Plant cell calcium‐rich environment enhances thermostability of recombinantly produced α‐amylase from the hyperthermophilic bacterium Thermotoga maritime

In the industrial processing of starch for sugar syrup and ethanol production, a liquefaction step is involved where starch is initially solubilized at high temperature and partially hydrolyzed with a thermostable and thermoactive α‐amylase. Most amylases require calcium as a cofactor for their activity and stability, therefore calcium, along with the thermostable enzyme, are typically added to the starch mixture during enzymatic liquefaction, thereby increasing process costs. An attractive alternative would be to produce the enzyme directly in the tissue to be treated. In a proof of concept study, tobacco cell cultures were used as model system to test in planta production of a hyperthermophilic α‐amylase from Thermotoga maritima. While comparable biochemical properties to recombinant production in Escherichia coli were observed, thermostability of the plant‐produced α‐amylase benefited significantly from high intrinsic calcium levels in the tobacco cells. The plant‐made enzyme retained 85% of its initial activity after 3 h incubation at 100°C, whereas the E. coli‐produced enzyme was completely inactivated after 30 min under the same conditions. The addition of Ca²⁺ or plant cell extracts from tobacco and sweetpotato to the E. coli‐produced enzyme resulted in a similar stabilization, demonstrating the importance of a calcium‐rich environment for thermostability, as well as the advantage of producing this enzyme directly in plant cells where calcium is readily available. Biotechnol. Bioeng. 2009; 104: 947–956. © 2009 Wiley Periodicals, Inc.     

Deciphering chemotaxis pathways using cross species comparisons

Background  

Chemotaxis is the process by which motile bacteria sense their chemical environment and move towards more favourable conditions. Escherichia coli utilises a single sensory pathway, but little is known about signalling pathways in species with more complex systems.     

不同致泻性大肠杆菌感染调控细胞信号通路的研究进展

腹泻性大肠杆菌是在全世界引起人类和动物疾病的主要病原之一,也给社会经济带来巨大损失。根据致病机理的不同,可将腹泻性大肠杆菌分为6种:肠致病性大肠杆菌、肠出血性大肠杆菌、肠凝集性大肠杆菌、产肠毒素大肠杆菌、扩散黏附性大肠杆菌和肠侵袭性大肠杆菌。不同致病型大肠杆菌侵入宿主的方式及引起的炎症反应有所不同。文章综合分析了致病机制不同的大肠杆菌在调控宿主细胞信号通路方式上的不同,从炎症级联反应方面阐述了不同致病类型大肠杆菌的感染特征,并探讨了炎症信号通路与病原感染、预防和治疗的关系,以期为腹泻性大肠杆菌致病机制及治疗方案的研究提供帮助。     

Temperature gradient-based high-cell density fed-batch fermentation for the production of pyruvate oxidase by recombinant E. coli

Pyruvate oxidase (PyOD) is a very powerful enzyme for clinical diagnostic applications and environmental monitoring. Influences of temperature on cell growth, plasmid stability, and PyOD expression during the PyOD fermentation process by recombinant Escherichia coli were investigated. Based on the influences of temperature on the physiological metabolism, a novel high-cell density fed-batch cultivation with gradient temperature decrease strategy for effective PyOD production was achieved, under which the biomass (OD₆₀₀) of recombinant E. coli could reach to 71 and the highest PyOD activity in broth could reach to 3,307 U/L in 26?hr fermentation.     

Ecology of coliphages in southern California coastal waters

Aims: This study aims to investigate the ecology of coliphages, an important microbial pollution indicator. Specifically, our experiments address (i) the ability of environmental Escherichia coli (E. coli) to serve as hosts for coliphage replication, and (ii) the temporal and spatial distribution of coliphages in coastal waters. Methods and Results: Water samples from three locations in California’s Newport Bay watershed were tested for the presence of coliphages every 2 weeks for an entire year. A total of nine E. coli strains isolated from various sources served as hosts for coliphage detection. Coliphage occurrence was significantly different between freshwater, estuarine and coastal locations and correlated with water temperature, salinity and rainfall in the watershed. The coliphages isolated on the environmental hosts had a broad host‐range relative to the coliphages isolated on an E. coli strain from sewage and a US EPA recommended strain for coliphage detection. Conclusions: Coliphage occurrence was related to the temperature, rainfall and salinity within the bay. The adaptation to a broad host‐range may enable the proliferation of coliphages in the aquatic environment. Significance and Impact of the Study: Understanding the seasonal variation of phages is useful for establishing a background level of coliphage presence in coastal waters. The broad host‐range of coliphages isolated on the environmental E. coli host calls for investigation of coliphage replication in the aquatic environment.     

Intracellular pH modulates quinary structure

NMR spectroscopy can provide information about proteins in living cells. pH is an important characteristic of the intracellular environment because it modulates key protein properties such as net charge and stability. Here, we show that pH modulates quinary interactions, the weak, ubiquitous interactions between proteins and other cellular macromolecules. We use the K10H variant of the B domain of protein G (GB1, 6.2 kDa) as a pH reporter in Escherichia coli cells. By controlling the intracellular pH, we show that quinary interactions influence the quality of in‐cell ¹⁵N–¹H HSQC NMR spectra. At low pH, the quality is degraded because the increase in attractive interactions between E. coli proteins and GB1 slows GB1 tumbling and broadens its crosspeaks. The results demonstrate the importance of quinary interactions for furthering our understanding of protein chemistry in living cells.     

High yield expression of leptospirosis vaccine candidates LigA and LipL32 in the methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Background  

Leptospirosis, a zoonosis caused by Leptospira spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in Escherichia coli have demonstrated promising results, albeit with variable efficacy. Pichia pastoris is an alternative host with several advantages for the production of recombinant proteins.     

Genetic analysis of the structure and function of RNase P fromE. coli

A brief review of the genetic studies on ribonuclease P (RNase P) fromEscherichia coli is presented. Temperature-sensitive mutants ofE. coli defective in tRNA processing were isolated by screening cells which were unable to synthesize a suppressor tRNA at restrictive temperature. Structural analysis of accumulated tRNA precursors showed that the isolated mutants were defective in RNase P activity. Analyses of the mutants revealed that the enzyme is essential for the synthesis of all tRNA molecules in cells and that the enzymes consists of two subunits. Analyses of the isolated mutants revealed a possible domain structure of the RNA subunit of the enzyme.Abbreviations E. coli Escherichia coli - RNase P ribonuclease P     

Synthesis and localisation of Escherichia coli UDP-glucose hydrolase (5′-nucleotidase), and demonstration of a cytoplasmic inhibitor of this enzyme in Salmonella typhimurium

A strain of Salmonella typhimurium has been constructed, by transfer of F-13 from Escherichia coli, which synthesises and secretes Escherichia coli UDPglucose hydrolase. Although Salmonella typhimurium does not normally contain a secreted (periplasmic) UDPglucose hydrolase, we find that it contains, like Escherichia coli, an intracellular inhibitor of the enzyme.     

Effect of growth temperature,induction, and molecular chaperones on the solubilization of over-expressed cellobiose phosphorylase from <Emphasis Type="Italic">Cellvibrio Gilvus</Emphasis> under <Emphasis Type="Italic">in vivo</Emphasis> conditions

In vivo folding of many proteins can be facilitated by growth temperature, extent of induction, and molecular chaperones, which prevent over-expressed protein from being trapped into insoluble inclusion bodies. In the present report, we describe the role of molecular chaperones and growth temperature on the solubilization of overexpressed Cellobiose Phosphorylase (CBP) in Escherichia coli. The growth of host at low temperature enhanced enzyme in soluble fraction. Similarly, induction of target gene at low level of IPTG also yielded higher enzyme in soluble fraction. The synergistic effect of low temperature and induction on the prevention of inclusion bodies was also evident from our results. In addition, co-expression of the target gene with two types of molecular chaperones (GroESL and KODHsp) was also attempted. However, none of these chaperones enhanced the solubilization under in vivo conditions. Nevertheless, effective role of low growth temperature coupled with low level of induction appeared to be an attractive feature for producing recombinant protein.     

Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose

5-Aminolevulinic acid (ALA) recently received much attention due to its potential applications in many fields. In this study, we developed a metabolic strategy to produce ALA directly from glucose in recombinant Escherichia coli via the C5 pathway. The expression of a mutated hemA gene, encoding a glutamyl-tRNA reductase from Salmonella arizona, significantly improved ALA production from 31.1 to 176 mg/L. Glutamate-1-semialdehyde aminotransferase from E. coli was found to have a synergistic effect with HemA^M from S. arizona on ALA production (2052 mg/L). In addition, we identified a threonine/homoserine exporter in E. coli, encoded by rhtA gene, which exported ALA due to its broad substrate specificity. The constructed E. coli DALA produced 4.13 g/L ALA in modified minimal medium from glucose without adding any other co-substrate or inhibitor. This strategy offered an attractive potential to metabolic production of ALA in E. coli.     

In vivo measurement of internal and global macromolecular motions in Escherichia coli

We present direct quasielastic neutron scattering measurements, in vivo, of macromolecular dynamics in Escherichia coli. The experiments were performed on a wide range of timescales to cover the large panel of internal and self-diffusion motions. Three major internal processes were extracted at physiological temperature: a fast picosecond process that corresponded to restricted jump diffusion motions and two slower processes that resulted from reorientational motions occurring in ∼40 ps and 90 ps, respectively. The analysis of the fast process revealed that the cellular environment leads to an appreciable increase in internal molecular flexibility and diffusive motion rates compared with those evaluated in fully hydrated powders. The result showed that the amount of cell water plays a decisive role in internal molecular dynamics. Macromolecular interactions and confinement, however, attenuate slightly the lubricating effect of water, as revealed by the decrease of the in vivo parameters compared with those measured in solution. The study demonstrated that standard sample preparations do not mimic accurately the physiological environment and suggested that intracellular complexity participates in functional dynamics necessary for biological activity. Furthermore, the method allowed the extraction of the self-diffusion of E. coli macromolecules, which presented similar parameters as those extracted for hemoglobin in red blood cells.     

Temperature regulation of membrane composition in the Firmicute,Enterococcus faecalis,parallels that of Escherichia coli

Both Enterococcus faecalis and Escherichia coli can undergo abrupt temperature transitions in nature. E. coli changes the composition of its phospholipid acyl chains in response to shifts growth temperature. This is mediated by a naturally temperature sensitive enzyme, FabF (3-ketoacyl-acyl carrier protein synthase II), that elongates the 16 carbon unsaturated acyl chain palmitoleate to the 18 carbon unsaturated acyl chain, cis-vaccenate. FabF is more active at low temperatures resulting in increased incorporation of cis-vaccenoyl acyl chains into the membrane phospholipids. This response to temperature is an intrinsic property of FabF and does not require increased synthesis of the enzyme. We report that the FabF of the very divergent bacterium, E. faecalis, has properties very similar to E. coli FabF and is responsible for changing E. faecalis membrane phospholipid acyl chain composition in response to temperature. Moreover, expression E. faecalis FabF in an E. coli ∆fabF strain restores temperature regulation to the E. coli strain.     

Reactor Performance in the Presence of Catalyst Deactivation

The main variable of enzymatic processes is often found to be the operating temperature. An increase in temperature leads to higher rates for the catalytic transformation. However, beyond a certain temperature catalyst deactivation is winning the game. Therefore, processes should be optimized in order to determine the temperature which leads to a minimal demand of enzyme preparation. For the prediction of such optimal reactor operation, modeling of the temperature dependence of the process has to be performed. Examples of such modeling are given for the hydrolysis of lactose in UHT milk by means of three different β‐galactosidases – those from Aspergillus oryzae, Kluyveromyces lactis, and Escherichia coli. The reaction kinetics for a constant initial lactose concentration can be described by a model of two parameters, of which only one depends on temperature. For the lactase of E. coli the reaction can be described as a simple reaction with first order kinetics. The deactivation mechanism includes a reversible as well as an irreversible path of denaturation. The temperature dependent parameters follow Arrhenius' and van't Hoff's law, respectively. On the basis of their particular reaction models all three enzymes can be compared with respect to their optimum use. The models have been verified under laboratory conditions and have shown their usefulness for the prediction of optimum operating variables. Quite remarkable features have been found for the lactase of E. coli.     

Effects of temperature shifts and oscillations on recombinant protein production expressed in Escherichia coli

Escherichia coli is widely used host for the intracellular expression of many proteins. However, in some cases also secretion of protein from periplasm was observed. Improvement of both intracellular and extracellular production of recombinant protein in E. coli is an attractive goal in order to reduce production cost and increase process efficiency and economics. Since heat shock proteins in E. coli were reported to be helpful for protein refolding and hindering aggregation, in this work different types of single and periodic heat shocks were tested on lab scale to enhance intracellular and extracellular protein production. A single heat shock prior to induction and different oscillatory temperature variations during the induction phase were executed. The results showed that these variations influence protein production negatively. In other words, 45 and 50 % reduction in extracellular protein production were observed for the single heat shock and oscillated temperature between 35 and 40 °C, respectively. However, the oscillatory temperature approach introduced in this study is recommended as a tool to quantitatively analyze the effects of inhomogeneous temperature on cell physiology and productivity in large-scale bioreactors.     

High-temperature cultivation and 5′ mRNA optimization are key factors for the efficient overexpression of thermostable Deinococcus geothermalis purine nucleoside phosphorylase in Escherichia coli

Overexpression of genes from thermophiles in Escherichia coli is an attractive approach towards the large-scale production of thermostable biocatalysts. However, various factors can challenge efficient heterologous protein expression – one example is the formation of stable 5′ mRNA secondary structures that can impede an efficient translation initiation.In this work, we describe the expression optimization of purine nucleoside phosphorylase from the thermophilic microbe Deinococcus geothermalis in E. coli. Poor expression levels caused by stable secondary 5′ mRNA structure formation were addressed by two different approaches: (i) increasing the cultivation temperature above the range used typically for recombinant protein expression and (ii) optimizing the 5′ mRNA sequence for reduced secondary structures in the translation initiation region.The increase of the cultivation temperature from 30 °C to 42 °C allowed a more than 10-fold increase of activity per cell and optimizing the 5′ mRNA gene sequence further increased the activity per cell 1.7-fold at 42 °C. Thus, the combination of high-temperature cultivation and 5′ sequence optimization is described as an effective approach to overcome poor expression levels resulting from stable secondary 5′ mRNA structure formation. We suggest that this method is especially suitable for improving the expression of proteins derived from thermophiles in E. coli.     

Enhanced production of penicillin G acylase from a recombinant Escherichia coli

Penicillin G acylase (pac) gene was cloned into a stable asd ⁺ vector (pYA292) and expressed in Escherichia coli. This recombinant strain produced 1000 units penicillin G acylase g^–1 cell dry wt, which is 23-fold more than that produced by parental Escherichia coli ATCC11105. This enzyme was purified to 16 units mg^–1 protein by a novel two-step process.     

A Genomically Modified Marker Strain of Escherichia coli

Contamination of the environment with human sewage represents a serious public health concern in which Escherichia coli plays a central role, either directly as a human pathogen or indirectly through its use as an indicator organism. There is thus an ongoing effort to better understand the behavior of E. coli within such environments. Useful to such studies is the ability to readily detect a specific E. coli population and distinguish it from similar indigenous bacteria. Herein, we report the construction of an E. coli strain (PCPHR) that expresses a Stable Artificial RNA (SAR) from the chromosomal rrnH operon. The SAR product is present in large numbers of copies/cell and thus provides an enhanced detection signal without significant effect on the wild-type growth rate. Detection can be accomplished by any of several routine molecular methods. Preliminary studies suggest SAR expression levels correlate positively with growth. PCPHR is immediately available for use as a marker strain for E. coli in application in the arena of public health or environmental studies. Received: 14 April 1998 / Accepted: 17 June 1998     

Environmental Escherichia coli: ecology and public health implications—a review

Escherichia coli is classified as a rod‐shaped, Gram‐negative bacterium in the family Enterobacteriaceae. The bacterium mainly inhabits the lower intestinal tract of warm‐blooded animals, including humans, and is often discharged into the environment through faeces or wastewater effluent. The presence of E. coli in environmental waters has long been considered as an indicator of recent faecal pollution. However, numerous recent studies have reported that some specific strains of E. coli can survive for long periods of time, and potentially reproduce, in extraintestinal environments. This indicates that E. coli can be integrated into indigenous microbial communities in the environment. This naturalization phenomenon calls into question the reliability of E. coli as a faecal indicator bacterium (FIB). Recently, many studies reported that E. coli populations in the environment are affected by ambient environmental conditions affecting their long‐term survival. Large‐scale studies of population genetics revealed the diversity and complexity of E. coli strains in various environments, which are affected by multiple environmental factors. This review examines the current knowledge on the ecology of E. coli strains in various environments with regard to its role as a FIB and as a naturalized member of indigenous microbial communities. Special emphasis is given on the growth of pathogenic E. coli in the environment, and the population genetics of environmental members of the genus Escherichia. The impact of environmental E. coli on water quality and public health is also discussed.     

The nucleoid-associated DNA-binding protein H-NS is required for the efficient adaptation of Escherichia coli K-12 to a cold environment

The hns gene is a member of the cold-shock regulon, indicating that the nucleoid-associated, DNA-binding protein H-NS plays an important role in the adaptation of Escherichia coli to low temperatures. We show here that the ability to cope efficiently with a cold environment (12°C and 25°C) is strongly impaired in E. coli strains carrying hns mutations. Growth inhibition is much more pronounced in strains carrying the hns-206 allele (an ampicillin resistance cassette inserted after codon 37) than in those carrying the hns-205 mutation (a Tn10 insertion located in codon 93). A protein fragment (H-NS^*) is synthesized in strains carrying the hns-205::Tn10 mutation, suggesting that this truncated polypeptide is partially functional in the cold adaptation process. Analysis of the growth properties of strains harbouring four different low-copy-number plasmid-encoded hns genes that result in the production of C-terminally truncated H-NS proteins supports this proposal. H-NS^* proteins composed of 133, 117 or 94 amino-terminal amino acids partially complemented the severe cold-sensitive growth phenotype of the hns-206 mutant. In contrast, synthesis of a truncated H-NS protein with only 75 amino-terminal amino acids was insufficient to restore growth at low temperature.