Amino acid composition of bulk protein and salt relationships of selected enzymes of Salinibacter ruber,an extremely halophilic bacterium

The extremely halophilic bacterium Salinibacter ruber was previously shown to have a high intracellular potassium content, comparable to that of halophilic Archaea of the family Halobacteriaceae. The amino acid composition of its bulk protein showed a high content of acidic amino acids, a low abundance of basic amino acids, a low content of hydrophobic amino acids, and a high abundance of serine. We tested the level of four cytoplasmic enzymatic activities at different KCl and NaCl concentrations. Nicotinamide adenine dinucleotide (NAD)-dependent isocitrate dehydrogenase functioned optimally at 0.5-2 M KCl, with rates of 60% of the optimum value at 3.3 M. NaCl provided less activation: 70% of the optimum rates in KCl were found at 0.2-1.2 M NaCl, and above 3 M NaCl, activity was low. We also detected nicotinamide adenine dinucleotide phosphate (NADP)-dependent isocitrate activity, which remained approximately constant between 0-3.2 M NaCl and increased with increasing KCl concentration. NAD-dependent malate dehydrogenase functioned best in the absence of salt, but rates as high as 25% of the optimal values were measured in 3-3.5 M KCl or NaCl. NAD-dependent glutamate dehydrogenase, assayed by the reductive amination of 2-oxoglutarate, showed low activity in the absence of salt. NaCl was stimulatory with optimum activity at 3-3.5 M. However, no activity was found above 2.5 M KCl. Although the four activities examined all function at high salt concentrations, the behavior of individual enzymes toward salt varied considerably. The results presented show that Salinibacter enzymes are adapted to function in the presence of high salt concentrations.     

Enhanced extracellular lipid accumulation in acidic environments

PURPOSE OF REVIEW: Binding of apolipoprotein B-100-containing lipoproteins (VLDL, IDL, and LDL) to proteoglycans and modifications of the lipoproteins, whether bound or unbound, are key processes in atherogenesis. The complex interplay between binding and modification has been studied at neutral pH conditions. It has been demonstrated that during atherogenesis the extracellular pH of the lesions decreases. We summarize findings suggesting that lipoprotein binding and modification are enhanced at acidic pH. RECENT FINDINGS: Many enzymes found in the arterial intima, such as secretory sphingomyelinase and cathepsins, are able to hydrolyze lipoproteins in vitro. These enzymes function optimally at slightly acidic pH (pH 5.5-6.5), and are likely to act on lipoproteins optimally in the acidic plaque areas. Also, the ability of human aortic proteoglycans to bind native VLDL, IDL, and LDL is dramatically increased at acidic pH; this binding can be further increased if these apolipoprotein B-100-containing particles are hydrolytically modified. SUMMARY: Recent in-vitro findings suggest that in areas of atherosclerotic arterial intima where the extracellular pH is decreased, binding of apolipoprotein B-100-containing lipoproteins to proteoglycans and modification of the lipoproteins by acidic enzymes are enhanced. The pH-induced amplification of these processes will lead to enhanced extracellular accumulation of lipoproteins and accelerated progression of the disease.     

Purification and Properties of Two Thermostable Alkaline Xylanases from an Alkaliphilic Bacillus sp.

Two xylanases, designated XylA and XylB, were purified from the culture supernatant of the alkaliphilic Bacillus sp. strain AR-009. The molecular masses of the two enzymes were estimated to be 23 kDa (XylA) and 48 kDa (XylB) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pHs for activity were 9 for XylA and 9 to 10 for XylB. The temperature optima for the activity of XylA were 60°C at pH 9 and 70°C at pH 8. XylB was optimally active at 75°C at pH 9 and 70°C at pH 8. Both enzymes were stable in a broad pH range and showed good stability when incubated at 60 and 65°C in pH 8 and 9 buffers.     

Characterization of proteolytic systems in human and rat urine

Activities of proteolytic enzymes were detected in rat and human urine by using [125 l] iodo-insulin B chain as a substrate. The pH optimum of human urine activity was in the acidic range (pH 2.0) whereas the rat urine had two pH optima, one at the acidic range similar to human urine and another at pH 7.5. The activities were linear with time and amount of enzyme. Study with various proteinase inhibitors revealed that the acidic pH activities of human and rat urine were apparently of carboxyl endopeptidases since they were totally inhibited by pepstatin 10-8M. The neutral pH proteolysis of rat urine was inhibited by chelating agents and therefore it was considered as a metalloendopeptidase activity. These findings show the difference between the content of urinary proteolytic enzymes in humans and in rats by using a sensitive and simple radioactive assay.     

Cleavage of peptide bonds bearing ionizable amino acids at P1 by serine proteases with hydrophobic S1 pocket

Enzymatic hydrolysis of the synthetic substrate succinyl-Ala-Ala-Pro-Xxx-pNA (where Xxx = Leu, Asp or Lys) catalyzed by bovine chymotrypsin (CHYM) or Streptomyces griseus protease B (SGPB) has been studied at different pH values in the pH range 3-11. The pH optima for substrates having Leu, Asp, and Lys have been found to be 7.5-8.0, 5.5-6.0, and ∼10, respectively. At the normally reported pH optimum (pH 7-8) of CHYM and SGPB, the substrate with Leu at the reactive site is more than 25,000-fold more reactive than that with Asp. However, when fully protonated, Asp is nearly as good a substrate as Leu. The pK values of the side chains of Asp and Lys in the hydrophobic S₁ pocket of CHYM and SGPB have been calculated from pH-dependent hydrolysis data and have been found to be about 9 for Asp and 7.4 and 9.7 for Lys for CHYM and SGPB, respectively. The results presented in this communication suggest a possible application of CHYM like enzymes in cleaving peptide bonds contributed by acidic amino acids between pH 5 and 6.     

AmyA, an α-Amylase with β-Cyclodextrin-Forming Activity, and AmyB from the Thermoalkaliphilic Organism Anaerobranca gottschalkii: Two α-Amylases Adapted to Their Different Cellular Localizations

Two α-amylase genes from the thermophilic alkaliphile Anaerobranca gottschalkii were cloned, and the corresponding enzymes, AmyA and AmyB, were investigated after purification of the recombinant proteins. Based on their amino acid sequences, AmyA is proposed to be a lipoprotein with extracellular localization and thus is exposed to the alkaline milieu, while AmyB apparently represents a cytoplasmic enzyme. The amino acid sequences of both enzymes bear high similarity to those of GHF13 proteins. The different cellular localizations of AmyA and AmyB are reflected in their physicochemical properties. The alkaline pH optimum (pH 8), as well as the broad pH range, of AmyA activity (more than 50% activity between pH 6 and pH 9.5) mirrors the conditions that are encountered by an extracellular enzyme exposed to the medium of A. gottschalkii, which grows between pH 6 and pH 10.5. AmyB, on the other hand, has a narrow pH range with a slightly acidic pH optimum at 6 to 6.5, which is presumably close to the pH in the cytoplasm. Also, the intracellular AmyB is less tolerant of high temperatures than the extracellular AmyA. While AmyA has a half-life of 48 h at 70°C, AmyB has a half-life of only about 10 min at that temperature, perhaps due to the lack of stabilizing constituents of the cytoplasm. AmyA and AmyB were very similar with respect to their substrate specificity profiles, clearly preferring amylose over amylopectin, pullulan, and glycogen. Both enzymes also hydrolyzed α-, β-, and γ-cyclodextrin. Very interestingly, AmyA, but not AmyB, displayed high transglycosylation activity on maltooligosaccharides and also had significant β-cyclodextrin glycosyltransferase (CGTase) activity. CGTase activity has not been reported for typical α-amylases before. The mechanism of cyclodextrin formation by AmyA is unknown.     

A direct continuous spectrophotometric assay for glycosidases with 3-nitro-2-pyridyl glycosides by tautomerization of 2-hydroxy-3-nitropyridine

Two kinds of 3-nitro-2-pyridyl glycosides were synthesized and evaluated as substrates for continuous spectrophotometric assay for glycosidases. The liberated aglycon, 2-hydroxy-3-nitropyridine, immediately tautomerized to 3-nitro-2(1H)-pyridone, causing an absorption shift of ca. 60 nm even under acidic conditions (pH 3-6). Consequently, the enzymatic hydrolysis of these glycosides was monitored continuously in the acidic to neutral pH range (pH 4-7), the optimum pH for most glycosidases. The absorbance of liberated aglycon increased linearly at 390 nm until 10% consumption of the substrate to enable the initial rate to be determined at once without terminating the reaction. The kinetic parameters for the hydrolysis of 3-nitro-2-pyridyl glycosides were obtained from the slopes of the progress curves and were compared with those obtained from the conventional discontinuous assay using p- and o-nitrophenyl glycosides as substrates. The kinetic parameters indicated that 3-nitro-2-pyridyl glycosides were more activated and specific substrates, but with less affinity to the enzymes than the corresponding nitrophenyl glycosides. Moreover, the absorbance shift by tautomerization should promise further applications to continuous spectrophotometric assays for other enzymes acting under acidic conditions, such as acid proteases and acid phosphatases.     

Purification and characterization of cellulases produced by two Bacillus strains

Cellulases produced by two Bacillus strains, CH43 and HR68, isolated from hot springs in Zimbabwe, were purified to homogeneity from culture supernatants. Both enzymes had molecular mass of 40 kDa and isoelectric point of 5.4. The enzymes also resembled each other in N-terminal amino acid sequence which was Ala-Gly-Thr-Lys-Thr-Pro-Val-Ala-Lys-Asn-Gly-Gln, showing 100% homology with that of endoglucanases from Bacillus subtilis belonging to glycoside hydrolase family five. The cellulases were optimally active in the pH range of 5-6.5. The optimum temperature was 65 and 70 degrees C for the endoglucanase of CH43 and HR68, respectively. The CH43 enzyme was stable at 50 degrees C in a pH range of 6-10, and HR68 at pH 6-8. Both the enzymes retained complete activity for at least 24 h at 50 degrees C. The enzymes showed highest activity with beta-glucan as substrate followed by carboxymethylcellulose. Significant activity was also observed with crystalline forms of cellulose such as filter paper and Avicel, particularly for HR68 cellulase. For carboxymethycellulose, the CH43 and HR68 cellulases had a Km of 1.5 and 1.7 mg ml(-1), respectively, and Vmax of 0.93 and 1.70 mmol glucose min(-1) mg protein(-1) respectively. The activity of the enzymes was not influenced by most metal ions at 1 mM concentration, but was increased by about 38% by Co2+. The inhibition by Hg2+ and Mn2+ was higher for CH43 than for HR68 enzyme. Ag+ inhibited the CH43 activity but stimulated the HR68 activity. The CH43 cellulase was inhibited by N-bromosuccinimide and iodoacetamide while HR68 was unaffected.     

Probing the active sites and mechanisms of rat metalloproteases meprin A and B

Meprin A and B are highly regulated, secreted and cell-surface homo- and hetero-oligomeric enzymes. Meprins are abundantly expressed in kidney and intestine. The multidomain alpha and beta subunits have high sequence identity, however they have very different substrate specificities, oligomerization potentials and are differentially regulated. Here we describe that meprin subunit activities are modulated differently by physico-chemical factors. Homo-oligomeric meprin B had an acidic pH optimum. The low pH protonation indicated the existence of at least two ionizable groups. An additional ionizable group generated a shoulder in the basic pH range. Homo-oligomeric meprin A had a neutral pH optimum and the activity curve revealed that two ionizable groups might be protonated at acidic pH similar to meprin B. Increasing the concentration of salt generally inhibited meprin B activity. Meprin A was inhibited at low salt concentrations but activated as salt was increased. This work has important implications in the elucidation of the catalytic mechanisms of meprins and other metalloproteases. In addition, the activity of meprin oligomers that arise in tissues will be affected by variations in pH and NaCl. This could have profound implications because meprins are exposed to a range of conditions in the extracellular milieu of renal and intestinal tissues and in inflammation and cancer.     

Glyoxylate Pathway in the Free-Living Stages of the Entomophilic Nematode Romanomermis culicivorax

Isocitrate lyase and malate synthetase, key enzymes of the glyoxylate cycle, were present in postparasites of the mermithid nematode Romanomermis culicivorax. Specific activities of enzymes were higher in adult postparasites than in newly emerged juveniles. Isocitrate lyase had a well-defined pH optimum (7.5), whereas malate synthetase functioned optimally over a broad range of alkaline pH (7.5-9.0). Substrate affinities of the two enzymes were measured.     

Methanol-Assimilation durch ein acidophiles Bacterium der Gattung Acetobacter

Generally, methylotrophic bacteria grow optimally in a pH range between 6 and 7.2. The assimilation of methanol can take place via several pathways. Acetobacter methanolicus preiers an acidic pH range for growth, the pH optimum is about 4, and it uses the FBP variant for methanol assimilation. The latter is interesting from a regulatory point of view because phosphofructokinase disappears during growth on glucose, which is assimilated via the hexosemonophosphate pathway. Since Entner-Doudoroff enzymes and phosphoketolase are absent in A. `ethanolicus as well as in non-methylotrophic Acetobacter and Gluconobacter species phosphofructokinase becomes a key enzyme of the assimilation of methanol. Although A. methanolicus uses the hexulosephosphate pathway the growth yield on methanol is smaller than with other “hexulosephosphate pathway bacteria” e. g. with obligate methanol assimilating bacteria. At first sight it may appear that the acidic optimum pH is responsible for the smaller growth yield and the discrepancy between the experimental and predicted values. The relationship between the dependence on and the protection from, high external proton concentration on the one hand and the causes of the low growth yield on the other are discussed. Accordingly, A. methanolicus and another heterotrophic acidophiles seem to be acidoresistant above all, their machinery guaranteeing the protection from the high proton concentration is responsible for the acidophily and the low growth efficiency is caused by a simple respiratory chain.     

How gastric lipase, an interfacial enzyme with a Ser-His-Asp catalytic triad, acts optimally at acidic pH

Gastric lipase is active under acidic conditions and shows optimum activity on insoluble triglycerides at pH 4. The present results show that gastric lipase also acts in solution on vinyl butyrate, with an optimum activity above pH 7, which suggests that gastric lipase is able to hydrolyze ester bonds via the classical mechanism of serine hydrolases. These results support previous structural studies in which the catalytic triad of gastric lipase was reported to show no specific features. The optimum activity of gastric lipase shifted toward lower pH values, however, when the vinyl butyrate concentration was greater than the solubility limit. Experiments performed with long-chain triglycerides showed that gastric lipase binds optimally to the oil-water interface at low pH values. To study the effects of the pH on the adsorption step independently from substrate hydrolysis, gastric lipase adsorption on solid hydrophobic surfaces was monitored by total internal reflection fluorescence (TIRF), as well as using a quartz crystal microbalance. Both techniques showed a pH-dependent reversible gastric lipase adsorption process, which was optimum at pH 5 (Kd = 6.5 nM). Lipase adsorption and desorption constants (ka = 147,860 M(-1) s(-1) and kd = 139 x 10(-4) s(-1) at pH 6) were estimated from TIRF experiments. These results indicate that the optimum activity of gastric lipase at acidic pH is only "apparent" and results from the fact that lipase adsorption at lipid-water interfaces is the pH-dependent limiting step in the overall process of insoluble substrate hydrolysis. This specific kinetic feature of interfacial enzymology should be taken into account when studying any soluble enzyme acting on an insoluble substrate.     

Alkaliphiles: Some Applications of Their Products for Biotechnology

The term “alkaliphile” is used for microorganisms that grow optimally or very well at pH values above 9 but cannot grow or grow only slowly at the near-neutral pH value of 6.5. Alkaliphiles include prokaryotes, eukaryotes, and archaea. Many different taxa are represented among the alkaliphiles, and some of these have been proposed as new taxa. Alkaliphiles can be isolated from normal environments such as garden soil, although viable counts of alkaliphiles are higher in samples from alkaline environments. The cell surface may play a key role in keeping the intracellular pH value in the range between 7 and 8.5, allowing alkaliphiles to thrive in alkaline environments, although adaptation mechanisms have not yet been clarified. Alkaliphiles have made a great impact in industrial applications. Biological detergents contain alkaline enzymes, such as alkaline cellulases and/or alkaline proteases, that have been produced from alkaliphiles. The current proportion of total world enzyme production destined for the laundry detergent market exceeds 60%. Another important application is the industrial production of cyclodextrin by alkaline cyclomaltodextrin glucanotransferase. This enzyme has reduced the production cost and paved the way for cyclodextrin use in large quantities in foodstuffs, chemicals, and pharmaceuticals. It has also been reported that alkali-treated wood pulp could be biologically bleached by xylanases produced by alkaliphiles. Other applications of various aspects of alkaliphiles are also discussed.     

Novel process for the production of cellulolytic enzymes

A novel process for the production of extracellular carboxymethylcellulase (CMCase) and xylanase by fermentation under nonaseptic or nonsterile conditions is described. The fermentation process is carried out under very acidic conditions of pH 2.0 by using a acidophilic cellulolytic fungus. Microbial contamination is avoided or minimized to an insignificant level under this acid pH condition. The culture medium for this production consists of a carbon source from cellulosics or lignocellulosics, such as Na-CMC, xylan, Avicel cellulose, cellulose powder, alpha-cellulose, sawdust, etc., or a mixture of the forementioned together with simple ingredients such as (NH(4))(2)SO(4), K(2)HPO(4), MgSO(4) and NaNO(3). The fermentation is carried out at room temperature (28-30 degrees C), under aerobic conditions, and without controlling the pH. The CMCase and xylanase produced are stable under very simple storage conditions, such as in the fresh culture medium not containing the substrate for a period of 3 days, at any temperature from 0 to 30 degrees C. These extracellular enzymes have an optimum pH around 3, with the best range of pH from 2.0 to 3.6, for any temperature between 15 and 60 degrees C. The optimum temperatures are 55 degrees C for CMCase activity and 25-50 degrees C for xylanase activity, at any pH between 2.0 and 5.2. The apparent Michaelis constants Km are 2.6 and 1.5 mg/mL for CMCase and xylanase of the culture filtrate, respectively.     

Isolation and Biochemical Characterization of Two Novel Metagenome-Derived Esterases

The metagenomes of uncultured microbial communities are rich sources for novel biocatalysts. In this study, esterase EstA3 was derived from a drinking water metagenome, and esterase EstCE1 was derived from a soil metagenome. Both esterases are approximately 380 amino acids in size and show similarity to β-lactamases, indicating that they belong to family VIII of the lipases/esterases. EstA3 had a temperature optimum at 50°C and a pH optimum at pH 9.0. It was remarkably active and very stable in the presence of solvents and over a wide temperature and pH range. It is active in a multimeric form and displayed a high level of activity against a wide range of substrates including one secondary ester, 7-[3-octylcarboxy-(3-hydroxy-3-methyl-butyloxy)]-coumarin, which is normally unreactive. EstCE1 was active in the monomeric form and had a temperature optimum at 47°C and a pH optimum at pH 10. It exhibited the same level of stability as EstA3 over wide temperature and pH ranges and in the presence of dimethyl sulfoxide, isopropanol, and methanol. EstCE1 was highly enantioselective for (+)-menthylacetate. These enzymes display remarkable characteristics that cannot be related to the original environment from which they were derived. The high level of stability of these enzymes together with their unique substrate specificities make them highly useful for biotechnological applications.     

Review of rotifers and crustaceans in highly acidic environments of pH values ≤3

A review of the literature on rotifers and crustacean zooplankton in highly acidic environments revealed that data from eleven aquatic environments on three continents (America, Europe, Japan) with a pH 3 are available. Seven sites are influenced by volcanism or weathering processes in the catchment area, four others originated from human mining activities. Species richness was generally low. Only 16 species are found and 1–11 species are reported for each area. These studies clearly show that small littoral or benthic rotifers predominate over crustaceans under highly acidic conditions. In the Lusatian mining area (Germany), all lakes are colonized by zooplankton, even the most acidic one with a pH of 2.3. The core community consists of the rotifers Cephalodella hoodi, C. gibba, Elosa worallii and Rotaria rotatoria, with C. hoodi and E. worallii the most abundant. Larger species, such as the rotifer Brachionus sericus or the cladoceran Chydorus sphaericus, occur at a pH close to 3. A similar pattern is reported from acidic mining lakes in Illinois, U.S.A. Many of these species can also be found in less acidic softwater or even alkaline environments due to the tolerance of a broad range of pH values. Elosa worallii and Brachionus sericus are probably the most acidophilic rotifer species, though at least the latter can also grow at neutral pH in the laboratory. Clear understanding of the pH limits of B. sericus in nature may also have been complicated by the fact that it has probably in the past been wrongly named as B. urceolaris (phenotype `sericus'). The typical B. urceolaris cannot tolerate extremely low pH. Overall, generalist species with a worldwide distribution seem to play the major role in the colonization of anthropogenic highly acidic lakes.     

Role of N-glycosylation in cathepsin E. A comparative study of cathepsin E with distinct N-linked oligosaccharides and its nonglycosylated mutant.

Cathepsin E (CE), a nonlysosomal, intracellular aspartic proteinase, exists in several molecular forms that are N-glycosylated with high-mannose and/or complex-type oligosaccharides. To investigate the role of N-glycosylation on the catalytic properties and molecular stability of CE, both natural and recombinant enzymes with distinct oligosaccharides were purified from different sources. An N-glycosylation minus mutant, that was constructed by site-directed mutagenesis (by changing asparagine residues to glutamine and aspartic acid residues at positions 73 and 305 in potential N-glycosylation sites of rat CE) and expressed in normal rat kidney cells, was also purified to homogeneity from the cell extracts. The kinetic parameters of the nonglycosylated mutant were found to be essentially equivalent to those of natural enzymes N-glycosylated with either high-mannose or complex-type oligosaccharides. In contrast, the nonglycosylated mutant showed lower pH and thermal stabilities than the glycosylated enzymes. The nonglycosylated mutant exhibited particular sensitivity to conversion to a monomeric form by 2-mercaptoethanol, as compared with those of the glycosylated enzymes. Further, the high-mannose-type enzymes were more sensitive to this agent than the complex-type proteins. A striking difference was found between the high-mannose and complex-type enzymes in terms of activation by ATP at a weakly acidic pH. At pH 5.5, the complex-type enzymes were stabilized by ATP to be restored to the virtual activity, whereas the high-mannose-type enzymes as well as the nonglycosylated mutant were not affected by ATP. These results suggest that N-glycosylation in CE is important for the maintenance of its proper folding upon changes in temperature, pH and redox state, and that the complex-type oligosaccharides contribute to the completion of the tertiary structure to maintain its active conformation in the weakly acidic pH environments.     

Intracellular pH Regulation in an Acidophilic Unicellular Alga, Cyanidium caldarium: 31P-NMR Determination of Intracellular pH

The intracellular pH of an acidophilic unicellular alga, Cyanidiumcaldarium, was determined as a function of external pH by ³¹Pnuclear magnetic resonance. The algal cells incubated underaerobic conditions or under anaerobic and illuminated conditionsmaintained the intracellular pH in the range from 6.8 to 7.0even when the external pH was changed from 1.2 to 8.4. Underanaerobic and dark conditions, however, the intracellular pHacidified at the acidic pH region of the external medium. Theacidified intracellular pH reversibly returned to neutral eitheron aeration or illumination. The results indicate that, in Cyanidiumcells growing in extremely acidic environments, an active H⁺efflux (H⁺ pump) which depends on metabolic activity (respirationor photosynthesis) is essential to maintain the intracellularpH at a constant physiological level against the passive H⁺leakage due to the steep pH gradient across the cell membrane. (Received March 19, 1986; Accepted July 17, 1986)     

Electrostatic role of aromatic ring stacking in the pH-sensitive modulation of a chymotrypsin-type serine protease, Achromobacter protease I.

Achromobacter protease I (API) has a unique region of aromatic ring stacking with Trp169-His210 in close proximity to the catalytic triad. This paper reveals the electrostatic role of aromatic stacking in the shift in optimum pH to the alkaline region, which is the highest pH range (8.5-10) among chymotrypsin-type serine proteases. The pH-activity profile of API showed a sigmoidal distribution that appears at pH 8-10, with a shoulder at pH 6-8. Variants with smaller amino acid residues substituted for Trp169 had lower pH optima on the acidic side by 0-0.9 units. On the other hand, replacement of His210 by Ala or Ser lowered the acidic rim by 1.9 pH units, which is essentially identical to that of chymotrypsin and trypsin. Energy minimization for the mutant structures suggested that the side-chain of Trp169 stacked with His210 was responsible for isolation of the electrostatic interaction between His210 and the catalytic Asp113 from solvent. The aromatic stacking regulates the low activity at neutral pH and the high activity at alkaline pH due to the interference of the hydrogen bonded network in the catalytic triad residues.     

Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella

During the course of infection, Salmonella enterica serovar Typhimurium must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments. Three low pH inducible amino acid decarboxylases were annotated in the genome of S. Typhimurium, AdiA, CadA and SpeF, which are specific for arginine, lysine and ornithine, respectively. In this study, we characterized and compared the contributions of those enzymes in response to acidic challenges. Individual mutants as well as a strain deleted for the three genes were tested for their ability (i) to survive an extreme acid shock, (ii) to grow at mild acidic pH and (iii) to infect the mouse animal model. We showed that the lysine decarboxylase CadA had the broadest range of activity since it both had the capacity to promote survival at pH 2.3 and growth at pH 4.5. The arginine decarboxylase AdiA was the most performant in protecting S. Typhimurium from a shock at pH 2.3 and the ornithine decarboxylase SpeF conferred the best growth advantage under anaerobiosis conditions at pH 4.5. We developed a GFP-based gene reporter to monitor the pH of the environment as perceived by S. Typhimurium. Results showed that activities of the lysine and ornithine decarboxylases at mild acidic pH did modify the local surrounding of S. Typhimurium both in culture medium and in macrophages. Finally, we tested the contribution of decarboxylases to virulence and found that these enzymes were dispensable for S. Typhimurium virulence during systemic infection. In the light of this result, we examined the genomes of Salmonella spp. normally responsible of systemic infection and observed that the genes encoding these enzymes were not well conserved, supporting the idea that these enzymes may be not required during systemic infection.