The glutaminase-dependent acid resistance system. Qualitative and quantitative assays and analysis of its distribution in enteric bacteria

Neutralophilic bacteria have developed several strategies to overcome the deleterious effects of acid stress. In particular, the amino acid-dependent systems are widespread, with their activities overlapping, covering a rather large pH range, from 6 to <2. Recent reports showed that an acid resistance (AR) system relying on the amino acid glutamine (AR2_Q), the most readily available amino acid in the free form, is operative in Escherichia coli, Lactobacillus reuteri and some Brucella species. This system requires a glutaminase active at acidic pH and the antiporter GadC to import L-glutamine and export either glutamate (the glutamine deamination product) or GABA. The latter occurs when the deamination of glutamine to glutamate, via acid-glutaminase (YbaS/GlsA), is coupled to the decarboxylation of glutamate to GABA, via glutamate decarboxylase (GadB), a structural component of the glutamate-dependent AR (AR2) system, together with GadC. Taking into account that AR2_Q could be widespread in bacteria and that until now assays based on ammonium ion detection were typically employed, this work was undertaken with the aim to develop assays that allow a straightforward identification of the acid-glutaminase activity in permeabilised bacterial cells (qualitative assay) as well as a sensitive method (quantitative assay) to monitor in the pH range 2.5-4.0 the transport of the relevant amino acids in vivo. The qualitative assay is colorimetric, rapid and reliable and provides several additional information, such as co-occurrence of AR2 and AR2_Q in the same bacterial species and assessment of the growth conditions that support maximal expression of glutaminase at acidic pH. The quantitative assay is HPLC-based and allows to concomitantly measure the uptake of glutamine and the export of glutamate and/or GABA via GadC in vivo and depending on the external pH. Finally, an extensive bioinformatic genome analysis shows that the gene encoding the glutaminase involved in AR2_Q is often nearby or in operon arrangement with the genes coding for GadC and GadB. Overall, our results indicate that AR2_Q is likely to be of prominent importance in the AR of enteric bacteria and that it modulates the enzymatic as well as antiport activities depending on the imposed acidic stress

Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system

In orally acquired bacteria, the ability to counteract extreme acid stress (pH < 2.5) ensures survival during transit through the animal host stomach. In several neutralophilic bacteria, the glutamate-dependent acid resistance system (GDAR) is the most efficient molecular system in conferring protection from acid stress. In Escherichia coli its structural components are either of the two glutamate decarboxylase isoforms (GadA, GadB) and the antiporter, GadC, which imports glutamate and exports γ-aminobutyrate, the decarboxylation product. The system works by consuming protons intracellularly, as part of the decarboxylation reaction, and exporting positive charges via the antiporter. Herein, biochemical and spectroscopic properties of GadB from Brucella microti (BmGadB), a Brucella species which possesses GDAR, are described. B. microti belongs to a group of lately described and atypical brucellae that possess functional gadB and gadC genes, unlike the most well-known "classical" Brucella species, which include important human pathogens. BmGadB is hexameric at acidic pH. The pH-dependent spectroscopic properties and activity profile, combined with in silico sequence comparison with E. coli GadB (EcGadB), suggest that BmGadB has the necessary structural requirements for the binding of activating chloride ions at acidic pH and for the closure of its active site at neutral pH. On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests that BmGadB does not undergo membrane recruitment at acidic pH, which was observed in EcGadB. The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved

The Glutaminase-dependent system confers extreme acid resistance to new species and atypical strains of Brucella

Neutralophilic bacteria have developed specific mechanisms to cope with the acid stress encountered in environments such as soil, fermented foods, and host compartments. In Escherichia coli, the glutamate decarboxylase (Gad)-dependent system is extremely efficient: it requires the concerted action of glutamate decarboxylase (GadA/GadB) and of the glutamate (Glu)/γ-aminobutyrate antiporter, GadC. Notably, this system is operative also in new strains/species of Brucella, among which Brucella microti, but not in the "classical" species, with the exception of marine mammals strains. Recently, the glutaminase-dependent system (named AR2_Q), relying on the deamination of glutamine (Gln) into Glu and on GadC activity, was described in E. coli. In Brucella genomes, a putative glutaminase (glsA)-coding gene is located downstream of the gadBC genes. We found that in B. microti these genes are expressed as a polycistronic transcript. Moreover, using a panel of Brucella genus-representative strains, we show that the AR2_Q system protects from extreme acid stress (pH =2.5), in the sole presence of Gln, only the Brucella species/strains predicted to have functional glsA and gadC. Indeed, mutagenesis approaches confirmed the involvement of glsA and gadC of B. microti in AR2_Q and that the acid-sensitive phenotype of B. abortus can be ascribed to a Ser248Leu substitution in GlsA, leading to loss of glutaminase activity. Furthermore, we found that the gene BMI_II339, of unknown function and downstream of the gadBC-glsA operon, positively affects Gad- and GlsA-dependent AR. Thus, we identified novel determinants that allow newly discovered and marine mammals Brucella strains to be better adapted to face hostile acidic environments. As for significance, this work may contribute to the understanding of the host preferences of Brucella species and opens the way to alternative diagnostic targets in epidemiological surveillance of brucellosis

Latex agglutination tests for selected Escherichia coli enzymes

Rapid latex agglutination assays for identifying tryptophanase, glutamate decarboxylase, and glucuronidase from Escherichia coli cell lysates were developed by using rabbit polyclonal antibodies elicited to commercial E. coli enzyme preparations. The latex agglutination tests had sensitivities of 77% for tryptophanase, 83% for glutamate decarboxylase, and 70% for glucuronidase. Specificities were 61% for tryptophanase, 57% for glutamate decarboxylase, and 82% for glucuronidase. The sensitivities and specificities were too low to warrant continued development of the assays;Development of the latex agglutination tests required extensive studies in attempts to reduce or eliminate nonspecific agglutination reactions. The effects of a wide variety of assay conditions on assay performance were studied. Latex particles of different affinities and with different surface charges, different blocking agents, assay buffers of different compositions, pH values, and ionic strengths, chaotropic agents, and different antibody preparations were all examined. A combination of 0.22 [mu]m diameter latex particles sensitized with both 0.5 and 1.0 mg/ml immunoglobulin and blocked with a [beta]-casein and glycerol mixture resulted in the production of the best reactive latex reagents. Immunoglobulin adsorption studies yielded valuable information that was used to optimize the binding of the antibodies to the latex particles. Four different antibody purification methods had no significant effect in the elimination of nonspecific agglutination reactions. Antibody characterization by polyacrylamide gel electrophoresis, Ouchterlony immunodiffusion, enzyme immunoassays, and Western blot analysis revealed that the antibody preparations were heterologous and reacted with many different proteins. The cross-reactions among the antibody preparations and nontarget proteins indicated that better methods of antibody production must be used to provide more specific reagents for the latex agglutination assay

Bacillus cereus responses to acid stress

Coping with acid environments is one of the prerequisites for the soil saprophytic and human pathogenic lifestyle of Bacillus cereus. This minireview highlights novel insights in the responses displayed by vegetative cells and germinating spores of B. cereus upon exposure to low pH as well as organic acids, including acetic acid, lactic acid and sorbic acid. Insights regarding the possible acid-inflicted damage, physiological responses and protective mechanisms have been compiled based on single cell fluorescence microscopy, flow cytometry and transcriptome analyses

Biochemical and biophysical characterization of Glutamate decarboxylase of bacterial origin

Bacteria are constantly exposed to harsh conditions during their life cycle. Thus, the development of molecular strategies to overcome stressful environments is required for their survival. The acid stress is considered one of the most severe life-threatening conditions for neutralophiles, i.e. microorganisms that live and replicate best at neutral pH. An acidic stress is frequently encountered in natural habitats and/or within the host during the infectious process. Therefore, if not properly counteracted, it can lead to death. In food-borne neutralophilic bacteria, the acid stress in mostly encountered during their transit through the host gastro-intestinal tract where the environmental pH fluctuates between 1.5-2.5 in the stomach and 4.5-5.0 in the distal intestine. In the last decades, several acid resistance molecular mechanisms have been identified; amongst them, the glutamate-dependent acid resistance mechanism is regarded the most potent. In Escherichia coli it relies on pyridoxal 5’-phosphate (PLP)-dependent glutamate decarboxylase GadA/B, which catalyze the proton-consuming decarboxylation of glutamate (L-Glu) to γ-aminobutyric acid (GABA), which is then exported by GadC, a Glu/GABA antiporter. The reaction consumes 1 H+/cycle. The pH-dependent biochemical properties of GadB from E. coli (EcGadB) have been studied in depth since the first publication of the purification protocol (De Biase et al., 1996), but several unanswered questions remain. As part of the research work leading to my PhD degree, I investigated EcGadB properties at different biochemical and biophysical levels, with regard to: i) the influence of PLP and of exposure to neutral-alkaline pH values on the quaternary structure of EcGadB; ii) the role of aspartate 86 in the pH-dependent control of EcGadB enzymatic activity. Moreover, I carried out a preliminary biochemical characterization of GadB from Mycobacterium tuberculosis to shed light on the hypothetical involvement of a Gad enzyme on either acid resistance within phagosome/phagolysosome in macrophages or as a key enzyme of the GABA-shunt, which provides succinate from α-ketoglutarate, thus refilling the otherwise incomplete TCA cycle. Finally, I focused on the antimicrobial activity of analogues of dicarboxylic acids, the results of which are under final assessment for deposit as an Italian patent

A Unified Radiometric Assay System for the Gaba-Glutamate Regulating Enzymes

The purpose of this paper was to develop a single assay system for the enzymes which regulate GABA and glutamate concentrations in brain and nerve tissue. Since all the enzymes produce L-glutamate, their activities were measured by coupling them to L-glutamate decarboxylase. Enzymatic activity was determined by measuring the release of co2 from radioactive substrates. The glutamate decarboxylase was obtained from a commercial acetone powder by simplifying existing procedures. The glutamate decarboxylase produced was of sufficient purity to be used in the coupled assays, which were checked with commercial preparations of each enzyme, where available, and with crude brain homogenates. All of the assays were shown to be linear with respect to both time and enzyme concentration, thus assuring the feasibility of the technique

X-ray crystallographic studies on Particulate Methane Monooxygenase, Thioredoxin A and Arginine Decarboxylase

The work presented in this thesis describes the X-ray crystallographic studies of particulate methane monoxygenase (pMMO) from Methylococcus capsulatus (Bath), thioredoxin A (BsTrxA) from Bacillus subtilis and arginine decarboxylase (AdiA) from Escherichia coli. 1. pMMO is a respiratory enzyme that catalyses the first step in the metabolic pathway in methanotrophic bacteria by converting methane to methanol. The crystal structure of this integral membrane protein was determined by molecular replacement to 3.5 Å resolution. The three metal sites in pMMO were confirmed to be a mononuclear copper site, a dinuclear copper site and a mononuclear zinc site. 2. Thioredoxin is a ubiquitous protein present in nearly all known organisms. Its purpose in the cell is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds to dithiols in a redox reaction. The crystal structure of an active site mutant of BsTrxA was determined by molecular replacement to 1.5 Å resolution. The structure shows a homodimer that resembles enzyme-substrate reaction intermediates. 3. AdiA is a vitamin B6-dependent enzyme that catalyses the decarboxylation of arginine into agmatine. It forms a part of an enzymatic system in E. coli that contribute to making this organism acid resistant. The structure of arginine decarboxylase (AdiA) from E. coli was determined by multiple isomorphous replacement and anomalous scattering (MIRAS) methods to 2.4 Å resolution. The structure revealed a ~800 kDa decamer composed as a pentamer of five homodimers. AdiA becomes active as the cellular environment becomes more acidic. The structure of AdiA suggests how functional decamers associate with decreasing pH or disassociates into inactive homodimers with increasing pH. The enzyme mechanism and determinants for substrate specificity are discussed within the framework of the structure and comparisons with related structures are made

Stukturelle und Biochemische Untersuchungen zweier Enzyme des Tetrapyrrol Stoffwechsels

The complex enzymatic catalysis involved in the forked tetrapyrrole biosynthesis is slowly becoming unravelled. During the last decades, work done on the biochemical and structural fields has contribute to the current knowledge about its several branching pathways. To gain further insight into the complex enzymatic catalysis involved in this metabolism, two enzymes were studied in the present work, namely uroporphyrinogen-III decarboxylase from Nicotiana tabacum and coproporphyrinogen-III oxidase from Escherichia coli. The functional relationships obtained from the structural and modelling analyses for Uroporphyrinogen-III Decarboxylase allowed the proposal for a refined catalytic mechanism. The purification of Oxygen-dependent Coproporphyrinogen-III Oxidase allowed the biochemical characterisation and the initialisation of structural studies.Trotz der großen Bedeutung der Tetrapyrrolbiosynthese für lebende Systeme ist die mechanistische Seite dieser Prozesse noch wenig verstanden. Ein Beitrag zur Klärung dieser offenen Fragen wurde im Rahmen dieser Arbeit versucht, in der zwei Enzyme, die Uroporphyrinogen-III Decarboxylase aus Nicotiana tabacum und die sauerstoffabhängige Coproporphyrinogen-III Oxidase aus Escherichia coli, strukturell und biochemisch untersucht wurden. Die Kristallstrukturanalyse der Uroporphyrinogen-III Decarboxylase aus N. tabacum erlaubt die weitere Differenzierung des katalytischen Mechanismus des Enzyms. Die Isolierung der sauertoffabhängigen Coproporphyrinogen-III Oxidase erlaubt die biochemische Charakterisierung und erste Kristallisationsversuche des Enzyms