Mathematical modeling of regulatory mechanisms in yeast colony development

In the present study, yeast colony development serves as a model system to study growth of fungal populations with negligible nutrient and signal transport within the mycelium. Mathematical simulations address the question whether colony development is governed by diffusional limitation of nutrients. A hybrid one-dimensional cellular automaton model was developed that describes growth of discrete cells based upon microscopic interaction rules in a continuous field of nutrient and messenger. The model is scaled for the geometry of the experimental setup, cell size, growth- and substrate uptake rates. Therefore, calculated cell density profiles and nutrient distributions can be compared to experimental results and the model assumptions can be verified. In the physiologically relevant parameter range, simulations show an exponentially declining cell density along the median axis of the colonies in case of a diffusion limited growth scenario. These results are in good agreement with cell density profiles obtained in cultivations of the yeast Candida boidinii with glucose as the limiting carbon source but stand in contrast to the constant cell density profile estimated for Yarrowia lipolytica grown under the same conditions. While from the comparison of experimental results and simulations a diffusion limited growth mechanism is proposed for glucose limited C. boidinii colonies, this hypothesis is rejected for the growth of Y. lipolytica. As an alternative, a quorum sensing model was developed that can explain the evolution of constant cell density profiles based on the effect of a not further characterized unstable or volatile messenger.     

Arabidopsis growth and defense are modulated by bacterial quorum sensing molecules

N-acyl-homoserine lactones (AHLs) play an important role in the communication within the rhizosphere; they serve as a chemical base for interactions within and between different species of Gram-negative bacteria. Not only bacteria, also plants perceive and react to AHLs with diverse responses. Here we describe a negative correlation between the length of AHLs’ lipid chains and the observed growth promotion in Arabidopsis thaliana. Moreover, we speculate on a positive correlation between the reinforcement of defense mechanisms and the length of the lipid moieties. Observation presented here may be of great importance for understanding of the complex interplay between plants and their environment, as well as for agronomic applications.     

Kinetics of the association/dissociation cycle of an ATP-binding cassette nucleotide-binding domain

Most ATP binding cassette (ABC) proteins are pumps that transport substrates across biological membranes using the energy of ATP hydrolysis. Functional ABC proteins have two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is unresolved. This is due in part to the limited kinetic information on NBD association and dissociation. Here, we show dimerization of a catalytically active NBD and follow in real time the association and dissociation of NBDs from the changes in fluorescence emission of a tryptophan strategically located at the center of the dimer interface. Spectroscopic and structural studies demonstrated that the tryptophan can be used as dimerization probe, and we showed that under hydrolysis conditions (millimolar MgATP), not only the dimer dissociation rate increases, but also the dimerization rate. Neither dimer formation or dissociation are clearly favored, and the end result is a dynamic equilibrium where the concentrations of monomer and dimer are very similar. We proposed that based on their variable rates of hydrolysis, the rate-limiting step of the hydrolysis cycle may differ among full-length ABC proteins.     

Combinational quorum sensing devices for dynamic control in cross-feeding cocultivation

Quorum sensing (QS) offers cell density dependent dynamic regulations in cell culture through devices such as synchronized lysis circuit (SLC) and metabolic toggle switch (MTS). However, there is still a lack of studies on cocultivation with a combination of different QS-based devices. Taking the production of isopropanol and salidroside as case studies, we have mathematically modeled a comprehensive set of QS-regulated cocultivation schemes and constructed experimental combinations of QS devices, respectively, to evaluate their feasibility and optimality for regulating growth competition and corporative production. Glucose split ratio is proposed for the analysis of competition between cell growth and targeted production. Results show that the combination of different QS devices across multiple members offers a new tool with the potential to effectively coordinate synthetic microbial consortia for achieving high product titer in cross-feeding cocultivation. It is also evident that the performance of such systems is significantly affected by dynamic characteristics of chosen QS devices, carbon source control and the operational settings. This study offers insights for future applications of combinational QS devices in synthetic microbial consortia.     

Molecular identification of aiiA homologous gene from endophytic Enterobacter species and in silico analysis of putative tertiary structure of AHL-lactonase

The aiiA homologous gene known to encode AHL- lactonase enzyme which hydrolyze the N-acylhomoserine lactone (AHL) quorum sensing signaling molecules produced by Gram negative bacteria. In this study, the degradation of AHL molecules was determined by cell-free lysate of endophytic Enterobacter species. The percentage of quorum quenching was confirmed and quantified by HPLC method (p < 0.0001). Amplification and sequence BLAST analysis showed the presence of aiiA homologous gene in endophytic Enterobacter asburiae VT65, Enterobacter aerogenes VT66 and Enterobacter ludwigii VT70 strains. Sequence alignment analysis revealed the presence of two zinc binding sites, "HXHXDH" motif as well as tyrosine residue at the position 194. Based on known template available at Swiss-Model, putative tertiary structure of AHL-lactonase was constructed. The result showed that novel endophytic strains of Enterobacter genera encode the novel aiiA homologous gene and its structural importance for future study.     

一种新的乙酰高丝氨酸内酯水解酶基因的克隆和表达

目的 获得新的降解革兰阴性细菌数量阈值感应信号分子乙酰高丝氨酸内酯类化合物(AHL)的水解酶基因。方法 选择性富集和培养土壤中耐热细菌,抽取细菌总DNA作为模板,特异性聚合酶链反应扩增乙酰高丝氨酸内酯水解酶基因,进行克隆和DNA序列分析及原核表达。结果 得到1个新的AHL水解酶基因,该基因与已知基因的核苷酸序列和对应的氨基酸序列同源性最高分别为87％和94％。该基因在原核表达系统中表达,得到了与预期相对分子质鲢(Mr)一致的蛋白质。结论 证实乙酰高丝氨酸内酯水解酶广泛存在于环境微生物中。为进一步研究提供条件。     

A genetically engineered whole-cell pigment-based bacterial biosensing system for quantification of N-butyryl homoserine lactone quorum sensing signal

N-Acyl homoserine lactone (AHL) is a widely conserved quorum sensing (QS) signal of Gram-negative bacteria and has received attention in fighting against human diseases and environmental pollution. However, a method for quantifying AHL is lacking although it is urgently required for diagnosis and bioprocess manipulation. This work screened out an aromatics degrader Pseudomonas aeruginosa for biosensing system development, which produced a blue–green pigment regulated by the RhlI–RhlR QS system. By taking advantage of the recognition of N-butyryl homoserine lactone (BHL, the signal molecule of RhlI–RhlR QS system and an AHL) by the product of rhlR, a new whole-cell biosensor P. aeruginosa ΔrhlIR/pYC-rhlR (rhlI⁻rhlR⁺⁺) was developed. It was constructed through abolishing its BHL production by in-frame deletion of rhlIR and over-expressing rhlR by introducing a multi-copy plasmid pYC-rhlR into ΔrhlIR. By using the pigment production which responded to exogenous BHL as biosensor output, BHL quantification in samples was simply done spectrophotometrically. Under optimum conditions, the calibration curve had the limit of detection (LOD), the 50% activation/effect concentration, the limit of quantification (LOQ), and the quantitative detection range of 1.3 nM, 2.77 ± 0.45 μM, 5.7 nM and 0.11–49.7 μM, respectively. The biosensor output was stable, culture samples could be stored 10 days under −20 °C, and this sensing system was resistant to interferences by toxic aromatic pollutants. It was successfully applied to environmental samples even without extraction. The new whole-cell biosensing system provided a simple, stable, toxic pollutants-tolerant, and cost-effective tool for quantitative investigation of the QS signals’ role in environmental processes.