707</i> (AH1598) compared to wild type (AH1709) grown to logarithmic stage (OD₆₀₀∼0.5) in rich medium followed by bile salt stress for 15 min.</p

Susceptibilities of <i>B. cereus</i> ATCC14579 (wild type) compared to its isogenic <i>Δbc4707</i> mutant.

§<p>represents the fold difference in MIC between the wild type <i>B. cereus</i> ATCC14579 and its isogenic <i>Δbc4707</i> mutant.</p

Relative expression of <i>bc4707</i> in stressed cells compared to unstressed cells.

<p>The expression level of <i>bc4707</i> was measured by qRTPCR and was normalized against the expression of16S RNA. The values shown are ratios of the normalized expression levels of cells stressed with antibiotics for 15 min compared to unstressed cells. Shown are averages and standard deviations of at least two independent experiemnts each done with two technical replicates.</p

Susceptibilities of <i>E. coli</i> DH5α <i>ΔacrAB</i> expressing <i>bc4707</i> (pBC4707) compared to its vector control (pTTQ18) under uninduced (LB (0 mM IPTG)) and induced (LB (0.05 mM IPTG)) conditions.

§<p>represents the fold difference in MIC between the <i>E. coli</i> DH5α <i>ΔacrAB</i> (pBC4707) strain compared to the vector control (DH5α <i>ΔacrAB</i> (pTTQ18)).</p