Bacterial RNA thermometers: molecular zippers and switches

Bacteria use complex strategies to coordinate temperature-dependent gene expression. Many genes encoding heat shock proteins and virulence factors are regulated by temperature-sensing RNA sequences, known as RNA thermometers (RNATs), in their mRNAs. For these genes, the 5' untranslated region of the mRNA folds into a structure that blocks ribosome access at low temperatures. Increasing the temperature gradually shifts the equilibrium between the closed and open conformations towards the open structure in a zipper-like manner, thereby increasing the efficiency of translation initiation. Here, we review the known molecular principles of RNAT action and the hierarchical RNAT cascade in Escherichia coli. We also discuss RNA-based thermosensors located upstream of cold shock and other genes, translation of which preferentially occurs at low temperatures and which thus operate through a different, more switch-like mechanism. Finally, we consider the potential biotechnological applications of natural and synthetic RNATs.     

RNA thermometers in bacteria: Role in thermoregulation

An array of external factors, an important one being temperature, decide the fate of survival in a microbe. The ability of microbes to sense external cues and to regulate the expression of genes accordingly is critical for its likely survival. Among a myriad of cellular defence mechanisms, a strategy to recuperate stress involves RNA regulatory elements. RNAs own a repertoire of functions in a cell as messengers, for transfer or as a component of ribosomes. A shift from its indigenous role is as regulators of gene expression, where in the cis-encoded RNA termed as “RNA Thermometers” play a pivotal role in translational level of gene expression. In this paper, we review the occurrence, the different types and molecular mechanism of gene regulation by RNATs, with a special focus limited to the domain Bacteria. We discuss the role of RNATs in mediating expression of temperature-responsive genes like heat shock/cold attributing in heat/cold shock response and a cascade of virulence genes to evade host defence mechanisms.     

Immediate stoichiometric appearance of β-galactosidase products in the medium of Escherichia coli cells incubated with lactose

Products of β-galactosidase action on lactose by intact E. coli cells appeared in the medium as soon as lactose was added and the amount of product was equal to the lactose used. No detectable levels of β-galactosidase were found in the medium and lactose was not significantly broken down unless lac permease was present. The appearance did not depend upon the presence of any of the commonly known galactose or glucose permease systems. The Km of product appearance from whole cells was equal to the K_t for lactose transport by lac permease. When the cells were broken the Km became the normal β-galactosidase Km.     

Messenger RNA potential and the delay before exponential decay of messages

Potential is a parameter of messenger RNA decay that measures the ability of a message population to continue to initiate complete translations. It can be measured from the start of induction using a specific inhibitor of translation-initiation. With very short inductions it can be seen that the potential of the β-galactosidase message is not lost at a constant exponential rate until a time (delay time) approximately equal to the time required to synthesize the β-galactosidase message. The capacity to make enzyme declines only after the induction lag and its subsequent decay shows a somewhat longer delay; unlike potential, capacity is also affected by the variable rate of ribosome movement. The delays in loss of potential and capacity are consistent with either of two mechanisms: (1) the message must be completed before attack at its starting end, or (2) two or more separate events (or hits) with specific time constants are needed to inactivate. Even at very low growth temperatures, functional decay kinetics are consistent with either mechanism, as is the mass decay of β-galactosidase message at 37 °C. Messages for anthranilate synthetase and galactoside acetyl-transferase do not require two hits to inactivate, but the data cannot determine if there is a delay equal to their synthesis time. Either β-galactosidase message is exceptional and, as opposed to other messages, requires two or more hits to be inactivated, or Escherichia coli messages generally do not commence to decay until they are completed.     

Decay of messenger ribonucleic acid from the lactose operon of Escherichia coli as a function of growth temperature

The inactivation rates of the first, β-galactosidase, and last, transacetylase, messages of the lactose operon of Escherichia coli were measured at different growth temperatures. The inactivation rate of each message appears to increase exponentially with temperature. The rate constant for this increase is almost twice as high for transacetylase message as it is for β-galactosidase message. The inactivation rate is more a direct function of growth temperature than of growth rate. At 15 °C transacetylase message is inactivated about 2.5 times more slowly than is β-galactosidase message. This difference is not paralleled by a different rate of chemical loss of the β-galactosidase message compared to the distal lac mRNA; all parts of the molecule appear to be lost at the same rate. This same pattern is observed in decay of the total mRNA; loss of capacity to direct peptide synthesis (functional inactivation) occurs at variable rates whereas loss of mRNA mass (chemical degradation) seems to occur at a uniform rate.We conclude that each message has a unique target for inactivation with a specifie temperature coefficient of sensitivity, and the inactivation of a message need not be associated with chemical destruction of the molecule.     

LacI(Ts)-regulated expression as an in situ intracellular biomolecular thermometer

In response to needs for in situ thermometry, a temperature-sensitive vector was adapted to report changes in the intracellular heat content of Escherichia coli in near-real time. This model system utilized vectors expressing increasing quantities of β-galactosidase in response to stepwise temperature increases through a biologically relevant range (22 to 45°C). As judged by calibrated fluorometric and colorimetric reporters, both whole E. coli cells and lysates expressed significant repeatable changes in β-galactosidase activity that were sensitive to temperature changes of less than 1°C (35 to 45°C). This model system suggests that changes in cellular heat content can be detected independently of the medium in which cells are maintained, a feature of particular importance where the medium is heterogeneous or nonaqueous, or otherwise has a low heat transfer capacity. We report here that the intracellular temperature can be reliably obtained in near-real time using reliable fluorescent reporting systems from cellular scales, with a 20°C range of detection and at least 0.7°C sensitivity between 35 and 45°C.     

The purification of beta-galactosidase-specific polysomes by affinity chromatography.

Affinity chromatography on a β-galactosidase substrate analog-Sepharose column was used to purify β-galactosidase-specific polysomes from E. coli. The purification was monitored by hybridization of [³H]uridine pulse-labeled RNA extracted from polysomes to p lac 5 DNA. A purification of at least 12-fold was obtained. Binding of lac polysomes to the column required the presence of Sepharose-bound substrate analog; salt and pH conditions favorable to β-galactosidase binding; and intact polyribosomes. It was calculated that 40–50% of the labeled mRNA recovered was lac RNA.     

Maximal Expression of Recombinant cDNAs in COS Cells for Use in Expression Cloning

In the process of establishing an expression cloning system for cell surface receptors we examined parameters which influence the expression of foreign genes in COS cells. The bacterial β-galactosidase gene was chosen as a reporter gene, since it permits the determination of (i) the fraction of cells transfected as well as (ii) the total activity of the synthesized enzyme in parallel experiments. This renders it possible to calculate the enzyme activity per individual cell. In transfected COS cells, the plasmid pXMgal directed a 20- and 10-fold higher β-galactosidase activity than pCH110 and pCDLgal, respectively. DEAE-dextran-mediated DNA uptake and protoplast fusion were found to result in higher expression rates than lipofection and electroporation. A coincubation of the cells with chloroquine during the DEAE-dextran transfection protocol caused, as reported, an increase of β-galactosidase positive cells but considerably reduced the total β-galactosidase activity. However, a 10% DMSO shock at the end of the transfection procedure simultaneously increased the number of transfected cells and the total β-galactosidase activity, thus maintaining the high expression per single cell. Using these optimized conditions, COS-1 cells expressed higher amounts of recombinant protein than COS-7 cells.     

Immobilization of β-galactosidase on modified polypropilene membranes

A new immobilized system: β-galactosidase-modified polypropylene membrane was created. It was obtained 13 different carriers by chemical modification of polypropylene membranes by two stages. The first stage is treatment with K(2)Cr(2)O(7) to receive carboxylic groups on membrane surface. The second stage is treatment with different modified agents ethylendiamine, hexamethylenediamine, hydrazine dihydrochloride, hydroxylamine, o-phenylenediamine, p-phenylenediamine, N,N'-dibenzyl ethylenediamine diacetate to receive amino groups. The quantity of the amino groups, carboxylic groups and the degree of hydrophilicity of unmodified and modified polypropilene membranes were determined. β-Galactosidase was chemically immobilized on the obtained carries by glutaraldehyde. The highest relative activity of immobilized enzyme was recorded at membrane modified with 10% hexamethylenediamine (Membrane 5) - 92.77%. The properties of immobilized β-galactosidase on different modified membranes - pH optimum, temperature optimum, pH stability and thermal stability were investigated and compared with those of free enzyme. The storage stability of all immobilized systems was studied. It was found that the most stable system is immobilized enzyme on Membrane 5. The system has kept 90% of its initial activity at 300th day (pH=6.8; 4°C). The stability of the free and immobilized β-galactosidase on the modified membrane 5 with 10% HMDA in aqueous solutions of alcohols - mono-, diol and triol was studied. The kinetics of enzymatic reaction of free and immobilized β-galactosidase on the modified membrane 5 at 20°C and 40°C and at the optimal pH for both forms of the enzyme were investigated. It was concluded that the modified agent - hexamethylenediamine, with long aliphatic chain ensures the best immobilized β-galactosidase system.     

Influence of lactose and lactate on growth and β-galactosidase activity of potential probiotic Propionibacterium acidipropionici

Dairy propionibacteria are microorganisms of interest for their role as starters in cheese technology and as well as their functions as probiotics. Previous studies have demonstrated that Propionibacterium acidipropionici metabolize lactose by a β-galactosidase that resists the gastrointestinal transit and the manufacture of a Swiss-type cheese, so that could be considered for their inclusion in a probiotic product assigned to intolerant individuals. In the present work we studied the effect of the sequential addition of lactose and lactate as first or second energy sources on the growth and β-galactosidase activity of P. acidipropionici Q4. The highest β-galactosidase activity was observed in a medium containing only lactate whereas higher final biomass was obtained in a medium with lactose. When lactate was used by this strain as a second energy source, a marked increase of the intracellular pyruvate level was observed, followed by lactate consumption and increase of specific β-galactosidase activity whereas lactose consumption became negligible. On the contrary, when lactose was provided as second energy source, lactic acid stopped to be metabolized, a decrease of the intracellular pyruvate concentration was observed and β-galactosidase activity sharply returned to a value that resembled the observed during the growth on lactose alone. Results suggest that the relative concentration of each substrate in the culture medium and the intracellular pyruvate level were decisive for both the choice of the energetic substrate and the β-galactosidase activity in propionibacteria. This information should be useful to decide the most appropriate vehicle to deliver propionibacteria to the host in order to obtain the highest β-galactosidase activity.     

Cold active β-galactosidase from Thalassospira sp. 3SC-21 to use in milk lactose hydrolysis: a novel source from deep waters of Bay-of-Bengal

The cold active β-galactosidase from psychrophilic bacteria accelerate the possibility of outperforming the current commercial β-galactosidase production from mesophilic sources. The present study is carried out to screen and isolate a cold active β-galactosidase producing bacterium from profound marine waters of Bay-of-Bengal and to optimize the factors for lactose hydrolysis in milk. Isolated bacterium 3SC-21 was characterized as marine psychrotolerant, halophile, gram negative, rod shaped strain producing an intracellular cold active β-galactosidase enzyme. Further, based upon the 16S rRNA gene sequence, bacterium 3SC-21 was identified as Thalassospira sp. The isolated strain Thalassospira sp. 3SC-21 had shown the enzyme activity between 4 and 20?°C at pH of 6.5 and the enzyme was completely inactivated at 45?°C. The statistical method, central composite rotatable design of response surface methodology was employed to optimize the hydrolysis of lactose and to reveal the interactions between various factors behind this hydrolysis. It was found that maximum of 80.18?% of lactose in 8?ml of raw milk was hydrolysed at pH of 6.5 at 20?°C in comparison to 40?% of lactose hydrolysis at 40?°C, suggesting that the cold active β-galactosidase from Thalassospira sp. 3SC-21would be best suited for manufacturing the lactose free dairy products at low temperature.     

The effect of high pressure homogenization on the activity of a commercial β-galactosidase

High pressure homogenization (HPH) has been proposed as a promising method for changing the activity and stability of enzymes. Therefore, this research studied the activity of β-galactosidase before and after HPH. The enzyme solution at pH values of 6.4, 7.0, and 8.0 was processed at pressures of up to 150?MPa, and the effects of HPH were determined from the residual enzyme activity measured at 5, 30, and 45?°C immediately after homogenization and after 1?day of refrigerated storage. The results indicated that at neutral pH the enzyme remained active at 30?°C (optimum temperature) even after homogenization at pressures of up to 150?MPa. On the contrary, when the β-galactosidase was homogenized at pH 6.4 and 8.0, a gradual loss of activity was observed, reaching a minimum activity (around 30?%) after HPH at 150?MPa and pH 8.0. After storage, only β-galactosidase that underwent HPH at pH 7.0 retained similar activity to the native sample. Thus, HPH did not affect the activity and stability of β-galactosidase only when the process was carried out at neutral pH; for the other conditions, HPH resulted in partial inactivation of the enzyme. Considering the use of β-galactosidase to produce low lactose milk, it was concluded that HPH can be applied with no deleterious effects on enzyme activity.     

STATISTICAL APPROACH FOR THE ENHANCED PRODUCTION OF COLD-ACTIVE β-GALACTOSIDASE FROM Thalassospira frigidphilosprofundus: A NOVEL MARINE PSYCHROPHILE FROM DEEP WATERS OF BAY OF BENGAL

In the present investigation Thalassospira frigidphilosprofundus, a novel species from the deep waters of the Bay of Bengal, was explored for the production of cold-active β-galactosidase by submerged fermentation using marine broth medium as the basal medium. Effects of various medium constituents, namely, carbon, nitrogen source, pH, and temperature, were investigated using a conventional one-factor-at-a-time method. It was found that lactose, yeast extract, and bactopeptones are the most influential components for β-galactosidase production. Under optimal conditions, the production of β-galactosidase was found to be 3,864 U/mL at 20 ± 2°C, pH 6.5 ± 0.2, after 48 hr of incubation. β-Galactosidase production was further optimized by the Taguchi orthogonal array design of experiments and the central composite rotatable design (CCRD) of response surface methodology. Under optimal experimental conditions the cold-active β-galactosidase enzyme production from Thalassospira frigidphilosprofundus was enhanced from 3,864 U/mL to 10,657 U/mL, which is almost three times higher than the cold-active β-galactosidase production from the well-reported psychrophile Pseudoalteromonas haloplanktis.     

Multiple β-galactosidase activities in petunia hybrida: Separation and characterization

Using isoelectrofocusing (IEF), multiple forms of Petunia β-galactosidase activity could be detected. The β-galactosidase pattern showed only minor tissue-specific differences. There were, however, species-specific differences. Zea mays, for instance, showed two bands which differed from the zones obtained with Petunia preparations. Petunia and corn leaves were mixed and extracted commonly. The species-specific activity patterns remained unchanged.Petunia preparations were inactivated by 8 Murea. Following dialysis, enzymatic activity and the Petunia-specific pattern were restored. The same holds true for a mixture of Petunia and E. coli β-galactosidase preparations. On refocusing isolated Petunia zones, untreated or inactivated by 8 M urea and reactivated by dialysis, the original mobilities were shown. Therefore, it seems highly improbable that the β-galactosidase pattern was due to artefacts. Using a Petunia line which was ‘pure’, also in respect to its β-galactosidase pattern, the four main bands were preparatively separated by IEF and characterized. They showed the same pH optimum (4.3), the same temperature optimum (55°), the same inactivation kinetics by urea, the same sensitivity against Cl^?, and closely related K_m. values. In sucrose gradient centrifugation they invariably showed S values of 8–10. The multiple activities could not be separated by zone electrophoresis using various carrier systems, or by gel filtration. It seems possible that they represent forms which differ only in isoelectric points, not in MW.