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Background: Flavin adenine dinucleotide (FAD) is a redox-active coenzyme that regulates several important enzymatic reactions during metabolism. FAD is used in the medicinal and food industries and FAD supplements have been used to treat some inheritable diseases. FAD can be biosynthesized from flavin mononucleotide (FMN) and adenosine triphosphate (ATP), catalyzed by FAD synthetase (FADS). Objective: The aim of this study was to heterologously express the gene encoding FADS from the flavinogenic yeast Candida famata (FADSCf) for biosynthesis of FAD. Methods: The sequence encoding FADSCf was retrieved and heterologously expressed in Escherichia coli. The structure and enzymatic properties of recombinant FADSCf were characterized. Results: FADSCf (279 amino acids) was successfully expressed in E. coli BL21 (DE3), with a theoretical molecular weight of 32299.79 Da and an isoelectric point of 6.09. Secondary structural analysis showed that the number of α-helices was 2-fold higher than the number of β-sheets, indicating that the protein was highly hydrophilic. Under fixed ATP concentration, FADSCf had a Km of 0.04737±0.03158 mM and a Vmax of 3.271±0.79 μM/min/mg. Under fixed FMN concentration, FADSCf had a Km of 0.1214±0.07464 mM and a Vmax of 2.6695±0.3715 μM/min/mg. Enzymatic reactions in vitro showed that expressed FADSCf could form 80 mM of FAD per mg of enzyme after 21 hours under the following conditions: 0.5 mM FMN, 5 mM ATP and 10 mM Mg2+. Conclusion: Under optimized conditions (0.5 mM FMN, 5 mM ATP and 10 mM Mg2+), the production of FAD reached 80 mM per mg of FADSCf after a 21-hour reaction. Our results indicate that purified recombinant FADSCf can be used for the biosynthesis of FAD.

Bacteria commonly exhibit social activities through acyl-homoserine lactones (AHLs)-based quorum sensing (QS) systems to form their unique social network. The sigma factor RpoS is an important regulator that controls QS system in different bacteria. However, the upstream of RpoS involving regulation on QS system remains unclear. In Escherichia coli RpoS is regulated by stringent starvation protein A (SspA), which is dependent of histone-like nucleoid structuring protein (H-NS). To date, the connection between SspA and QS system is essentially unknown. Here, we characterized a typical LuxI/LuxR-type QS system in marine bacterium Pseudoalteromonas sp. T1lg65 which can produce four types of AHLs. The luxI encoding AHLs synthase and luxR encoding AHLs-responsive receptor are co-transcribed, providing advantages in rapidly amplifying QS signaling. Notably, SspA positively regulated luxI/luxR transcription by activating RpoS expression, which is mediated by H-NS. Interestingly, LuxR in turn positively regulated SspA expression. Therefore, SspA and QS system constitute a mutual positive regulation loop in T1lg65. In view of the crucial roles of SspA and QS system in environmental adaption, we believe that the improvement of bacterial tolerance to marine environments could be related to rapidly tuning SspA-involved QS programming.

Despite the conservative DNA sequences among LuxI (Acyl Homoserine Lactones synthase gene) homologs, structure-product relationship of AHL synthase remains to be elucidated. In this study, through degenerate primers and in vitro expression methods, we collected the information of the gene sequences and AHL profiles from nine LuxIs among Ensifer adhaerens strains. The chromosome-encoded LuxI (C-LuxI) distinguished themselves from the plasmid-encoded ones (P-LuxI) not only in the DNA sequences, but also in AHL profiles. The C-LuxIs produced only C14-HSL, while the P-LuxIs produced predominantly C8-HSL and 3-oxo-C8-HSL. Sequence-product relationship analysis updated our recognition of the role of T140 (EsaI) in the 3-oxo-HSL production. Computational calculation based on 3D structures of these LuxIs revealed the linear relationship between the chain length and the affinity of amides to AHL synthase in C-LuxI, which was not found in the P-LuxI. We hereby proposed the linear docking affinity as a criterion for the prediction of long-chain AHL production by an AHL synthase. This study extends our understanding on the structure-product relationship of AHL synthases and revealed the distinct chromosome and plasmid origin of this enzyme among E. adhaerens .

Prodiginines are a family of red-pigmented secondary metabolites with multiple biological activities. The biosynthesis of prodiginines is affected by various physiological and environmental factors. Thus, prodiginine biosynthesis regulation is highly complex and multifaceted. Although the regulatory mechanism for prodiginine biosynthesis has been extensively studied in Serratia and Streptomyces species, little is known about that in the marine betaproteobacterium Pseudoalteromonas. In this study, we report that stringent starvation protein A (SspA), an RNA polymerase-associated regulatory protein, is required for the biosynthesis of prodiginine in Pseudoalteromonas sp. strain R3. The strain lacking sspA (ΔsspA) fails to produce prodiginine, which resulted from the downregulation of the prodiginine biosynthetic gene (pig) cluster. The effect of SspA on prodiginine biosynthesis is independent of histone-like nucleoid structuring protein (H-NS) and RpoS (σS). Further analysis demonstrates that the ΔsspA strain has a significant decrease in the transcription of the siderophore biosynthesis gene (pvd) cluster, leading to the inhibition of siderophore production and iron uptake. The ΔsspA strain regains the ability to synthesize prodiginine by cocultivation with siderophore producers or the addition of iron. Therefore, we conclude that SspA-regulated prodiginine biosynthesis is due to decreased siderophore levels and iron deficiency. We further show that the iron homeostasis master regulator Fur is also essential for pig transcription and prodiginine biosynthesis. Overall, our results suggest that SspA indirectly regulates the biosynthesis of prodiginine, which is mediated by the siderophore-dependent iron uptake pathway. IMPORTANCE The red-pigmented prodiginines are attracting increasing interest due to their broad biological activities. As with many secondary metabolites, the biosynthesis of prodiginines is regulated by both environmental and physiological factors. At present, studies on the regulation of prodiginine biosynthesis are mainly restricted to Serratia and Streptomyces species. This work focused on the regulatory mechanism of prodiginine biosynthesis in Pseudoalteromonas sp. R3. We found that stringent starvation protein A (SspA) positively regulates prodiginine biosynthesis via affecting the siderophore-dependent iron uptake pathway. The connections among SspA, iron homeostasis, and prodiginine biosynthesis were investigated. These findings uncover a novel regulatory mechanism for prodigiosin biosynthesis.

Photodynamic therapy (PDT) as a rising platform of the cancer treatment method is receiving increased attention. Through systematic evaluation of halogen substitution on aza-4,4-difluoro-4-bora-3a,...

Members of the Sphingomonadales are renowned for their ability to degrade polycyclic aromatic hydrocarbons (PAHs). However, little is known about the regulatory mechanisms of the degradative pathway. Using cross-feeding bioassay, a functional LuxI/LuxR-type acyl-homoserine lactone (AHL)-mediated quorum sensing (QS) system was identified from Croceicoccus naphthovorans PQ-2, a member of the order Sphingomonadales. Inactivation of the QS system resulted in a significant decrease in PAHs degradation. The QS system positively controlled the expression of three PAH-degrading genes (ahdA1e, xylE and xylG) and a regulatory gene ardR, which are located on the large plasmid. Interestingly, the transcription levels of these three PAH-degrading genes were significantly down-regulated in the ardR mutant. In addition, bacterial cell surface hydrophobicity and cell morphology were altered in the QS-deficient mutant. Therefore, the QS system in strain PQ-2 positively regulates PAH degradation via two mechanisms: (i) by induction of PAH-degrading genes directly and/or indirectly, and (ii) by an increase of bacterial cell surface hydrophobicity. The findings of this study improve our understanding of how the QS system influences the degradation of PAHs, therefore facilitating the development of new strategies for the bioremediation of PAHs.

In rod-shaped Gram-negative bacteria, penicillin binding protein 1a (PBP1a) and 1b (PBP1b) form peptidoglycan-synthesizing complexes with the outer membrane lipoprotein LpoA and LpoB, respectively. Escherichia coli mutants lacking PBP1b/LpoB are sicker than those lacking PBP1a/LpoA. However, we previously found that mutants lacking PBP1a/LpoA but not PBP1b/LpoB are deleterious in Shewanella oneidensis. Here, we show that S. oneidensis PBP1a (SoPBP1a) contains conserved signature motifs with its E. coli counterpart, EcPBP1a. Although EcPBP1a play a less prominent role in E. coli, it is capable of substituting for the SoPBP1a in a manner dependent on SoLpoA. In S. oneidensis, expression of PBP1b is lower than PBP1a, and therefore the additional expression of SoPBP1b at low levels can functionally compensate for the absence of SoPBP1a. Importantly, S. oneidensis PBP1a variants lacking either glycosyltransferase (GTase) or transpeptidase (TPase) activity fail to maintain normal morphology and cell envelope integrity. Similarly, SoPBP1b variants also fail to compensate for the loss of SoPBP1a. Furthermore, overproduction of variants of SoPBP1a, but not SoPBP1b, has detrimental effects on cell morphology in S. oneidensis wild type cells. Overall, our results indicate that the combined enzymatic activities of SoPBP1a are essential for cell wall homeostasis.

Polymyxins are cationic antimicrobial peptides used as the last-line therapy to treat multidrug-resistant Gram-negative bacterial infections. The bactericidal activity of polymyxins against Gram-negative bacteria relies on the electrostatic interaction between the positively charged polymyxins and the negatively charged lipid A of lipopolysaccharide (LPS). Given that Gram-positive bacteria lack an LPS-containing outer membrane, it is generally acknowledged that polymyxins are less active against Gram-positive bacteria. However, Gram-positive bacteria produce negatively charged teichoic acids, which may act as the target of polymyxins. More and more studies suggest that polymyxins have potential as a treatment for Gram-positive bacterial infection. This mini-review discusses recent advances in the mechanism of the antibacterial activity and resistance of polymyxins in Gram-positive bacteria.Key Points• Teichoic acids play a key role in the action of polymyxins on Gram-positive bacteria.• Polymyxin kills Gram-positive bacteria by disrupting cell surface and oxidative damage.• Modification of teichoic acids and phospholipids contributes to polymyxin resistance in Gram-positive bacteria.• Polymyxins have potential as a treatment for Gram-positive bacterial infection.

Piezoelectric actuator has the advantages of high rigidity, wide bandwidth, fast response and high resolution. Therefore, they are widely used in many micro and nano positioning applications. However, the hysteresis characteristic in the piezoelectric actuator (PEA) seriously affects its positioning accuracy and even causes instability. In this paper, a modified Prandtl-Ishlinskii (MPI) model, which can describe the rate asymmetric hysteresis of piezoelectric actuator, is studied. The hysteresis compensation is realized by using the rate dependent Prandtl-Iishlinskii model based on the improved Prandtl-Iishlinskii hysteresis model and the hysteresis characteristics of the driver measured in the laboratory under the frequency input of up to 100 Hz. In order to further reduce the error of feedforward compensation, a sliding mode controller is designed. The stability of the control system is proved by Lyapunov theory. The experimental results show that the linear error of the system is reduced from 10% to less than 1%, and the tracking error can also be reduced by 90%., Electrical engineering; Physics; Piezoelectric actuator, Precision positioning, Hysteresis, Prandtl-Ishlinskii.

Polymyxins are nonribosomal peptide antibiotics used as the last-resort drug for treatment of multidrug-resistant Gram-negative bacteria. However, strains that are resistant to polymyxins have emerged in many countries. Although several mechanisms for polymyxin resistance have been well described, there is little knowledge on the hydrolytic mechanism of polymyxin. Here, we identified a polymyxin-inactivating enzyme from Bacillus licheniformis strain DC-1 which was produced and secreted into the medium during entry into stationary phase. After purification, sequencing, and heterologous expression, we found that the alkaline protease Apr is responsible for inactivation of polymyxins. Analysis of inactivation products demonstrated that Apr cleaves polymyxin E at two peptide bonds: one is between the tripeptide side chain and the cyclic heptapeptide ring, the other between l-Thr and l-α-γ-diaminobutyric acid (l-Dab) within the cyclic heptapeptide ring. Apr is highly conserved among several genera of Gram-positive bacteria, including Bacillus and Paenibacillus. It is noteworthy that two peptidases S8 from Gram-negative bacteria shared high levels of sequence identity with Apr. Our results indicate that polymyxin resistance may result from inactivation of antibiotics by hydrolysis.

Bacteria usually produce, release and detect quorum sensing (QS)-based signal molecules, and successively orchestrate gene expression in respond to environmental changes. Pseudoalteromonas are typical marine bacteria, but knowledge on their QS systems is extremely fragmentary. In this study, genome sequencing of Pseudoalteromonas sp. R3 was performed. Accordingly, a QS working model including three sets of hierarchically organized QS systems was proposed in strain R3. Among them, the typical LuxI/R-type QS system using acyl-homoserine lactones (AHLs) as signal molecules was characterized. Sequence similarity analysis indicated luxI encoding AHLs synthase is novel. The luxR encoding AHLs receptor is directly adjacent to luxI downstream. Notably, mutagenesis demonstrated LuxI and LuxR affect each other at transcriptional level, and both control the AHLs formation. Interestingly, it was found that LuxI/R-type QS system positively involves resistance to streptomycin. Thin-layer chromatography analysis showed strain R3 can produce 3-OH-C6-HSL and C8-HSL, which was supported by heterologous expression of LuxI in Escherichia coli. Sequence alignment analysis indicated that the N-terminal region of LuxI is more conservative than the C-terminal region, revealing the importance of N-terminal region in AHLs synthesis. The obtained findings enrich our knowledge on LuxI/R-type QS system in Pseudoalteromonas and its regulation on adaptation to marine environments.

Homocamptothecin is emerging as an important topoisomerase I inhibitor originating in natural product camptothecin. We report the modifications and SAR of homocamptothecin on position C10 to develop potent topoisomerase I inhibitors for anticancer drug discovery. Based on click chemistry, twenty-one 1,2,3-triazole-substituted homocamptothecin derivatives were readily synthesized in two steps. For A549, cycloalkyl- and alkyl-substituted compounds 6j, 6l, and 6o revealed highly antiproliferative inhibitory activities with IC50 value of 30, 30, and 50 nm, respectively. In addition, cyclopropyl 6j exhibited greater Topo I inhibitory activity than 20(S)-Camptothecin, which indicated suitability for further drug development.

The commonly believed mechanism of colistin against Gram-negative bacteria is to cause cell membrane lysis, whereas the mechanism of colistin against Gram-positive bacteria is extremely fragmented. In this study, we found that colistin treatment on Bacillus subtilis WB800, Paenibacillus polymyxa C12 and Paenibacillus polymyxa ATCC842 enhances not only the activities of α-ketoglutaric dehydrogenase and malate dehydrogenase in tricarboxylic acid (TCA) cycle, but also the relative expression levels of their encoding genes. Additionally, the oxaloacetate concentration also increases. Interestingly, the analysis of the relative expression of genes specific for respiratory chain showed that colistin treatment stimulates the respiratory chain in Gram-positive bacteria. Accordingly, the NAD+/NADH ratio increases and the oxidative level is then boosted up. As a result, the intensive oxidative damages are induced in Gram-positive bacteria and cells are killed. Notably, both rotenone and oligomycin, respectively, inhibiting NADH dehydrogenase and phosphorylation on respiratory chain can downgrade oxidative stress formation, thus alleviating the colistin-induced killing of Gram-positive cells. Besides, thiourea-based scavenging for reactive oxygen species also rescues the colistin-subjected cells. These data collectively demonstrate that colistin stimulates both TCA cycle and respiratory chain in Gram-positive bacteria, leading to the enhancement of NADH metabolism and resulting in the generation of oxidative damages in Gram-positive cells. Our studies provide a better understanding of antibacterial mechanism of colistin against Gram-positive bacteria, which is important for knowledge on bacterial resistance to colistin happening via the inhibition of respiratory chain and manipulation of its production.

A Gram-strain-negative, coccoid bacterium, lacking bacteriochlorophyll, designated strain T1lg56T, was isolated from a sediment sample collected from Ximen island mangrove forest, Zhejiang province, China. Cells were halotolerant, and catalase- and oxidase-positive. Growth was observed at 18–42 °C (optimum, 35 °C), at pH 6.0–9.5 (optimum, pH 6.5) and in the presence of 0–15 % (w/v) NaCl (optimum, 2–5 %). The major cellular fatty acids were C18 : 1ω7c and C16 : 0. The polar lipid profile of strain T1lg56T consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylmonomethylethanolamine, two unidentified phospholipids and five unidentified lipids. Ubiquinone-10 was the predominant respiratory quinone. The assimilation of the substrates in the API 20NE kit was positive in strain T1lg56T. The DNA G+C content of strain T1lg56T was 67.2 mol%. 16S rRNA gene sequence analysis indicated that strain T1lg56T was a member of family Rhodobacteraceae and was closely related to Poseidonocella pacifica KMM 9010T, with 95.7 % similarity to the type strain. Phylogenetic analysis showed that strain T1lg56T formed a separate evolutionary branch, and was parallel to other related genera of Rhodobacteraceae . Its phylogenetic distinctiveness and distinguishing phenotypic characteristics supported that strain T1lg56T represents a novel genus of the family Rhodobacteraceae , for which the name Mangrovicoccus ximenensis gen. nov., sp. nov. is proposed. The type strain is T1lg56T (=CCTCC AB 2016238T=KCTC 52623T).

Production of chromosome-encoded β-lactamases confers resistance to β-lactams in many Gram-negative bacteria. Some inducible β-lactamases, especially the class C β-lactamase AmpC in Enterobacteriaceae, share a common regulatory mechanism, the ampR-ampC paradigm. Induction of ampC is intimately linked to peptidoglycan recycling, and the LysR-type transcriptional regulator AmpR plays a central role in the process. However, our previous studies have demonstrated that the expression of class D β-lactamase gene blaA in Shewanella oneidensis is distinct from the established paradigm since an AmpR homolog is absent and major peptidoglycan recycling enzymes play opposite roles in β-lactamase expression. Given that lytic transglycosylases (LTs), a class of peptidoglycan hydrolases cleaving the β-1,4 glycosidic linkage in glycan strands of peptidoglycan, can disturb peptidoglycan recycling, and thus may affect induction of blaA. In this study, we investigated impacts of such enzymes on susceptibility to β-lactams. Deletion of three LTs (SltY, MltB and MltB2) increased β-lactam resistance, while four other LTs (MltD, MltD2, MltF, and Slt2) seemed dispensable to β-lactam resistance. The double LT mutants ΔmltBΔmltB2 and ΔsltYΔmltB2 had β-lactam resistance stronger than any of the single mutants. Deletion of ampG (encoding permease AmpG) and mrcA (encoding penicillin binding protein 1a, PBP1a) from both double LT mutants further increased the resistance to β-lactams. Notably, all increased β-lactam resistance phenotypes were in accordance with enhanced blaA expression. Although significant, the increase in β-lactamase activity after inactivating LTs is much lower than that produced by PBP1a inactivation. Our data implicate that LTs play important roles in blaA expression in S. oneidensis.

High molecular weight penicillin-binding proteins (PBPs) are responsible for the biosynthesis of peptidoglycan. In Escherichia coli, PBP1a and PBP1b form multienzyme peptidoglycan-synthesizing complexes with outer membrane lipoproteins LpoA and LpoB, respectively. The two complexes appear to be largely redundant, although their distinct physiological roles remain unclear. PBP1a/LpoA and PBP1b/LpoB also exist in Shewanella oneidensis strain MR-1, but effects of the two complexes on aerobic growth and β-lactam resistance are quite different. In this study, the phenotypes of strains lacking a certain complex in S. oneidensis were compared. Deletion of PBP1a/LpoA caused aberrant cell morphology (including branches and bulges), enhanced sensitivity to various envelope stresses and outer membrane permeability. On the contrary, strains lacking PBP1b/LpoB displayed phenotypes similar to the wild type.

Polymyxin E or colistin, produced by Paenibacillus polymyxa, is an important antibiotic against Gram-negative pathogens. The objective of this study is to evaluate the effect of starch in fermentation medium on colistin biosynthesis in P. polymyxa. The results indicated that replacement of glucose by starch stimulated colistin production and biosynthesis rate. Overall, the stimulation extent was starch concentration-dependent. As expected, addition of starch induced the expression of amyE encoding amylase and increased amylase activity in fermentation solution. Additionally, replacement of glucose by starch resulted in residue reducing sugar and pH of fermentation mixture low relative to glucose as the sole sugar source. At the molecular level, it was found that replacement of glucose by starch has enhanced the relative expression level of ccpA encoding catabolite control protein A. Therefore, the repression of starch utilization by glucose could be probably relieved. In addition, use of starch stimulated the expression of regulatory gene spo0A but repressed the expression of another regulatory gene abrB. As a result, the expression of genes directly involved in colistin biosynthesis and secretion increased, indicating that at the transcriptional level spo0A and abrB played opposite roles in regulating colistin biosynthesis in P. polymyxa. Taken together, our data demonstrated that starch instead of glucose can promote colistin production probably by affecting the expression of colistin biosynthesis-related genes, as well as reducing the repression of glucose to a secondary metabolic product.

To date, the mechanisms underlying the flavoprotein l-amino acid oxidase (LAAO) accumulation in cells remain unclear. In this study, using LAAO-producer Pseudoalteromonas spp. as model organisms, we found that the cell biomass is negatively associated with LAAO accumulation, whereas the LAAO accumulation is positively associated with the reactive oxygen species (ROS)-scavenging capability. The expression levels of ROS-scavenging-associated genes were up-regulated with LAAO accumulation in Pseudoalteromonas cells, which is presumably due to the requirement for the removal of LAAO-induced ROS. Exogenous H2O2 exposure experiment supported that the ROS-scavenging system is associated with LAAO accumulation in Pseudoalteromonas. All these observations indicate that ROS-scavenging capacity determines LAAO accumulation in bacterial cells. Our results shed a light on understanding the mechanism underlying controlling and adapting to LAAO accumulation in Pseudoalteromonas. Besides, our findings are critical to the improvement of heterologous expression of active LAAO in the future.

A Gram-stain-negative, coccoid, yellow, non-motile, aerobic bacterium, designated strain S36T, was isolated from soil of the Xixi wetland in Zhejiang province, PR China. Phylogenetic analysis, based on 16S rRNA gene sequences, revealed that strain S36T could represent a novel species of genus Altererythrobacter showing highest similarity to Altererythrobacter atlanticus 26DY36T (96.31 % 16S rRNA gene sequence similarity). The temperature, pH and NaCl concentration ranges for growth were 10–37 °C (optimum 32 °C), pH 5.0–10.0 (optimum pH 7.0) and 0.5–3 % (optimum 1 %, w/v), respectively. The predominant respiratory quinone of strain S36T was Q-10. The major fatty acids were C16 : 0, C17 : 1ω6c, C18 : 1ω7c and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). The G+C content of the genomic DNA was 62.7 mol%. These data all support the affiliation of strain S36T to the genus Altererythrobacter . The polar lipids profile of strain S36T comprised diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified phospholipids and two unidentified glycolipids. The results of physiological and biochemical tests allowed differentiation of strain S36T from other members of the genus Altererythrobacter . Therefore, strain S36T represents a novel species of the genus Altererythrobacter , for which the name Altererythrobacter xixiisoli sp. nov. is proposed; the type strain is S36T (=CGMCC 1.12804T=NBRC 110413T).

Methanopyrus spp. are usually isolated from harsh niches, such as high osmotic pressure and extreme temperature. However, the molecular mechanisms for their environmental adaption are poorly understood. Archaeal species is commonly considered as primitive organism. The evolutional placement of archaea is a fundamental and intriguing scientific question. We sequenced the genomes of Methanopyrus strains SNP6 and KOL6 isolated from the Atlantic and Iceland, respectively. Comparative genomic analysis revealed genetic diversity and instability implicated in niche adaption, including a number of transporter- and integrase/transposase-related genes. Pan-genome analysis also defined the gene pool of Methanopyrus spp., in addition of ~120-Kb genomic region of plasticity impacting cognate genomic architecture. We believe that Methanopyrus genomics could facilitate efficient investigation/recognition of archaeal phylogenetic diverse patterns, as well as improve understanding of biological roles and significance of these versatile microbes.

It is known that some bacteria, especially members of the family Rhizobiaceae, have multiple N-acyl homoserine lactones (AHL) synthase genes and produce multiple AHL signals. However, how bacteria selectively utilize these multiple genes and signals to cope with changing environments is poorly understood. Ensifer adhaerens is an important microorganism in terms of biotechnology, ecology and evolutionary. In this study, we investigated the AHL-based QS system of E. adhaerens X097 and its response to different lifestyles or nutrients. Draft genome sequence data indicated that X097 harbored three distinct AHL synthase genes (ensI1, 2, 3) and seven luxR homologs, which was different from other E. adhaerens strains. In vitro expression indicated that plasmid-encoded ensI1 and ensI2 directed production of multiple AHLs, while chromosome-encoded ensI3 only directed production of C14-HSL. Predicted three dimensional structure of EnsI3 was quite different from that of EnsI1 and EnsI2. X097 produced different AHL profiles in Luria-Bertani (LB) and NFB medium, under biofilm and planktonic lifestyle, respectively. Notably, expression of ensI1 and ensI2 but not ensI3 is hypersensitive to different lifestyles and nutrients. The hypersensitive response of plasmid-encoded AHL synthase genes to different culture conditions may shed a light on the phylogenetic development of AHL synthase genes in Rhizobiaceae family.

Historically, the colistin has been thought to kill bacteria through membrane lysis. Here, we present an alternative mechanism that colistin induces rapidPaenibacillus polymyxadeath through reactive oxygen species production. This significantly augments our understanding of the mechanism of colistin action, which is critical knowledge toward the yield development of colistin in the future.

Quorum sensing (QS) enables single-celled bacteria to communicate with chemical signals in order to synchronize group-level bacterial behavior. Pseudoalteromonas are marine bacteria found in versatile environments, of which QS regulation for their habitat adaptation is extremely fragmentary. To distinguish genes required for QS regulation in Pseudoalteromonas, comparative genomics was deployed to define the pan-genomics for twelve isolates and previously-sequenced genomes, of which acyl-homoserine lactone (AHL)-based QS traits were characterized. Additionally, transposon mutagenesis was used to identify the essential QS regulatory genes in the selected Pseudoalteromonas isolate. A remarkable feature showed that AHL-based colorization intensity of biosensors induced by Pseudoalteromonas most likely correlates with QS regulators genetic heterogeneity within the genus. This is supported by the relative expression levels of two of the main QS regulatory genes (luxO and rpoN) analyzed in representative Pseudoalteromonas isolates. Notably, comprehensive QS regulatory schema and the working model proposed in Pseudoalteromonas seem to phylogenetically include the network architectures derived from Escherichia coli, Pseudomonas, and Vibrio. Several associated genes were mapped by transposon mutagenesis. Among them, a right origin-binding protein-encoding gene (robp) was functionally identified as a positive QS regulatory gene. This gene lies on a genomic instable region and exists in the aforementioned bioinformatically recruited QS regulatory schema. The obtained data emphasize that the distinctly- and hierarchically-organized mechanisms probably target QS association in Pseudoalteromonas dynamic genomes, thus leading to bacterial ability to accommodate their adaption fitness and survival advantages.

Aim The aim of this study was to describe and evaluate a surgical technique for the treatment of mallet finger fractures using a K-wire stabilization of the distal interphalangeal (DIP) joint and another K-wire pressing the bone fragment. Methods Between June 2007 and March 2014, 41 patients (28 men, 13 women) with isolated closed mallet finger fracture were treated using two K-wires. In the cohort, the mean joint surface involvement was 44.3% (range: 28–62%). With a mean period of 23.6 months, patient follow-up lasted 13–34 months. The fingers were evaluated for loss of extension and flexion of the (DIP) joints. The results were graded using Crawford's criteria. Results Union of all fractures took place at an average of 5.5 weeks after the surgical procedure. Average extension lag was 4°, and active flexion 71°. According to the Crawford rating scale, 35 fingers were excellent, four were good, one was fair, and one was poor. Conclusions Pressing fixation of the bone fragment with the end of a K-wire was a useful technique in the treatment of mallet finger fractures. Type of study/level of evidence Therapeutic IV.

The gamma-aminobutyric acid type-A (GABA(A)) receptor plays a major role in inhibitory neurotransmissions. Intronic SNPs and haplotypes in GABRB2, the gene for GABA(A) receptor beta(2) subunit, are associated with schizophrenia and correlated with the expression of two alternatively spliced beta(2) isoforms. In the present study, using chimpanzee as an ancestral reference, high frequencies were observed for the derived (D) alleles of the four SNPs rs6556547, rs187269, rs1816071 and rs1816072 in GABRB2, suggesting the occurrence of positive selection for these derived alleles. Coalescence-based simulation showed that the population frequency spectra and the frequencies of H56, the haplotype having all four D alleles, significantly deviated from neutral-evolution expectation in various demographic models. Haplotypes containing the derived allele of rs1816072 displayed significantly less diversity compared to haplotypes containing its ancestral allele, further supporting positive selection. The variations in DD-genotype frequencies in five human populations provided a snapshot of the evolutionary history, which suggested that the positive selections of the D alleles are recent and likely ongoing. The divergence between the DD-genotype profiles of schizophrenic and control samples pointed to the schizophrenia-relevance of positive selections, with the schizophrenic samples showing weakened selections compared to the controls. These DD-genotypes were previously found to increase the expression of beta(2), especially its long isoform. Electrophysiological analysis showed that this long beta(2) isoform favored by the positive selections is more sensitive than the short isoform to the inhibition of GABA(A) receptor function by energy depletion. These findings represent the first demonstration of positive selection in a schizophrenia-associated gene.