A marine-derived fatty acid targets the cell membrane of Gram-positive bacteria.

IMPORTANCE: With the lack of new antibiotics in the drug discovery pipeline, coupled with accelerated evolution of antibiotic resistance, new sources of antibiotics that target pathogens of clinical importance are paramount. Here, we use bacterial cytological profiling to identify the mechanism of action of the monounsaturated fatty acid (Z)-13-methyltetra-4-decenoic acid isolated from the marine bacterium Olleya marilimosa with antibacterial effects against Gram-positive bacteria. The fatty acid antibiotic was found to rapidly destabilize the cell membrane by pore formation and membrane aggregation in Bacillus subtilis, suggesting that this fatty acid may be a promising adjuvant used in combination to enhance antibiotic sensitivity.

Hydroxychloroquine-Loaded Chitosan Nanoparticles Induce Anticancer Activity in A549 Lung Cancer Cells: Design, BSA Binding, Molecular Docking, Mechanistic, and Biological Evaluation.

The current study describes the encapsulation of hydroxychloroquine, widely used in traditional medicine due to its diverse pharmacological and medicinal uses, in chitosan nanoparticles (CNPs). This work aims to combine the HCQ drug with CS NPs to generate a novel nanocomposite with improved characteristics and bioavailability. HCQ@CS NPs are roughly shaped like roadways and have a smooth surface with an average size of 159.3 ± 7.1 nm, a PDI of 0.224 ± 0.101, and a zeta potential of +46.6 ± 0.8 mV. To aid in the development of pharmaceutical systems for use in cancer therapy, the binding mechanism and affinity of the interaction between HCQ and HCQ@CS NPs and BSA were examined using stopped-flow and other spectroscopic approaches, supplemented by molecular docking analysis. HCQ and HCQ@CS NPs binding with BSA is driven by a ground-state complex formation that may be accompanied by a non-radiative energy transfer process, and binding constants indicate that HCQ@CS NPs-BSA was more stable than HCQ-BSA. The stopped-flow analysis demonstrated that, in addition to increasing BSA affinity, the nanoformulation HCQ@CS NPS changes the binding process and may open new routes for interaction. Docking experiments verified the development of the HCQ-BSA complex, with HCQ binding to site I on the BSA structure, primarily with the amino acids, Thr 578, Gln 579, Gln 525, Tyr 400, and Asn 404. Furthermore, the nanoformulation HCQ@CS NPS not only increased cytotoxicity against the A549 lung cancer cell line (IC50 = 28.57 ± 1.72 µg/mL) compared to HCQ (102.21 ± 0.67 µg/mL), but also exhibited higher antibacterial activity against both Gram-positive and Gram-negative bacteria when compared to HCQ and chloramphenicol, which is in agreement with the binding constants. The nanoformulation developed in this study may offer a viable therapy option for A549 lung cancer.

Antibacterial and immunoregulatory activity of an antimicrobial peptide hepcidin in loach (Misgurnus anguillicaudatus).

Antimicrobial peptides (AMPs) are members of humoral immunity and particpate in resisting microbial invasion. In this study, an AMP gene hepcidin was obtained from the oriental loach Misgurnus anguillicaudatus and named Ma-Hep. This Ma-Hep encodes a peptide of 90 amino acids, with a predicted active peptide segment (Ma-sHep) of 25 amino acids at C terminus. Stimulation by a bacterial pathogen Aeromonas hydrophila resulted in significant up-regulation of Ma-Hep transcripts in loach midgut, head kidney, and gill. Ma-Hep and Ma-sHep proteins were expressed in Pichia pastoris and their antibacterial activity was examined. Results showed that Ma-sHep possessed stronger antibacterial activity against various Gram-positive and Gram-negative bacteria, compared to Ma-Hep. Scanning electron microscopy showed that Ma-sHep might kill bacteria by destroying bacterial cell membranes. Moreover, we found that Ma-sHep had an inhibitory effect on blood cell apoptosis induced by A. hydrophila and facilitated the bacterial phagocytosis and clearance in loach. Histopathological analysis indicated Ma-sHep could protect liver and gut of loach from bacterial infection. Ma-sHep has high thermal stability and PH stability, which is conducive to further feed addition. Feed supplemented with Ma-sHep expressing yeast improved the intestinal flora of loach by increasing the dominant bacteria and decreasing the harmful bacteria. Feed supplemented with Ma-sHep expressing yeast also regulated the expression of inflammatory related factors in various tissues of loach and reduced the mortality of loach upon bacterial infection. These findings show that the antibacterial peptide Ma-sHep is involved in the antibacterial defense of loach and can be used as a candidate for new antimicrobial agents in aquaculture.

Effects of grape seed oil nanoemulsion on physicochemical and antibacterial properties of gelatin­sodium alginate film blends.

The present study aimed to evaluate the impact of incorporating grape seed oil (GSO) nanoemulsion (NE) at varying concentrations into the film matrix on the physicochemical and antimicrobial properties of the resulting films. In this study, ultrasonic treatment was used to prepare GSO-NE, and different levels (2, 4, and 6%) of nanoemulsioned GSO were incorporated into gelatin (Ge)/sodium alginate (SA)-based films to produce films with improved physical and antibacterial properties. The results revealed that incorporation of GSO-NE at 6% concentration decreased the tensile strength (TS) and puncture force (PF) significantly (p < 0.05). The whiteness index (WI) of the films decreased from 63.4 to 47.79, while the total color change (ΔE) increased significantly (p < 0.05) with the increase in GSO-NE concentration. Thermogravimetric analysis (TGA) results showed that GSO-NE at different concentrations had improved the thermal stability of Ge/SA-based films. The incorporation of GSO-NE into the films led to the formation of a slightly porous structure. The incorporation of GSO-NE at 4 and 6 % concentrations decreased the water vapor permeability (WVP), moisture content (MC) %, and water solubility (WS) % significantly (p < 0.05). All composite films exhibited hydrophobic surfaces with contact angles θ > 90°. Ge/SA/GSO-NE films were found to be effective against both Gram-positive and Gram-negative bacteria. The prepared active films containing GSO-NE had a high potential for preventing food spoilage in food packaging.

Gold(I) selenium N-heterocyclic carbene complexes as potent antibacterial agents against multidrug-resistant gram-negative bacteria via inhibiting thioredoxin reductase.

Multidrug-resistant (MDR) Gram-negative bacteria have become a global threat to human life and health, and novel antibiotics are urgently needed. The thioredoxin (Trx) system can be used as an antibacterial target to combat MDR bacteria. Here, we found that two active gold(I) selenium N-heterocyclic carbene complexes H7 and H8 show more promising antibacterial effects against MDR bacteria than auranofin. Both H7 and H8 irreversibly inhibit the bacterial TrxR activity via targeting the redox-active motif, abolishing the capacity of TrxR to quench reactive oxygen species (ROS) and finally leading to oxidative stress. The increased cellular superoxide radical levels impact a variety of functions necessary for bacterial survival, such as cellular redox balance, cell membrane integrity, amino acid metabolism, and lipid peroxidation. In vivo data present much better antibacterial activity of H7 and H8 than auranofin, promoting the wound healing and prolonging the survival time of Carbapenem-resistant Acinetobacter baumannii (CRAB) induced peritonitis. Most notably in this study, we revealed the influence of gold(I) complexes on both the Trx system and the cellular metabolic states to better understand their killing mechanism and to support further antibacterial drug design.

Lichen Depsidones with Biological Interest.

Depsidones are some of the most abundant secondary metabolites produced by lichens. These compounds have aroused great pharmacological interest due to their activities as antioxidants, antimicrobial, and cytotoxic agents. Hence, this paper aims to provide up-to-date knowledge including an overview of the potential biological interest of lichen depsidones. So far, the most studied depsidones are fumarprotocetraric acid, lobaric acid, norstictic acid, physodic acid, salazinic acid, and stictic acid. Their pharmacological activities have been mainly investigated in in vitro studies and, to a lesser extent, in in vivo studies. No clinical trials have been performed yet. Depsidones are promising cytotoxic agents that act against different cell lines of animal and human origin. Moreover, these compounds have shown antimicrobial activity against both Gram-positive and Gram-negative bacteria and fungi, mainly Candida spp. Furthermore, depsidones have antioxidant properties as revealed in oxidative stress in vitro and in vivo models. Future research should be focused on further investigating the mechanism of action of depsidones and in evaluating new potential actions as well as other depsidones that have not been studied yet from a pharmacological perspective. Likewise, more in vivo studies are prerequisite, and clinical trials for the most promising depsidones are encouraged.

Tremella fuciformis Polysaccharides Inhibited Colonic Inflammation in Dextran Sulfate Sodium-Treated Mice via Foxp3+ T Cells, Gut Microbiota, and Bacterial Metabolites.

Tremella fuciformis is an edible medicinal mushroom, and its polysaccharide components are found to confer various health benefits. This study identified the protective effects of polysaccharides of Tremella fuciformis (TPs) against dextran sulfate sodium (DSS)-induced colitis in mice. High dose of TPs (HTPs) could prevent the colon from shortening, reduce activity of colonic myeloperoxidase and serum diamine oxidase (DAO), decrease the concentration of D-lactate, and alleviate the colonic tissue damage in colitic mice. HTPs treatment stimulated Foxp3+T cells, and promoted the production of anti-inflammatory cytokines whereas it reduced the production of pro-inflammatory and the portion of immunoglobulin A (IgA)-coated bacteria, which was related to modulation of immune responses. 16S rRNA sequencing analysis showed that TPs could significantly increase gut community diversity, and restore the relative abundances of Lactobacillus, Odoribacter, Helicobacter, Ruminococcaceae, and Marinifilaceae. According to metabolomic analysis, HTPs induced specific microbial metabolites akin to that in normal mice. Tyrosine biosynthesis, tryptophan metabolism, and bile acid metabolism were influenced in the HTPs group compared with those in the DSS group. HTPs could alleviate DSS-induced colitis by immunoregulation and restored the gut microbiota and microbial metabolites. The results indicated that HTPs have potential to be developed as a food supplement to ameliorate intestinal diseases.

Quercetin inhibits carbapenemase and efflux pump activities among carbapenem-resistant Gram-negative bacteria.

Rapid dissemination of carbapenem-resistant Gram-negative bacteria (CRGNB) is a global threat. Quercetin is known for its antimicrobial activity. In this study, carbapenemase and efflux pump inhibitory activities of quercetin were demonstrated against carbapenem-resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. Further, molecular docking was performed to elucidate molecular mechanisms of such inhibition. CRGNB, expressing one of the carbapenemases, demonstrated significant inhibition of carbapenemase activity when pre-incubated with 64 µg/ml quercetin. Moreover, acrB overexpressing enterobacterial isolates exhibited significant inhibition of efflux activity upon quercetin treatment. Molecular docking studies revealed stability of quercetin-carbapenemase complexes. (i) Virtual superimposition of quercetin onto meropenem, (ii) proximity of quercetin to attacking nucleophile and (iii) involvement of same amino acids that stabilize both meropenem and quercetin - indicated competition between quercetin and meropenem for ligand binding. Although quercetin and PAßN, a standard efflux pump inhibitor, docked at both central cavity and periplasmic drug binding sites of AcrB, they did not virtually superimpose on each other. However, sufficient release of Gibb's free energy and involvement of same set of amino acids in PAßN and quercetin stability predicted quercetin's efflux pump inhibitory potential. Hence, quercetin could be potential adjuvant therapeutics for CRGNB-mediated infections.

Low relapse rate of urinary tract infections from extended-spectrum beta-lactamase-producing bacteria in young children.

BACKGROUND: Extended-spectrum-beta-lactamase (ESBL)-producing bacteria are an increasingly important cause of urinary tract infections (UTIs) worldwide. We evaluated clinical characteristics and associated risk factors of UTIs in young children according to ESBL-producing status and relapse rates. METHODS: All urinary culture results in patients younger than 2 years old were assessed, and only children with febrile UTIs from gram-negative bacterial infections were reviewed. RESULTS: Of 845 episodes evaluated, 146 (17.3%) were caused by ESBL-positive bacteria. Significant differences were observed in previous UTIs, use of antibiotics or history of hospitalization within previous 3 months, and underlying urinary abnormalities between the ESBL UTI and non-ESBL UTI groups. After 2 weeks of treatment completion, UTI relapse occurred in 2.7% of children in the ESBL group and 1.1% of children in the non-ESBL group (P = 0.13). In the ESBL UTI group, relapse rate was not significantly different between patients treated with susceptible antibiotics and those treated with non-susceptible but clinically effective antibiotics. CONCLUSIONS: Previous history of UTI, antibiotic treatment, or hospitalization within previous 3 months and underlying disease are risk factors for ESBL UTI in children under 24 months of age. However, relapse rate was < 3% regardless of in vitro susceptibility of the treating antibiotics, as long as the antibiotics were clinically effective. We cautiously propose that we may continue the use of initial empirical antibiotics when a definite clinical response is observed, although further study is necessary to confirm the findings of this study.

Bioactive compounds and biological properties of Brazilian stingless bee honey have a strong relationship with the pollen floral origin.

Multivariate data analysis feasibility for the evaluation of Brazilian stingless bee honey (SBH) by pollen spectrum, bioactive compounds content, physicochemical, antioxidant and antimicrobial analysis was investigated. Levels of total and individual phenolics content were analyzed by HPLC-PDA. The antioxidant capacity was performed by 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH), oxygen radical absorption capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays. The total phenolic compounds from the thirty-two SBH was positively correlated with the antioxidant capacity. Bioactive compounds such as p-coumaric acid, quercetin, and hesperetin were identified in all the samples. Brazilian SBH shows more effective antibacterial activity against Gram-negative bacteria (E. coli and S. Typhimurium) compared to Gram-positive ones. Results also revealed that SBH could reach up to 45% higher antioxidant and biological activities than the traditional Apis mellifera honey. Chemometrics shows that chemical and biological properties of SBH have a strong relationship with the pollen botanical origin. Principal component analysis (PCA) grouped the honey into three categories with predominant pollen from Verbenaceae, Asteraceae and Sapindaceae families, confirming that SBH belonging to the same floral origin present similar characteristics.

Toll-like receptor 4: A promising therapeutic target for pneumonia caused by Gram-negative bacteria.

Gram-negative bacteria (GNB) emerge as important pathogens causing pulmonary infection, which can develop into sepsis due to bacterial resistance to antibiotics. GNB pneumonia poses a huge social and economic burden all over the world. During GNB infection in the lung, Toll-like receptor 4 (TLR4) can form a complex with MD2 and CD14 after recognizing lipopolysaccharide of GNB, initiate the MyD88- and TRIF-dependent signalling pathways and stimulate host non-specific immune response. In this review, we summarize recent progress in our understanding of the role of TLR4 in GNB pneumonia. The latest experimental results, especially in TLR4 knockout animals, suggest a promising potential of targeting TLR4 signalling pathway for the treatment of GNB pneumonia. Furthermore, we highlight the benefits of Traditional Chinese Medicine as novel candidates for the therapy of GNB pneumonia due to the modulation of TLR4 signalling pathway. Finally, we discuss the promise and challenge in the development of TLR4-based drugs for GNB pneumonia.

Chemical composition and bioactive properties of the wild edible plant Raphanus raphanistrum L.

Recently, there has been an interest regarding the consumption of wild edible plants in modern diets. However, there is still scarce information about several wild vegetables traditionally consumed. Therefore, this work aims on documenting the nutritional and chemical composition of wild radish (Raphanus raphanistrum L.), as well as its bioactive potential. Results showed that wild radish is a potential source of beneficial compounds, including vitamin E, polyunsaturated fatty acid (particularly α-linolenic acid) and different phenolic compounds, in which fourteen phenolics were identified, with kaempferol-3,7-O-di-rhamnoside being the most abundant. The bioactive potential was exploited using hydroethanolic and decoction extracts. Both proved to inhibit several Gram-positive and Gram-negative bacteria and revealed antioxidant activity, while cytotoxicity against non-tumor cell was not observed. In general, results evidence the interest in recovering the use of this wild vegetable as part of a varied diet, which can bring several health benefits.

Deciphering the Role of Intramolecular Networking in Cholic Acid-Peptide Conjugates on the Lipopolysaccharide Surface in Combating Gram-Negative Bacterial Infections.

The presence of lipopolysaccharide and emergence of drug resistance make the treatment of Gram-negative bacterial infections highly challenging. Herein, we present the synthesis and antibacterial activities of cholic acid-peptide conjugates (CAPs), demonstrating that valine-glycine dipeptide-derived CAP 3 is the most effective antimicrobial. Molecular dynamics simulations and structural analysis revealed that a precise intramolecular network of CAP 3 is maintained in the form of evolving edges, suggesting intramolecular connectivity. Further, we found high conformational rigidity in CAP 3 that confers maximum perturbations in bacterial membranes relative to other small molecules. Interestingly, CAP 3-coated catheters did not allow the formation of biofilms in mice, and treatment of wound infections with CAP 3 was able to clear the bacterial infection. Our results demonstrate that molecular conformation and internal connectivity are critical parameters to describe the antimicrobial nature of compounds, and the analysis presented here may serve as a general principle for the design of future antimicrobials.

Gram-negative bacilli-derived peptide antibiotics developed since 2000.

Gram-negative bacilli such as Pseudomonas spp., Pseudoalteromonas sp., Angiococcus sp., Archangium sp., Burkholderia spp., Chromobacterium sp., Chondromyces sp., Cystobacter sp., Jahnella sp., Janthinobacterium sp., Lysobacter spp., Paraliomyxa sp., Photobacterium spp., Photorhabdus sp., Pontibacter sp., Ruegeria sp., Serratia sp., Sorangium sp., Sphingomonas sp., and Xenorhabdus spp. produce an enormous array of short peptides of 30 residues or fewer that are potential pharmaceutical drugs and/or biocontrol agents. The need for novel lead antibiotic compounds is urgent due to increasing drug resistance, and this review summarises 150 Gram-negative bacilli-derived compounds reported since 2000, including 40 cyclic lipopeptides from Pseudomonas spp.; nine aromatic peptides; eight glycopeptides; 45 different cyclic lipopeptides; 24 linear lipopeptides; eight thiopeptides; one lasso peptide; ten typical cyclic peptides; and five standard linear peptides. The current and potential therapeutic applications of these peptides, including structures and antituberculotic, anti-cyanobacterial, antifungal, antibacterial, antiviral, insecticidal, and antiprotozoal activities are discussed.

Nanomaterial-microbe cross-talk: physicochemical principles and (patho)biological consequences.

The applications of nanoparticles (NPs) are increasing exponentially in consumer products, biotechnology and biomedicine, and humans, as well as the environment, are increasingly being exposed to NPs. Analogously, various (pathogenic) microorganisms are present at all the major exposure and entry sites for NPs in the human body as well as in environmental habitats. However, the field has just started to explore the complex interplay between NPs and microbes and the (patho)biological consequences. Based on recent insights, herein, we critically reviewed the available knowledge about the interaction of NPs with microbes and the analytical investigations including the latest intravital imaging tools. We have commented on how the NPs' characteristics influence complex formation with microorganisms, presented the underlying physicochemical forces, and provided examples of how this knowledge can be used to rationally control the NP-microbe interaction. We concluded by discussing the role of the biomolecule corona in NP-microbe crosstalk and speculated the impact of NP-microbe complex formation on the (patho)biological outcome and fate of microbial pathogens. The presented insights will not only support the field in engineering NPs with improved anti-microbial activity but also stimulate research on the biomedical and toxicological relevance of nanomaterial-microbiome complex formation for the anthropocene in general.

High-Throughput Flow Cytometry Screening of Multidrug Efflux Systems.

The resistance nodulation cell division (RND) family of proteins are inner membrane transporters that associate with periplasmic adaptor proteins and outer membrane porins to affect substrate transport from the cytosol and periplasm in Gram-negative bacteria. Various structurally diverse compounds are substrates of RND transporters. Along with their notable role in antibiotic resistance, these transporters are essential for niche colonization, quorum sensing, and virulence as well as for the removal of fatty acids and bile salts. As such, RNDs are an attractive target for antimicrobial development. However, while enhancing the utility of antibiotics with an RND inhibitor is an appealing concept, only a small core of chemotypes has been identified as efflux pump inhibitors (EPIs). Thus, our key objective is the development and validation of an efflux profiling and discovery strategy for RND model systems. Here we describe a flow cytometric dye accumulation assay that uses fluorescein diacetate (FDA) to interrogate the model Gram-negative pathogens Escherichia coli, Franscisella tularensis, and Burkholderia pseudomallei. Fluorochrome retention is increased in the presence of known efflux inhibitors and in RND deletion strains. The assay can be used in a high-throughput format to evaluate efflux of dye-substrate candidates and to screen chemical libraries for novel EPIs. Triaged compounds that inhibit efflux in pathogenic strains are tested for growth inhibition and antibiotic potentiation using microdilution culture plates in a select agent Biosafety Level-3 (BSL3) environment. This combined approach demonstrates the utility of flow cytometric analysis for efflux activity and provides a useful platform in which to characterize efflux in pathogenic Gram-negative bacteria. Screening small molecule libraries for novel EPI candidates offers the potential for the discovery of new classes of antibacterial compounds.

Characterization of the Serralysin-Like Gene of 'Candidatus Liberibacter solanacearum' Associated with Potato Zebra Chip Disease.

The nonculturable bacterium 'Candidatus Liberibacter solanacearum' is the causative agent of zebra chip disease in potato. Computational analysis of the 'Ca. L. solanacearum' genome revealed a serralysin-like gene based on conserved domains characteristic of genes encoding metalloprotease enzymes similar to serralysin. Serralysin and other serralysin family metalloprotease are typically characterized as virulence factors and are secreted by the type I secretion system (T1SS). The 'Ca. L. solanacearum' serralysin-like gene is located next to and divergently transcribed from genes encoding a T1SS. Based on its relationship to the T1SS and the role of other serralysin family proteases in circumventing host antimicrobial defenses, it was speculated that a functional 'Ca. L. solanacearum' serralysin-like protease could be a potent virulence factor. Gene expression analysis showed that, from weeks 2 to 6, the expression of the 'Ca. L. solanacearum' serralysin-like gene was at least twofold higher than week 1, indicating that gene expression stays high as the disease progresses. A previously constructed serralysin-deficient mutant of Serratia liquefaciens FK01, an endophyte associated with insects, as well as an Escherichia coli lacking serralysin production were used as surrogates for expression analysis of the 'Ca. L. solanacearum' serralysin-like gene. The LsoA and LsoB proteins were expressed as both intact proteins and chimeric S. liquefaciens-'Ca. L. solanacearum' serralysin-like proteins to facilitate secretion in the S. liquefaciens surrogate and as intact proteins or as a truncated LsoB protein containing just the putative catalytic domains in the E. coli surrogate. None of the 'Ca. L. solanacearum' protein constructs expressed in either surrogate demonstrated proteolytic activity in skim milk or zymogram assays, or in colorimetric assays using purified protein, suggesting that the 'Ca. L. solanacearum' serralysin-like gene does not encode a functional protease, or at least not in our surrogate systems.

Human resistin protects against endotoxic shock by blocking LPS-TLR4 interaction.

Helminths trigger multiple immunomodulatory pathways that can protect from sepsis. Human resistin (hRetn) is an immune cell-derived protein that is highly elevated in helminth infection and sepsis. However, the function of hRetn in sepsis, or whether hRetn influences helminth protection against sepsis, is unknown. Employing hRetn-expressing transgenic mice (hRETNTg+) and recombinant hRetn, we identify a therapeutic function for hRetn in lipopolysaccharide (LPS)-induced septic shock. hRetn promoted helminth-induced immunomodulation, with increased survival of Nippostrongylus brasiliensis (Nb)-infected hRETNTg+ mice after a fatal LPS dose compared with naive mice or Nb-infected hRETNTg- mice. Employing immunoprecipitation assays, hRETNTg+Tlr4-/- mice, and human immune cell culture, we demonstrate that hRetn binds the LPS receptor Toll-like receptor 4 (TLR4) through its N terminal and modulates STAT3 and TBK1 signaling, triggering a switch from proinflammatory to anti-inflammatory responses. Further, we generate hRetn N-terminal peptides that are able to block LPS proinflammatory function. Together, our studies identify a critical role for hRetn in blocking LPS function with important clinical significance in helminth-induced immunomodulation and sepsis.

Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorus predation.

Modification of essential bacterial peptidoglycan (PG)-containing cell walls can lead to antibiotic resistance; for example, ß-lactam resistance by L,D-transpeptidase activities. Predatory Bdellovibrio bacteriovorus are naturally antibacterial and combat infections by traversing, modifying and finally destroying walls of Gram-negative prey bacteria, modifying their own PG as they grow inside prey. Historically, these multi-enzymatic processes on two similar PG walls have proved challenging to elucidate. Here, with a PG-labelling approach utilizing timed pulses of multiple fluorescent D-amino acids, we illuminate dynamic changes that predator and prey walls go through during the different phases of bacteria:bacteria invasion. We show formation of a reinforced circular port-hole in the prey wall, L,D-transpeptidaseBd-mediated D-amino acid modifications strengthening prey PG during Bdellovibrio invasion, and a zonal mode of predator elongation. This process is followed by unconventional, multi-point and synchronous septation of the intracellular Bdellovibrio, accommodating odd- and even-numbered progeny formation by non-binary division.

Efficient computation of transfer free energies of amino acids in beta-barrel membrane proteins.

MOTIVATION: Transmembrane beta-barrel proteins (TMBs) serve a multitude of essential cellular functions in Gram-negative bacteria, mitochondria and chloroplasts. Transfer free energies (TFEs) of residues in the transmembrane (TM) region provides fundamental quantifications of thermodynamic stabilities of TMBs, which are important for the folding and the membrane insertion processes, and may help in understanding the structure-function relationship. However, experimental measurement of TFEs of TMBs is challenging. Although a recent computational method can be used to calculate TFEs, the results of which are in excellent agreement with experimentally measured values, this method does not scale up, and is limited to small TMBs. RESULTS: We have developed an approximation method that calculates TFEs of TM residues in TMBs accurately, with which depth-dependent transfer free energy profiles can be derived. Our results are in excellent agreement with experimental measurements. This method is efficient and applicable to all bacterial TMBs regardless of the size of the protein. AVAILABILITY AND IMPLEMENTATION: An online webserver is available at http://tanto.bioe.uic.edu/tmb-tfe . CONTACT: : jliang@uic.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.