In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes

Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC₂; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC₂, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg²⁺. We were able to purify a full complex, RbsABC₂, in the presence of stable, transition state mimics (ATP, Mg²⁺, and VO₄); a RbsAC complex in the presence of ADP and Mg²⁺; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC₂ shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters.     

Characterization of the Pivotal Carbon Metabolism of Streptococcus suis Serotype 2 under ex Vivo and Chemically Defined in Vitro Conditions by Isotopologue Profiling

Streptococcus suis is a neglected zoonotic pathogen that has to adapt to the nutritional requirements in the different host niches encountered during infection and establishment of invasive diseases. To dissect the central metabolic activity of S. suis under different conditions of nutrient availability, we performed labeling experiments starting from [¹³C]glucose specimens and analyzed the resulting isotopologue patterns in amino acids of S. suis grown under in vitro and ex vivo conditions. In combination with classical growth experiments, we found that S. suis is auxotrophic for Arg, Gln/Glu, His, Leu, and Trp in chemically defined medium. De novo biosynthesis was shown for Ala, Asp, Ser, and Thr at high rates and for Gly, Lys, Phe, Tyr, and Val at moderate or low rates, respectively. Glucose degradation occurred mainly by glycolysis and to a minor extent by the pentose phosphate pathway. Furthermore, the exclusive formation of oxaloacetate by phosphoenolpyruvate (PEP) carboxylation became evident from the patterns in de novo synthesized amino acids. Labeling experiments with S. suis grown ex vivo in blood or cerebrospinal fluid reflected the metabolic adaptation to these host niches with different nutrient availability; however, similar key metabolic activities were identified under these conditions. This points at the robustness of the core metabolic pathways in S. suis during the infection process. The crucial role of PEP carboxylation for growth of S. suis in the host was supported by experiments with a PEP carboxylase-deficient mutant strain in blood and cerebrospinal fluid.     

伦敦白胶和茶：一种既快又安全的用于透射电子显微镜的细菌样品制备方法

【目的】检测乌龙茶提取物是否可作为电子染色剂取代醋酸双氧铀用于细菌细胞染色,使其能在透射电子显微镜下进行观察。【方法】利用伦敦白胶对细菌样品(大肠杆菌和金黄色葡萄球菌)进行胶块的制备,再在复染铅与不复染铅这两种情况下对超薄切片样品进行3种不同染色剂的电子染色,之后在透射电子显微镜下观察比较其不同之处。这3种不同的染色剂分别是醋酸双氧铀、0.05%乌龙茶提取物以及0.1%乌龙茶提取物。首先将带有超薄切片样品的铜网悬浮于不同的待比较染液中10?15 min,若需进一步用柠檬酸铅复染,则将经3次蒸馏水冲洗过后的铜网再次悬浮于柠檬酸铅染液中8?10 min。【结果】复染铅的情况下,在透射电子显微镜下无论是大肠杆菌还是金黄色葡萄球菌,利用3种电子染色剂进行染色的结果均非常相似。【结论】实验结果表明,在观察细菌结构中,乌龙茶提取物可以替代醋酸双氧铀进行透射电子显微镜样品的电子染色。     

吲哚作为细菌细胞间信号分子的研究进展

吲哚广泛存在于自然界,目前已知超过145种革兰氏阳性和阴性细菌能产生吲哚,其中包括许多病原菌。随着细菌密度感应系统及其信号分子作用机制研究的深入,吲哚已被证实是肠道病原菌如致病性大肠杆菌、迟缓爱德华氏菌、霍乱弧菌等一类细胞间重要的信号分子,并参与细菌的多种生理活动,如毒力、抗药性、生物膜形成、运动性、质粒稳定性、抗酸性、孢子产生等。更为重要的是,吲哚及其衍生物还参与协调菌群竞争,有益于人体肠道菌群平衡和免疫系统。本文在吲哚作为细胞间信号分子参与迟缓爱德华氏菌的毒力、抗药性、生物膜形成和运动性的研究基础上,对近年来吲哚作为细菌细胞间信号分子的研究进展进行了综述。随着吲哚作用机制的进一步揭示,将有助于新型抗病原菌感染策略的研发和生物工程方面的应用。     

Higher order assembling of the mycobacterial polar growth factor DivIVA/Wag31

DivIVA or Wag31, which is an essential pole organizing protein in mycobacteria, can self-assemble at the negatively curved side of the membrane at the growing pole to form a higher order structural scaffold for maintaining cellular morphology and localizing various target proteins for cell-wall biogenesis. The structural organization of polar scaffold formed by polymerization of coiled-coil rich Wag31, which is implicated in the anti-tubercular activities of amino-pyrimidine sulfonamides, remains to be determined. A single-site phosphorylation in Wag31 regulates peptidoglycan biosynthesis in mycobacteria. We report biophysical characterizations of filaments formed by mycobacterial Wag31 using circular dichroism, atomic force microscopy and small angle solution X-ray scattering. Atomic force microscopic images of the wild-type, a phospho-mimetic (T73E) and a phospho-ablative (T73A) form of Wag31 show mostly linear filament formation with occasional curving, kinking and apparent branching. Solution X-ray scattering data indicates that the phospho-mimetic forms of the Wag31 polymers are on average more compact than their phospho-ablative counterparts, which is likely due to the extent of bending/branching. Observed structural features in this first view of Wag31 filaments suggest a basis for higher order Wag31 scaffold formation at the pole.     

Antibacterial activity of singly and doubly modified salinomycin derivatives

The increasing challenge of antibiotic resistance stimulates the search for novel antibacterial agents, especially such that would be effective against multi-drug resistant bacterial strains. Fortunately, natural compounds are excellent sources of potentially new drug leads. Particularly interesting in this context are polyether antibiotic salinomycin (SAL) and its semi-synthetic derivatives, as they exhibit large spectrum of bioactivity. We synthesized and evaluated the antibacterial activity of a series of SAL analogs; four singly (2–3, 15, 17) and two doubly modified (16, 18) derivatives were found to show excellent inhibitory activity not only against planktonic Gram(+) bacterial cells, but also towards select strains of methicillin-resistant staphylococci with the MIC values of 1–4 µg mL⁻¹. Of note, the most promising candidates were more effective in preventing bacterial biofilm formation than unmodified SAL and a commonly used antibiotic – ciprofloxacin. Furthermore, we proved that rational modification of C20 hydroxyl of SAL may reduce genotoxic properties of the obtained analogs. Mechanistically, the structure-activity relationship studies suggested that electroneutral transport mechanism could be beneficial in terms of ensuring high antibacterial activity of SAL derivatives.     

Bacterial cellulose membrane – A new support carrier for yeast immobilization for ethanol fermentation

The aim of the work was to study the properties of the bacterial cellulose membrane (BCM) and the feasibility of using it as a new, environmentally friendly support carrier for yeast cell immobilization. It was observed that the morphology of BCM varied with different cultivation methods and the scanning electron microscopy (SEM) images confirmed that the yeast cells were entrapped in the porous network of BCM obtained from the static culture and stabilized by the cross-linked fibrils. Particularly, the research confirmed the effectiveness of yeast immobilization in BCM reflected by the high yield of alcohol (9.7% v/v, a 21.25% increase of those using free cells) and the high stability. The specific rate of ethanol production by the immobilized cells in BCM was 2.1 g g⁻¹ h⁻¹, 31.3% greater than that of the suspended cells. Results implied that applying BCM as the support carrier had little adverse effects on cell viability and proliferation. Instead, it facilitated the product leakage and nutrients transportation through the porous network.     

Dynamics in the bacterial community-level physiological profiles and hydrological characteristics of constructed wetland mesocosms during start-up

The objective of this work was to study the effect of plant presence (Phragmites australis) and inoculant origin on wetland mesocosm start-up dynamics. Eight mesocosms were studied based on a duplicated 2² factorial design tracking bacterial community and hydrological changes during an 8 month start-up period. The mesocosms were characterized in terms of their hydrological character based on evapotranspiration (ET), porosity, and a dispersion coefficient. The microbiological regime was characterized using a microbial activity measure and community-level physiological profiling (CLPP) employing BIOLOG™ ECO plates. CLPP-related indices such as substrate richness, substrate diversity, over-all community profile, and community divergence are also presented. It was found that mesocosm porosities decreased over time as a result of media-related biofilm development. This biofilm development also contributed to a substantial increase in the dispersion coefficient in the mesocosms over the start-up period. Dispersion coefficients in planted systems reached values of ∼50-55 cm²/min whereas in the unplanted systems values of ∼30-35 cm²/min were observed. Bacterial community divergence in the mesocosms was quantified using a Euclidean-based divergence metric. All mesocosms showed a sharp increase in community divergence until day 75, at which point a steady state was reached. The interstitial communities were also characterized in terms of similarity based on the experimental design treatments. Four stages of mesocosm development were identified that can be described by an initial community state based on the origins of the initial inoculum [days 0-6]; a dynamic period where adjustments and shifts in the bacterial community occurred in all mesocosms [days 7-26]; a period where all interstitial CLPPs were quite similar [days 27-73]; and finally a shift towards unplanted and planted mesocosm CLPP groupings [days 74-232].