Regulation of an antibiotic resistance efflux pump by quorum sensing and a TetR-family repressor in Chromobacterium subtsugae.

The soil bacterium Chromobacterium substugae uses a single LuxI-R-type quorum-sensing system, CviI-R, to regulate genes in a cell density-dependent manner. CviI synthesizes the signal N-hexanoyl-homoserine lactone (C6-HSL) and CviR is a C6-HSL-responsive cytoplasmic transcription regulator. C6-HSL-bound CviR activates dozens of genes, for example the cdeAB-oprM cluster coding for an efflux pump conferring antibiotic resistance. The cdeAB-oprM genes are also regulated by an antibiotic-responsive transcription factor, CdeR, which represses expression of these genes. We are interested in understanding how C. subtsugae integrates different environmental cues to regulate antibiotic resistance. In this study, we sought to delineate the mechanism of regulation of the cdeAB-oprM genes by CviR and CdeR. In recombinant E. coli, the cdeA promoter is activated by CviR and repressed by CdeR. We identify non-overlapping sequence elements in the cdeA promoter that are required for CviR activation and CdeR repression, respectively. We also examined the role of CdeR in modulating cdeA activation by C6-HSL in C. subtsugae. We show that CviR and CdeR can independently modulate transcription from the cdeA promoter in C. subtsugae, consistent with the conclusion that CviR and CdeR regulate the cdeAB-oprM genes by interacting directly with different binding sites in the cdeA promoter. These results contribute to a molecular understanding of how the cdeAB-oprM genes are regulated and provide new insight into how C. subtsugae integrates different environmental cues to regulate antibiotic resistance.

An Amino Acid Substitution in Elongation Factor EF-G1A Alters the Antibiotic Susceptibility of Pseudomonas aeruginosa LasR-Null Mutants.

The opportunistic bacterium Pseudomonas aeruginosa uses the LasR-I quorum-sensing system to increase resistance to the aminoglycoside antibiotic tobramycin. Paradoxically, lasR-null mutants are commonly isolated from chronic human infections treated with tobramycin, suggesting there may be a mechanism that permits the emergence of lasR-null mutants under tobramycin selection. We hypothesized that some other genetic mutations that emerge in these isolates might modulate the effects of lasR-null mutations on antibiotic resistance. To test this hypothesis, we inactivated lasR in several highly tobramycin-resistant isolates from long-term evolution experiments. In some of these isolates, inactivating lasR further increased resistance, compared with decreasing resistance of the wild-type ancestor. These strain-dependent effects were due to a G61A nucleotide polymorphism in the fusA1 gene encoding amino acid substitution A21T in the translation elongation factor EF-G1A. The EF-G1A mutational effects required the MexXY efflux pump and the MexXY regulator ArmZ. The fusA1 mutation also modulated ΔlasR mutant resistance to two other antibiotics, ciprofloxacin and ceftazidime. Our results identify a gene mutation that can reverse the direction of the antibiotic selection of lasR mutants, a phenomenon known as sign epistasis, and provide a possible explanation for the emergence of lasR-null mutants in clinical isolates. IMPORTANCE One of the most common mutations in Pseudomonas aeruginosa clinical isolates is in the quorum sensing lasR gene. In laboratory strains, lasR disruption decreases resistance to the clinical antibiotic tobramycin. To understand how lasR mutations emerge in tobramycin-treated patients, we mutated lasR in highly tobramycin-resistant laboratory strains and determined the effects on resistance. Disrupting lasR enhanced the resistance of some strains. These strains had a single amino acid substitution in the translation factor EF-G1A. The EF-G1A mutation reversed the selective effects of tobramycin on lasR mutants. These results illustrate how adaptive mutations can lead to the emergence of new traits in a population and are relevant to understanding how genetic diversity contributes to the progression of disease during chronic infections.

Cross-species activation of hydrogen cyanide production by a promiscuous quorum-sensing receptor promotes Chromobacterium subtsugae competition in a dual-species model.

Many saprophytic bacteria have LuxR-I-type acyl-homoserine lactone (AHL) quorum-sensing systems that may be important for competing with other bacteria in complex soil communities. LuxR AHL receptors specifically interact with cognate AHLs to cause changes in expression of target genes. Some LuxR-type AHL receptors have relaxed specificity and are responsive to non-cognate AHLs. These promiscuous receptors might be used to sense and respond to AHLs produced by other bacteria by eavesdropping. We are interested in understanding the role of eavesdropping during interspecies competition. The soil saprophyte Chromobacterium subtsugae has a single AHL circuit, CviR-I, which produces and responds to N-hexanoyl-HSL (C6-HSL). The AHL receptor CviR can respond to a variety of AHLs in addition to C6-HSL. In prior studies we have utilized a coculture model with C. subtsugae and another soil saprophyte, Burkholderia thailandensis. Using this model, we previously showed that promiscuous activation of CviR by B. thailandensis AHLs provides a competitive advantage to C. subtsugae. Here, we show that B. thailandensis AHLs activate transcription of dozens of genes in C. subtsugae, including the hcnABC genes coding for production of hydrogen cyanide. We show that hydrogen cyanide production is population density-dependent and demonstrate that the cross-induction of hydrogen cyanide by B. thailandensis AHLs provides a competitive advantage to C. subtsugae. Our results provide new information on C. subtsugae quorum sensing and are the basis for future studies aimed at understanding the role of eavesdropping in interspecies competition.

Tobramycin adaptation alters the antibiotic susceptibility of Pseudomonas aeruginosa quorum sensing-null mutants.

The opportunistic bacterium Pseudomonas aeruginosa uses the LasR-I quorum sensing system to increase resistance to the aminioglycoside antibiotic tobramycin. Paradoxically, lasR-null mutants are commonly isolated from chronic human infections treated with tobramycin, suggesting there may be a mechanism allowing the lasR-null mutants to persist under tobramycin selection. We hypothesized that the effects of inactivating lasR on tobramycin resistance might be dependent on the presence or absence of other gene mutations in that strain, a phenomenon known as epistasis. To test this hypothesis, we inactivated lasR in several highly tobramycin-resistant isolates from long-term evolution experiments. We show that the effects of ΔlasR on tobramycin resistance are strain dependent. The effects can be attributed to a point mutation in the gene encoding the translation elongation factor fusA1 (G61A nucleotide substitution), which confers a strong selective advantage to lasR-null PA14 under tobramycin selection. This fusA1 G61A mutation results in increased activity of the MexXY efflux pump and expression of the mexXY regulator ArmZ. The fusA1 mutation can also modulate ΔlasR mutant resistance to two other antibiotics, ciprofloxacin and ceftazidime. Our results demonstrate the importance of epistatic gene interactions on antibiotic susceptibility of lasR-null mutants. These results support of the idea that gene interactions might play a significant role in the evolution of quorum sensing in P. aeruginosa.

Secondary metabolites from the Burkholderia pseudomallei complex: structure, ecology, and evolution.

Bacterial secondary metabolites play important roles in promoting survival, though few have been carefully studied in their natural context. Numerous gene clusters code for secondary metabolites in the genomes of members of the Bptm group, made up of three closely related species with distinctly different lifestyles: the opportunistic pathogen Burkholderia pseudomallei, the non-pathogenic saprophyte Burkholderia thailandensis, and the host-adapted pathogen Burkholderia mallei. Several biosynthetic gene clusters are conserved across two or all three species, and this provides an opportunity to understand how the corresponding secondary metabolites contribute to survival in different contexts in nature. In this review, we discuss three secondary metabolites from the Bptm group: bactobolin, malleilactone (and malleicyprol), and the 4-hydroxy-3-methyl-2-alkylquinolines, providing an overview of each of their biosynthetic pathways and insight into their potential ecological roles. Results of studies on these secondary metabolites provide a window into how secondary metabolites contribute to bacterial survival in different environments, from host infections to polymicrobial soil communities.

Charging ain't all bad: Complex physics in DyScO3.

Although charging is ubiquitous in electron microscopy, its effects are typically avoided or ignored. However, avoiding charging is not possible in some materials, e.g. lanthanide scandates with well-ordered surfaces positively charge immensely under electron beam illumination because of their electronic structure, and ignoring charging can leave new science undiscovered. In this work, a combination of rapidly acquired electron energy loss spectra and cross-correlation were used to understand and overcome charging effects in DyScO3. A 5.4 eV band gap was extracted from the charging-corrected loss spectrum, in good agreement with previously reported band gaps, and a 3.8 eV in-gap peak was attributed to surface states via comparison with density functional theory calculations. Additionally, ultraviolet photoelectron spectroscopy measurements indicated that under some conditions well-annealed DyScO3 surfaces charge negatively causing upward band bending associated with occupied surface states in the gap. As was previously found in the case of positive charging under electron beam illumination with in-situ flexoelectric bending observations, the magnitude of negative charging under ultraviolet illumination is Zener tunneling limited in well-annealed DyScO3.

Direct Observation of Large Flexoelectric Bending at the Nanoscale in Lanthanide Scandates.

There is a growing interest in the flexoelectric effect, since at the nanoscale it is predicted to be very large. However, there have been no direct observations of flexoelectric bending consistent with current theoretical work that implies strains comparable to or exceeding the yield strains of typical materials. Here we show a direct observation of extraordinarily large, two-dimensional reversible bending at the nanoscale in dysprosium scandate due to the converse flexoelectric effect, with similar results for terbium and gadolinium scandate. Within a transmission electron microscope, thin features bend up to 90° with radii of curvature of about 1 µm, corresponding to very large nominal strains. Analysis including independent experimental determination of the flexoelectric coefficient is semiquantitatively consistent with interpreting the results as due to flexoelectricity. These results experimentally demonstrate large flexoelectric bending at the nanoscale.

Epidemiological Profile and Risk Factors for Acquiring HBV and/or HCV in HIV-Infected Population Groups in Nepal.

HBV and HCV infections are widespread among the HIV-infected individuals in Nepal. The goals of this study were to investigate the epidemiological profile and risk factors for acquiring HBV and/or HCV coinfection in disadvantaged HIV-positive population groups in Nepal. We conducted a retrospective study on blood samples from HIV-positive patients from the National Public Health Laboratory at Kathmandu to assay for HBsAg, HBeAg, and anti-HCV antibodies, HIV viral load, and CD4+ T cell count. Among 579 subjects, the prevalence of HIV-HBV, HIV-HCV, and HIV-HBV-HCV coinfections was 3.62%, 2.93%, and 0.34%, respectively. Multivariate regression analysis indicated that spouses of HIV-positive migrant labourers were at significant risk for coinfection with HBV infection, and an age of >40 years in HIV-infected individuals was identified as a significant risk factor for HCV coinfection. Overall our study indicates that disadvantaged population groups such as intravenous drug users, migrant workers and their spouses, female sex workers, and men who have sex with HIV-infected men are at a high and persistent risk of acquiring viral hepatitis. We conclude that Nepalese HIV patients should receive HBV and HCV diagnostic screening on a regular basis.

When does atomic resolution plan view imaging of surfaces work?

Surface structures that are different from the corresponding bulk, reconstructions, are exceedingly difficult to characterize with most experimental methods. Scanning tunneling microscopy, the workhorse for imaging complex surface structures of metals and semiconductors, is not as effective for oxides and other insulating materials. This paper details the use of transmission electron microscopy plan view imaging in conjunction with image processing for solving complex surface structures. We address the issue of extracting the surface structure from a weak signal with a large bulk contribution. This method requires the sample to be thin enough for kinematical assumptions to be valid. The analysis was performed on two sets of data, c(6×2) on the (100) surface and (3×3) on the (111) surface of SrTiO3, and was unsuccessful in the latter due to the thickness of the sample and a lack of inversion symmetry. The limits and the functionality of this method are discussed.

Identification of hyperhalogens in Ag(n)(BO2)(m) (n = 1-3, m = 1-2) clusters: anion photoelectron spectroscopy and density functional calculations.

The electronic and structural properties of neutral and anionic Agn(BO2)m (n = 1-3, m = 1-2) clusters are investigated by using mass-selected anion photoelectron spectroscopy and density functional theory calculations. Agreement between the measured and calculated vertical detachment energies (VDEs) allows us to validate the equilibrium geometries of [Agn(BO2)m](-) clusters obtained from theory. The ground state structures of anionic Ag2(BO2) and Agn(BO2)2 (n = 1-3) clusters are found to be very different from those of their neutral counterparts. The structures of anionic clusters are chain-like while those of the neutral clusters are closed-rings. The presence of multiple isomers for [Ag2(BO2)2](-) and [Ag3(BO2)2](-) in the cluster beam has also been confirmed. Several of these clusters are found to be hyperhalogens.

Electronic and magnetic properties of manganese and iron atoms decorated with BO2 superhalogens.

Using density functional theory based calculations, we have systematically studied the equilibrium geometries, relative stabilities, and electronic and magnetic properties of Fe and Mn atoms interacting with a varying number of BO(2) moieties. These clusters are found to exhibit hyperhalogen behavior with electron affinities as high as 6.9 eV once the number of BO(2) moieties exceed the nominal valences of these transition metals toms, namely 2 for both Fe and Mn. In all cases the transition metal atoms retain a sizable spin magnetic moment, even exceeding their free atom values at certain compositions. We also note that when more than two BO(2) moieties are bound to neutral Fe and Mn atoms, they tend to dimerize. In the case of negative ions, this process occurs at n ≥ 3, thus leading to different neutral and anionic ground state geometries. The effect of these structural changes in the interpretation of photoelectron spectroscopy experiments is discussed.

(PbS)32: a baby crystal.

Theoretical calculations based on density functional theory have found (PbS)(32) to be the smallest cubic cluster for which its inner (PbS)(4) core enjoys bulk-like coordination. Cubic (PbS)(32) is thus a "baby crystal," i.e., the smallest cluster, exhibiting sixfold coordination, that can be replicated to obtain the bulk crystal. The calculated dimensions of the (PbS)(32) cluster further provide a rubric for understanding the pattern of aggregation when (PbS)(32) clusters are deposited on a suitable surface, i.e., the formation of square and rectangular, crystalline nano-blocks with predictable dimensions. Experiments in which mass-selected (PbS)(32) clusters were soft-landed onto a highly ordered pyrolytic graphite surface and the resulting aggregates imaged by scanning tunneling microscopy provide evidence in direct support of the computational results.

Structural evolution and stabilities of neutral and anionic clusters of lead sulfide: joint anion photoelectron and computational studies.

The geometric and electronic structures of both neutral and negatively charged lead sulfide clusters, (PbS)(n)/(PbS)(n)(-) (n = 2-10) were investigated in a combined anion photoelectron spectroscopy and computational study. Photoelectron spectra provided vertical detachment energies (VDEs) for the cluster anions and estimates of electron affinities (EA) for their neutral cluster counterparts, revealing a pattern of alternating EA and VDE values in which even n clusters exhibited lower EA and VDE values than odd n clusters up until n = 8. Computations found neutral lead sulfide clusters with even n to be thermodynamically more stable than their immediate (odd n) neighbors, with a consistent pattern also being found in their HOMO-LUMO gaps. Analysis of neutral cluster dissociation energies found the Pb(4)S(4) cube to be the preferred product of the queried fragmentation processes, consistent with our finding that the lead sulfide tetramer exhibits enhanced stability; it is a magic number species. Beyond n = 10, computational studies showed that neutral (PbS)(n) clusters in the size range, n = 11-15, prefer two-dimensional stacking of face-sharing lead sulfide cubical units, where lead and sulfur atoms possess a maximum of five-fold coordination. The preference for six-fold coordination, which is observed in the bulk, was not observed at these cluster sizes. Taken together, the results show a preference for the formation of slightly distorted, fused cuboids among small lead sulfide clusters.