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1.  Mechanisms of acid resistance in enterohemorrhagic Escherichia coli. 
Enterohemorrhagic strains of Escherichia coli must pass through the acidic gastric barrier to cause gastrointestinal disease. Taking into account the apparent low infectious dose of enterohemorrhagic E. coli, 11 O157:H7 strains and 4 commensal strains of E. coli were tested for their abilities to survive extreme acid exposures (pH 3). Three previously characterized acid resistance systems were tested. These included an acid-induced oxidative system, an acid-induced arginine-dependent system, and a glutamate-dependent system. When challenged at pH 2.0, the arginine-dependent system provided more protection in the EHEC strains than in commensal strains. However, the glutamate-dependent system provided better protection than the arginine system and appeared equally effective in all strains. Because E. coli must also endure acid stress imposed by the presence of weak acids in intestinal contents at a pH less acidic than that of the stomach, the ability of specific acid resistance systems to protect against weak acids was examined. The arginine- and glutamate-dependent systems were both effective in protecting E. coli against the bactericidal effects of a variety of weak acids. The acids tested include benzoic acid (20 mM; pH 4.0) and a volatile fatty acid cocktail composed of acetic, propionic, and butyric acids at levels approximating those present in the intestine. The oxidative system was much less effective. Several genetic aspects of E. coli acid resistance were also characterized. The alternate sigma factor RpoS was shown to be required for oxidative acid resistance but was only partially involved with the arginine- and glutamate-dependent acid resistance systems. The arginine decarboxylase system (including adi and its regulators cysB and adiY) was responsible for arginine-dependent acid resistance. The results suggest that several acid resistance systems potentially contribute to the survival of pathogenic E. coli in the different acid stress environments of the stomach (pH 1 to 3) and the intestine (pH 4.5 to 7 with high concentrations of volatile fatty acids). Of particular importance to the food industry was the finding that once induced, the acid resistance systems will remain active for prolonged periods of cold storage at 4 degrees C.
PMCID: PMC168100  PMID: 8795195
2.  Variation in Acid Resistance among Shiga Toxin-Producing Clones of Pathogenic Escherichia coli†  
Pathogenic strains of Escherichia coli, such as E. coli O157:H7, have a low infectious dose and an ability to survive in acidic foods. These bacteria have evolved at least three distinct mechanisms of acid resistance (AR), including two amino acid decarboxylase-dependent systems (arginine and glutamate) and a glucose catabolite-repressed system. We quantified the survival rates for each AR mechanism separately in clinical isolates representing three groups of Shiga toxin-producing E. coli (STEC) clones (O157:H7, O26:H11/O111:H8, and O121:H19) and six commensal strains from ECOR group A. Members of the STEC clones were not significantly more acid resistant than the commensal strains when analyzed using any individual AR mechanism. The glutamate system provided the best protection in a highly acidic environment for all groups of isolates (<0.1 log reduction in CFU/ml per hour at pH 2.0). Under these conditions, there was notable variation in survival rates among the 30 O157:H7 strains, which depended in part on Mg2+ concentration. The arginine system provided better protection at pH 2.5, with a range of 0.03 to 0.41 log reduction per hour, compared to the oxidative system, with a range of 0.13 to 0.64 log reduction per hour. The average survival rate for the O157:H7 clonal group was significantly less than that of the other STEC clones in the glutamate and arginine systems and significantly less than that of the O26/O111 clone in the oxidative system, indicating that this clonal group is not exceptionally acid resistant with these specific mechanisms.
PMCID: PMC1087542  PMID: 15870339
3.  Acid Resistance Systems Required for Survival of Escherichia coli O157:H7 in the Bovine Gastrointestinal Tract and in Apple Cider Are Different 
Escherichia coli O157:H7 is a highly acid-resistant food-borne pathogen that survives in the bovine and human gastrointestinal tracts and in acidic foods such as apple cider. This property is thought to contribute to the low infectious dose of the organism. Three acid resistance (AR) systems are expressed in stationary-phase cells. AR system 1 is σS dependent, while AR systems 2 and 3 are glutamate and arginine dependent, respectively. In this study, we sought to determine which AR systems are important for survival in acidic foods and which are required for survival in the bovine intestinal tract. Wild-type and mutant E. coli O157:H7 strains deficient in AR system 1, 2, or 3 were challenged with apple cider and inoculated into calves. Wild-type cells, adapted at pH 5.5 in the absence of glucose (AR system 1 induced), survived well in apple cider. Conversely, the mutant deficient in AR system 1, shown previously to survive poorly in calves, was susceptible to apple cider (pH 3.5), and this sensitivity was shown to be caused by low pH. Interestingly, the AR system 2-deficient mutant survived in apple cider at high levels, but its shedding from calves was significantly decreased compared to that of wild-type cells. AR system 3-deficient cells survived well in both apple cider and calves. Taken together, these results indicate that E. coli O157:H7 utilizes different acid resistance systems based on the type of acidic environment encountered.
PMCID: PMC492388  PMID: 15294816
4.  Role of rpoS in Acid Resistance and Fecal Shedding of Escherichia coli O157:H7 
Acid resistance (AR) is important to survival of Escherichia coli O157:H7 in acidic foods and may play a role during passage through the bovine host. In this study, we examined the role in AR of the rpoS-encoded global stress response regulator ςS and its effect on shedding of E. coli O157:H7 in mice and calves. When assayed for each of the three AR systems identified in E. coli, an rpoS mutant (rpoS::pRR10) of E. coli O157:H7 lacked the glucose-repressed system and possessed reduced levels of both the arginine- and glutamate-dependent AR systems. After administration of the rpoS mutant and the wild-type strain (ATCC 43895) to ICR mice at doses ranging from 101 to 104 CFU, we found the wild-type strain in feces of mice given lower doses (102 versus 103 CFU) and at a greater frequency (80% versus 13%) than the mutant strain. The reduction in passage of the rpoS mutant was due to decreased AR, as administration of the mutant in 0.05 M phosphate buffer facilitated passage and increased the frequency of recovery in feces from 27 to 67% at a dose of 104 CFU. Enumeration of E. coli O157:H7 in feces from calves inoculated with an equal mixture of the wild-type strain and the rpoS mutant demonstrated shedding of the mutant to be 10- to 100-fold lower than wild-type numbers. This difference in shedding between the wild-type strain and the rpoS mutant was statistically significant (P ≤ 0.05). Thus, ςS appears to play a role in E. coli O157:H7 passage in mice and shedding from calves, possibly by inducing expression of the glucose-repressed RpoS-dependent AR determinant and thus increasing resistance to gastrointestinal stress. These findings may provide clues for future efforts aimed at reducing or eliminating this pathogen from cattle herds.
PMCID: PMC91873  PMID: 10653728
5.  Arginine-Agmatine Antiporter in Extreme Acid Resistance in Escherichia coli 
Journal of Bacteriology  2003;185(22):6556-6561.
The process of arginine-dependent extreme acid resistance (XAR) is one of several decarboxylase-antiporter systems that protects Escherichia coli and possibly other enteric bacteria from exposure to the strong acid environment of the stomach. Arginine-dependent acid resistance depends on an intracellular proton-utilizing arginine α-decarboxylase and a membrane transport protein necessary for delivering arginine to and removing agmatine, its decarboxylation product, from the cytoplasm. The arginine system afforded significant protection to wild-type E. coli cells in our acid shock experiments. The gene coding for the transport protein is identified here as a putative membrane protein of unknown function, YjdE, which we now name adiC. Strains from which this gene is deleted fail to mount arginine-dependent XAR, and they cannot perform coupled transport of arginine and agmatine. Homologues of this gene are found in other bacteria in close proximity to homologues of the arginine decarboxylase in a gene arrangement pattern similar to that in E coli. Evidence for a lysine-dependent XAR system in E. coli is also presented. The protection by lysine, however, is milder than that by arginine.
PMCID: PMC262112  PMID: 14594828
6.  Arginine-Dependent Acid Resistance in Salmonella enterica Serovar Typhimurium 
Journal of Bacteriology  2006;188(15):5650-5653.
Salmonella enterica serovar Typhimurium does not survive a pH 2.5 acid challenge under conditions similar to those used for Escherichia coli (J. W. Foster, Nat. Rev. Microbiol. 2:898-907, 2004). Here, we provide evidence that S. enterica serovar Typhimurium can display arginine-dependent acid resistance (AR) provided the cells are grown under anoxic conditions and not under the microaerobic conditions used for assessment of AR in E. coli. The role of the arginine decarboxylase pathway in Salmonella AR was shown by the loss of AR in mutants lacking adiA, which encodes arginine decarboxylase; adiC, which encodes the arginine-agmatine antiporter; or adiY, which encodes an AraC-like regulator. Transcription of adiA and adiC was found to be dependent on AdiY, anaerobiosis, and acidic pH.
PMCID: PMC1540025  PMID: 16855258
7.  Loss of topoisomerase I function affects the RpoS-dependent and GAD systems of acid resistance in Escherichia coli 
Microbiology (Reading, England)  2005;151(Pt 8):2783-2791.
Acid resistance (AR) for Escherichia coli is important for its survival in the human gastrointestinal tract and involves three systems. The first AR system is dependent on the sigma factor RpoS. The second system (GAD system) requires glutamate decarboxylase isoforms encoded by the gadA and gadB genes. The third system (ARG system) requires arginine decarboxylase encoded by adiA. Loss of topoisomerase I function from topA deletion or Tn10 insertion mutations lowered the resistance to killing by pH 2 or 2.5 treatment by 10 to >100 fold. The RpoS and GAD systems were both affected by the topA mutation but the ARG system of acid resistance was not affected. Northern blot analysis showed that induction of gadA and gadB transcription in stationary phase and at pH 5.5 was decreased in the topA mutant. Western blot analysis showed that the topA mutation did not affect accumulation of RpoS, GadX or GadW proteins. Topoisomerase I could have a direct influence on transcription of acid resistance genes. This influence did not involve R-loop formation as the overexpression of RNase H did not alleviate the decrease of acid resistance from the topA mutation. The effect of the topA mutation could be suppressed by the hns mutation so topoisomerase I might be required to counteract the effect of H-NS protein on gene expression in addition to its influence on RpoS-dependent transcription.
PMCID: PMC1361560  PMID: 16079354
8.  Characterization of Enterohemorrhagic Escherichia coli Strains Based on Acid Resistance Phenotypes†  
Infection and Immunity  2005;73(8):4993-5003.
Acid resistance is perceived to be an important property of enterohemorrhagic Escherichia coli strains, enabling the organisms to survive passage through the acidic environment of the stomach so that they may colonize the mammalian gastrointestinal tract and cause disease. Accordingly, the organism has developed at least three genetically and physiologically distinct acid resistance systems which provide different levels of protection. The glutamate-dependent acid resistance (GDAR) system utilizes extracellular glutamate to protect cells during extreme acid challenges and is believed to provide the highest protection from stomach acidity. In this study, the GDAR system of 82 pathogenic E. coli isolates from 34 countries and 23 states within the United States was examined. Twenty-nine isolates were found to be defective in inducing GDAR under aerobic growth conditions, while five other isolates were defective in GDAR under aerobic, as well as fermentative, growth conditions. We introduced rpoS on a low-copy-number plasmid into 26 isolates and were able to restore GDAR in 20 acid-sensitive isolates under aerobic growth conditions. Four isolates were found to be defective in the newly discovered LuxR-like regulator GadE (formerly YhiE). Defects in other isolates could be due to a mutation(s) in a gene(s) with an as yet undefined role in acid resistance since GadE and/or RpoS could not restore acid resistance. These results show that in addition to mutant alleles of rpoS, mutations in gadE exist in natural populations of pathogenic E. coli. Such mutations most likely alter the infectivity of individual isolates and may play a significant role in determining the infective dose of enterohemorrhagic E. coli.
PMCID: PMC1201262  PMID: 16041014
9.  Control of Acid Resistance in Escherichia coli 
Journal of Bacteriology  1999;181(11):3525-3535.
Acid resistance (AR) in Escherichia coli is defined as the ability to withstand an acid challenge of pH 2.5 or less and is a trait generally restricted to stationary-phase cells. Earlier reports described three AR systems in E. coli. In the present study, the genetics and control of these three systems have been more clearly defined. Expression of the first AR system (designated the oxidative or glucose-repressed AR system) was previously shown to require the alternative sigma factor RpoS. Consistent with glucose repression, this system also proved to be dependent in many situations on the cyclic AMP receptor protein. The second AR system required the addition of arginine during pH 2.5 acid challenge, the structural gene for arginine decarboxylase (adiA), and the regulator cysB, confirming earlier reports. The third AR system required glutamate for protection at pH 2.5, one of two genes encoding glutamate decarboxylase (gadA or gadB), and the gene encoding the putative glutamate:γ-aminobutyric acid antiporter (gadC). Only one of the two glutamate decarboxylases was needed for protection at pH 2.5. However, survival at pH 2 required both glutamate decarboxylase isozymes. Stationary phase and acid pH regulation of the gad genes proved separable. Stationary-phase induction of gadA and gadB required the alternative sigma factor ςS encoded by rpoS. However, acid induction of these enzymes, which was demonstrated to occur in exponential- and stationary-phase cells, proved to be ςS independent. Neither gad gene required the presence of volatile fatty acids for induction. The data also indicate that AR via the amino acid decarboxylase systems requires more than an inducible decarboxylase and antiporter. Another surprising finding was that the ςS-dependent oxidative system, originally thought to be acid induced, actually proved to be induced following entry into stationary phase regardless of the pH. However, an inhibitor produced at pH 8 somehow interferes with the activity of this system, giving the illusion of acid induction. The results also revealed that the AR system affording the most effective protection at pH 2 in complex medium (either Luria-Bertani broth or brain heart infusion broth plus 0.4% glucose) is the glutamate-dependent GAD system. Thus, E. coli possesses three overlapping acid survival systems whose various levels of control and differing requirements for activity ensure that at least one system will be available to protect the stationary-phase cell under naturally occurring acidic environments.
PMCID: PMC93821  PMID: 10348866
10.  Genes of the GadX-GadW Regulon in Escherichia coli 
Journal of Bacteriology  2003;185(10):3190-3201.
Acid in the stomach is thought to be a barrier to bacterial colonization of the intestine. Escherichia coli, however, has three systems for acid resistance, which overcome this barrier. The most effective of these systems is dependent on transport and decarboxylation of glutamate. GadX regulates two genes that encode isoforms of glutamate decarboxylase critical to this system, but additional genes associated with the glutamate-dependent acid resistance system remained to be identified. The gadX gene and a second downstream araC-like transcription factor gene, gadW, were mutated separately and in combination, and the gene expression profiles of the mutants were compared to those of the wild-type strain grown in neutral and acidified media under conditions favoring induction of glutamate-dependent acid resistance. Cluster and principal-component analyses identified 15 GadX-regulated, acid-inducible genes. Reverse transcriptase mapping demonstrated that these genes are organized in 10 operons. Analysis of the strain lacking GadX but possessing GadW confirmed that GadX is a transcriptional activator under acidic growth conditions. Analysis of the strain lacking GadW but possessing GadX indicated that GadW exerts negative control over three GadX target genes. The strain lacking both GadX and GadW was defective in acid induction of most but not all GadX target genes, consistent with the roles of GadW as an inhibitor of GadX-dependent activation of some genes and an activator of other genes. Resistance to acid was decreased under certain conditions in a gadX mutant and even more so by combined mutation of gadX and gadW. However, there was no defect in colonization of the streptomycin-treated mouse model by the gadX mutant in competition with the wild type, and the gadX gadW mutant was a better colonizer than the wild type. Thus, E. coli colonization of the mouse does not appear to require glutamate-dependent acid resistance.
PMCID: PMC154079  PMID: 12730179
11.  Sigma Factor N, Liaison to an ntrC and rpoS Dependent Regulatory Pathway Controlling Acid Resistance and the LEE in Enterohemorrhagic Escherichia coli 
PLoS ONE  2012;7(9):e46288.
Enterohemorrhagic Escherichia coli (EHEC) is dependent on acid resistance for gastric passage and low oral infectious dose, and the locus of enterocyte effacement (LEE) for intestinal colonization. Mutation of rpoN, encoding sigma factor N (σN), dramatically alters the growth-phase dependent regulation of both acid resistance and the LEE. This study reports on the determinants of σN-directed acid resistance and LEE expression, and the underlying mechanism attributable to this phenotype. Glutamate-dependent acid resistance (GDAR) in TW14359ΔrpoN correlated with increased expression of the gadX-gadW regulatory circuit during exponential growth, whereas upregulation of arginine-dependent acid resistance (ADAR) genes adiA and adiC in TW14359ΔrpoN did not confer acid resistance by the ADAR mechanism. LEE regulatory (ler), structural (espA and cesT) and effector (tir) genes were downregulated in TW14359ΔrpoN, and mutation of rpoS encoding sigma factor 38 (σS) in TW14359ΔrpoN restored acid resistance and LEE genes to WT levels. Stability, but not the absolute level, of σS was increased in TW14359ΔrpoN; however, increased stability was not solely attributable to the GDAR and LEE expression phenotype. Complementation of TW14359ΔrpoN with a σN allele that binds RNA polymerase (RNAP) but not DNA, did not restore WT levels of σS stability, gadE, ler or GDAR, indicating a dependence on transcription from a σN promoter(s) and not RNAP competition for the phenotype. Among a library of σN enhancer binding protein mutants, only TW14359ΔntrC, inactivated for nitrogen regulatory protein NtrC, phenocopied TW14359ΔrpoN for σS stability, GDAR and ler expression. The results of this study suggest that during exponential growth, NtrC-σN regulate GDAR and LEE expression through downregulation of σS at the post-translational level; likely by altering σS stability or activity. The regulatory interplay between NtrC, other EBPs, and σN–σS, represents a mechanism by which EHEC can coordinate GDAR, LEE expression and other cellular functions, with nitrogen availability and physiologic stimuli.
PMCID: PMC3459932  PMID: 23029465
12.  Acid- and Base-Induced Proteins during Aerobic and Anaerobic Growth of Escherichia coli Revealed by Two-Dimensional Gel Electrophoresis 
Journal of Bacteriology  1999;181(7):2209-2216.
Proteins induced by acid or base, during long-term aerobic or anaerobic growth in complex medium, were identified in Escherichia coli. Two-dimensional gel electrophoresis revealed pH-dependent induction of 18 proteins, nine of which were identified by N-terminal sequencing. At pH 9, tryptophan deaminase (TnaA) was induced to a high level, becoming one of the most abundant proteins observed. TnaA may reverse alkalinization by metabolizing amino acids to produce acidic products. Also induced at high pH, but only in anaerobiosis, was glutamate decarboxylase (GadA). The gad system (GadA/GadBC) neutralizes acidity and enhances survival in extreme acid; its induction during anaerobic growth may help protect alkaline-grown cells from the acidification resulting from anaerobic fermentation. To investigate possible responses to internal acidification, cultures were grown in propionate, a membrane-permeant weak acid which acidifies the cytoplasm. YfiD, a homologue of pyruvate formate lyase, was induced to high levels at pH 4.4 and induced twofold more by propionate at pH 6; both of these conditions cause internal acidification. At neutral or alkaline pH, YfiD was virtually absent. YfiD is therefore a strong candidate for response to internal acidification. Acid or propionate also increased the expression of alkyl hydroperoxide reductase (AhpC) but only during aerobic growth. At neutral or high pH, AhpC showed no significant difference between aerobic and anaerobic growth. The increase of AhpC in acid may help protect the cell from the greater concentrations of oxidizing intermediates at low pH. Isocitrate lyase (AceA) was induced by oxygen across the pH range but showed substantially greater induction in acid or in base than at pH 7. Additional responses observed included the induction of MalE at high pH and induction of several enzymes of sugar metabolism at low pH: the phosphotransferase system components ManX and PtsH and the galactitol fermentation enzyme GatY. Overall, our results indicate complex relationships between pH and oxygen and a novel permeant acid-inducible gene, YfiD.
PMCID: PMC93635  PMID: 10094700
13.  YjdE (AdiC) Is the Arginine:Agmatine Antiporter Essential for Arginine-Dependent Acid Resistance in Escherichia coli 
Journal of Bacteriology  2003;185(15):4402-4409.
To survive in extremely acidic conditions, Escherichia coli has evolved three adaptive acid resistance strategies thought to maintain internal pH. While the mechanism behind acid resistance system 1 remains enigmatic, systems 2 and 3 are known to require external glutamate (system 2) and arginine (system 3) to function. These latter systems employ specific amino acid decarboxylases and putative antiporters that exchange the extracellular amino acid substrate for the intracellular by-product of decarboxylation. Although GadC is the predicted antiporter for system 2, the antiporter specific for arginine/agmatine exchange has not been identified. A computer-based homology search revealed that the yjdE (now called adiC) gene product shared an overall amino acid identity of 22% with GadC. A series of adiC mutants isolated by random mutagenesis and by targeted deletion were shown to be defective in arginine-dependent acid resistance. This defect was restored upon introduction of an adiC+-containing plasmid. An adiC mutant proved incapable of exchanging extracellular arginine for intracellular agmatine but maintained wild-type levels of arginine decarboxylase protein and activity. Western blot analysis indicated AdiC is an integral membrane protein. These data indicate that the arginine-to-agmatine conversion defect of adiC mutants was at the level of transport. The adi gene region was shown to be organized into two transcriptional units, adiAY and adiC, which are coordinately regulated but independently transcribed. The data also illustrate that the AdiA decarboxylase:AdiC antiporter system is designed to function only at acid levels sufficient to harm the cell.
PMCID: PMC165756  PMID: 12867448
14.  Enzymes Released from Escherichia coli With the Aid of a Servall Cell Fractionator 
Applied Microbiology  1963;11(6):467-471.
The release and stability of the enzymes S-adenosylhomocysteine nucleosidase, lysine decarboxylase, arginine decarboxylase, glutamic decarboxylase, formic hydrogenlyase, formic oxidase, and glucose oxidase from Escherichia coli during disruption of the organisms in a Servall-Ribi refrigerated cell fractionator were examined. With the possible exception of arginine decarboxylase, maximal activity was retained by all the enzymes reported here when the cell suspensions were processed at pressures necessary for rupture of all the organisms (15,000 to 25,000 psi). Considerable variation in the stability of different enzymes liberated by disruption at higher pressures (45,000 to 55,000 psi) was observed. It is reasonable to assume that mechanical forces rather than effects of temperature are responsible for inactivation of these enzymes.
PMCID: PMC1058031  PMID: 14075044
15.  pH-Dependent Modulation of Cyclic AMP Levels and GadW-Dependent Repression of RpoS Affect Synthesis of the GadX Regulator and Escherichia coli Acid Resistance 
Journal of Bacteriology  2003;185(23):6852-6859.
Extreme acid resistance is a remarkable property of virulent and avirulent Escherichia coli. The ability to resist environments in which the pH is 2.5 and below is predicted to contribute significantly to the survival of E. coli during passage through the gastric acid barrier. One acid resistance system imports glutamate from acidic environments and uses it as a proton sink during an intracellular decarboxylation reaction. Transcription of the genes encoding the glutamate decarboxylases and the substrate-product antiporter required for this system is induced under a variety of conditions, including the stationary phase and a low pH. Acid induction during log-phase growth in minimal medium appears to occur through multiple pathways. We recently demonstrated that GadE, the essential activator of the genes, was itself acid induced. In this report we present evidence that there is a regulatory loop involving cross-repression of two AraC-like regulators, GadX and GadW, that can either assist or interfere with GadE activation of the gad decarboxylase and antiporter genes, depending on the culture conditions. Balancing cross-repression appears to be dependent on cAMP and the cAMP regulator protein (CRP). The control loop involves the GadX protein repressing the expression of gadW and the GadW protein repressing or inhibiting RpoS, which is the alternative sigma factor that drives transcription of gadX. CRP and cAMP appear to influence GadX-GadW cross-repression from outside the loop by inhibiting production of RpoS. We found that GadW represses the decarboxylase genes in minimal medium and that growth under acidic conditions lowers the intracellular cAMP levels. These results indicate that CRP and cAMP can mediate pH control over gadX expression and, indirectly, expression of the decarboxylase genes. Mutational or physiological lowering of cAMP levels increases the level of RpoS and thereby increases the production of GadX. Higher GadX levels, in turn, repress gadW and contribute to induction of the gad decarboxylase genes. The presence of multiple pH control pathways governing expression of this acid resistance system is thought to reflect different environmental routes to a low pH.
PMCID: PMC262709  PMID: 14617649
16.  Influence of Carbohydrate Starvation and Arginine on Culturability and Amino Acid Utilization of Lactococcus lactis subsp. lactis† 
Two strains of Lactococcus lactis subsp. lactis were used to determine the influence of lactose and arginine on viability and amino acid use during carbohydrate starvation. Lactose provided energy for logarithmic-phase growth, and amino acids such as arginine provided energy after carbohydrate exhaustion. Survival time, cell numbers, and ATP concentrations increased with the addition of arginine to the basal medium. By the onset of lactose exhaustion, the concentrations of glycine-valine and glutamate had decreased by as much as 67% in L. lactis ML3, whereas the serine concentration increased by 97% during the same period. When no lactose was added, the concentrations of these amino acids remained constant. Similar trends were observed for L. lactis 11454. Without lactose or arginine, L. lactis ML3 was nonculturable on agar but was viable after 2 days, as measured by fluorescent viability stains and intracellular ATP levels. However, L. lactis 11454 without lactose or arginine remained culturable for at least 14 days. These data suggest that lactococci become viable but nonculturable in response to carbohydrate depletion. Additionally, these data indicate that amino acids other than arginine facilitate the survival of L. lactis during carbohydrate starvation.
PMCID: PMC91077  PMID: 9925598
17.  Outer and Inner Membrane Proteins Compose an Arginine-Agmatine Exchange System in Chlamydophila pneumoniae▿ † 
Journal of Bacteriology  2008;190(22):7431-7440.
Most chlamydial strains have a pyruvoyl-dependent decarboxylase protein that converts l-arginine to agmatine. However, chlamydiae do not produce arginine, so they must import it from their host. Chlamydophila pneumoniae has a gene cluster encoding a putative outer membrane porin (CPn1033 or aaxA), an arginine decarboxylase (CPn1032 or aaxB), and a putative cytoplasmic membrane transporter (CPn1031 or aaxC). The aaxC gene was expressed in Escherichia coli producing an integral cytoplasmic membrane protein that catalyzed the exchange of l-arginine for agmatine. Expression of the aaxA gene produced an outer membrane protein that enhanced the arginine uptake and decarboxylation activity of cells coexpressing aaxB and aaxC. This chlamydial arginine/agmatine exchange system complemented an E. coli mutant missing the native arginine-dependent acid resistance system. These cells survived extreme acid shock in the presence of l-arginine. Biochemical and evolutionary analysis showed the aaxABC genes evolved convergently with the enteric arginine degradation system, and they could have a different physiological role in chlamydial cells. The chlamydial system uniquely includes an outer membrane porin, and it is most active at a higher pH from 3 to 5. The chlamydial AaxC transporter was resistant to cadaverine, l-lysine and l-ornithine, which inhibit the E. coli AdiC antiporter.
PMCID: PMC2576674  PMID: 18790867
18.  Arginine metabolism in infected cell cultures as a marker character for the differentiation of orthopoxviruses. 
The Journal of Hygiene  1984;93(2):213-223.
Arginine has been shown to be essential for the replication of several orthopoxviruses in mouse sarcoma 180 cells and in chick embryo fibroblast cultures. Both host systems are characterized by their inabilities to utilize citrulline for the biosynthesis of arginine due to deficiencies in the requisite cellular enzymes and cell multiplication is absolutely dependent on the availability of exogenous arginine. Virus replication in such cells maintained with citrulline results from the induction of virus-specific enzymes. Significant virus yields in the absence of exogenous arginine or citrulline can arise from the replenishment of intracellular amino acid pools by increased utilization of arginyl residues in cellular proteins. The extent of the phenotypic expression of these characters in infected cells permitted significant discrimination between the viruses examined. Distinctions could be drawn between rabbitpox, ectromelia, cowpox, buffalopox and vaccinia strains. However, cowpox could not be distinguished from other viruses isolated from diseased animals in European zoos.
PMCID: PMC2129447  PMID: 6094662
19.  Fur-Dependent Detoxification of Organic Acids by rpoS Mutants during Prolonged Incubation under Aerobic, Phosphate Starvation Conditions▿  
Journal of Bacteriology  2008;190(16):5567-5575.
The activity of amino acid-dependent acid resistance systems allows Escherichia coli to survive during prolonged incubation under phosphate (Pi) starvation conditions. We show in this work that rpoS-null mutants incubated in the absence of any amino acid survived during prolonged incubation under aerobic, Pi starvation conditions. Whereas rpoS+ cells incubated with glutamate excreted high levels of acetate, rpoS mutants grew on acetic acid. The characteristic metabolism of rpoS mutants required the activity of Fur (ferric uptake regulator) in order to decrease the synthesis of the small RNA RyhB that might otherwise inhibit the synthesis of iron-rich proteins. We propose that RpoS (σS) and the small RNA RyhB contribute to decrease the synthesis of iron-rich proteins required for the activity of the tricarboxylic acid (TCA) cycle, which redirects the metabolic flux toward the production of acetic acid at the onset of stationary phase in rpoS+ cells. In contrast, Fur activity, which represses ryhB, and the lack of RpoS activity allow a substantial activity of the TCA cycle to continue in stationary phase in rpoS mutants, which decreases the production of acetic acid and, eventually, allows growth on acetic acid and Pi excreted into the medium. These data may help explain the fact that a high frequency of E. coli rpoS mutants is found in nature.
PMCID: PMC2519383  PMID: 18556786
20.  Polyamine Metabolism in Potassium-Deficient Bacteria 
Journal of Bacteriology  1972;112(3):1213-1221.
The metabolism of polyamines was studied in K+-dependent strains of Escherichia coli. When these stringent organisms were in a medium containing Na+ instead of K+, protein synthesis was arrested, but synthesis of ribonucleic acid continued as it would in a relaxed organism. The Na+ medium inhibited synthesis of spermidine and S-adenosylmethionine. However, the synthesis of putrescine was accelerated at least five- to eightfold. Exogenous ornithine doubled even this rate of putrescine synthesis but did not increase the low level of putrescine synthesis in the K+ medium. In K+ or Na+ media, with or without 0.3 mm arginine, putrescine was derived almost entirely from ornithine via ornithine decarboxylase. Addition of spermidine (5 mm) to a Na+ culture markedly inhibited putrescine synthesis. The ornithine decarboxylase of an extract of a K−-dependent strain prepared at low ionic strength was separated from ribosomes, deoxyribonucleic acid, and associated polyamines by centrifugation, and from many ions by ultrafiltration and fractionation on Sephadex G-100. Addition of Na+ and K+ salts to 200 mm was markedly inhibitory. The combined reductions both in synthesis of the inhibitor spermidine and in intracellular ionic strength may explain the in vivo activation of this enzyme.
PMCID: PMC251551  PMID: 4565534
21.  Metabolism of Glutamic Acid in a Mutant of Escherichia coli 
Journal of Bacteriology  1965;90(5):1304-1307.
Vender, Joyce (Indiana University, Bloomington), Kunthala Jayaraman, and H. V. Rickenberg. Metabolism of glutamic acid in a mutant of Escherichia coli. J. Bacteriol. 90:1304–1307. 1965.—A mutant strain of Escherichia coli W1485 was selected for its ability to utilize glutamic acid as the sole source of carbon. Growth of the mutant on glutamic acid led to the repression of glutamic acid dehydrogenase formation. The mutant differed from the wild-type strain in that glutamic decarboxylase activity was absent from the mutant under conditions of growth which supported the formation of this enzyme in the parent strain. Evidence is presented which suggests that loss of the decarboxylase activity results in the acquisition of the ability to utilize glutamic acid as sole source of carbon; a pathway of glutamate utilization via transamination is proposed.
PMCID: PMC315817  PMID: 5321484
22.  Characterization of an Acid-Dependent Arginine Decarboxylase Enzyme from Chlamydophila pneumoniae▿  
Journal of Bacteriology  2007;189(20):7376-7383.
Genome sequences from members of the Chlamydiales encode diverged homologs of a pyruvoyl-dependent arginine decarboxylase enzyme that nonpathogenic euryarchaea use in polyamine biosynthesis. The Chlamydiales lack subsequent genes required for polyamine biosynthesis and probably obtain polyamines from their host cells. To identify the function of this protein, the CPn1032 homolog from the respiratory pathogen Chlamydophila pneumoniae was heterologously expressed and purified. This protein self-cleaved to form a reactive pyruvoyl group, and the subunits assembled into a thermostable (αβ)3 complex. The mature enzyme specifically catalyzed the decarboxylation of l-arginine, with an unusually low pH optimum of 3.4. The CPn1032 gene complemented a mutation in the Escherichia coli adiA gene, which encodes a pyridoxal 5′-phosphate-dependent arginine decarboxylase, restoring arginine-dependent acid resistance. Acting together with a putative arginine-agmatine antiporter, the CPn1032 homologs may have evolved convergently to form an arginine-dependent acid resistance system. These genes are the first evidence that obligately intracellular chlamydiae may encounter acidic conditions. Alternatively, this system could reduce the host cell arginine concentration and produce inhibitors of nitric oxide synthase.
PMCID: PMC2168457  PMID: 17693492
23.  Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and beta-D-glucuronidase. 
Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and beta-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various other gram-negative and -positive bacteria found in water. The genotypic assays were performed with hybridization probes generated by PCR amplification of 670- and 623-bp segments of the gadA/B (GAD) and uidA (GUD) genes, respectively. The GAD enzymes catalyze the alpha-decarboxylation of L-glutamic acid to yield gamma-aminobutyric acid and carbon dioxide, which are detected in the phenotypic assay by a pH-sensitive indicator dye. The phenotypic assay for GUD involves the transformation of 4-methylumbelliferyl-beta-D-glucuronide to the fluorogenic compound 4-methylumbelliferone. The GAD phenotypic assay detected the majority of the E. coli strains tested, whereas a number of these strains, including all representatives of the O157:H7 serotype and several nonpathogenic E. coli strains, gave negative results in the GUD assay. Both phenotypic assays detected some but not all strains from each of the four Shigella species. A strain of Citrobacter freundii was also detected by the GUD assay but not by the GAD assay. All E. coli and Shigella strains were detected with both the gadA/B and uidA probes. A few Escherichia fergusonii strains gave weak hybridization signals in response to both probes at 65 degrees C but not at 68 degrees C. None of the other bacterial species tested were detected by either probe. These results were consistent with previous reports which have indicated that the GAD phenotypic assay detects a wider range of E. coli strains than does the GUD assay and is also somewhat more specific for this species. The genotypic assays for the two enzymes were found to be equivalent in both of these respects and superior to both of the phenotypic assays in terms of the range of E. coli strains and isolates detected.
PMCID: PMC168131  PMID: 8795225
24.  Sensing and Adaptation to Low pH Mediated by Inducible Amino Acid Decarboxylases in Salmonella 
PLoS ONE  2011;6(7):e22397.
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.
PMCID: PMC3143133  PMID: 21799843
25.  Arginine Control of Transcription of argECBH Messenger Ribonucleic Acid in Escherichia coli 
Journal of Bacteriology  1972;110(3):945-954.
The level of messenger ribonucleic acid specific for the argECBH gene cluster (arg-mRNA) of Escherichia coli was measured by deoxyribonucleic acid-ribonucleic acid hybridization in a number of strains. During the first 10 min after removal of arginine (derepression), the rate of arg-mRNA accumulation was six to ten times greater than that found in arginine-repressed argR+ cells. In the absence of arginine, l-canavanine (200 μg/ml) repressed arg-mRNA synthesis to a level only 20 to 30% lower than that found after arginine deprivation. High levels of arg-mRNA were produced by argR− strains with or without added arginine. Within about 2 min after arginine addition to argR+ cells, the rate of synthesis of arg-mRNA reached the repressed level. Likewise, 2.5 min after rifampin addition, all transcription of arg-mRNA was completed. These data are consistent with the view that arginine signals repression by inhibiting the initiation of transcription of arg-mRNA mediated in some way by the argR gene. The kinetics of arg-mRNA accumulation and the kinetics of completion of transcription together with the profile of hybridizable arg-mRNA in sucrose density gradients (major component 16S) suggest that the argECBH gene cluster is transcribed in short pieces rather than as a single unit.
PMCID: PMC247514  PMID: 4555419

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