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During its progression from the nasopharynx to other sterile and non-sterile niches of its human host, Streptococcus pneumoniae must cope with changes in temperature. We hypothesised that the temperature adaptation is an important facet of pneumococcal survival in the host. Here, we evaluated the effect of temperature on pneumococcus and studied the role of glutamate dehydrogenase (GdhA) in thermal adaptation associated with virulence and survival. Microarray analysis revealed a significant transcriptional response to changes in temperature, affecting the expression of 252 genes in total at 34°C and 40°C relative to at 37°C. One of the differentially regulated genes was gdhA, which is upregulated at 40°C and downregulated at 34°C relative to 37°C. Deletion of gdhA attenuated the growth, cell size, biofilm formation, pH survival, and biosynthesis of proteins associated with virulence in a temperature-dependent manner. Moreover, deletion of gdhA stimulated formate production irrespective of temperature fluctuation. Finally, ΔgdhA grown at 40°C was less virulent compare to other temperatures or than the wild type at the same temperature in a Galleria mellonella infection model, suggesting that GdhA is required for pneumococcal virulence at elevated temperature.

Listeria monocytogenes is a fastidious bacterial pathogen that can utilize only a limited number of nitrogen sources for growth. Both glutamine and ammonium are common nitrogen sources used in listerial defined growth media, but little is known about the regulation of their uptake or utilization. The functional role of L. monocytogenes GlnR, the transcriptional regulator of nitrogen metabolism genes in low-G+C Gram-positive bacteria, was determined using transcriptome sequencing and real-time reverse transcription-PCR experiments. The GlnR regulon included transcriptional units involved in ammonium transport (amtB glnK) and biosynthesis of glutamine (glnRA) and glutamate (gdhA) from ammonium. As in other bacteria, GlnR proved to be an autoregulatory repressor of the glnRA operon. Unexpectedly, GlnR was most active during growth with ammonium as the nitrogen source and less active in the glutamine medium, apparently because listerial cells perceive growth with glutamine as a nitrogen-limiting condition. Therefore, paradoxically, expression of the glnA gene, encoding glutamine synthetase, was highest in the glutamine medium. For the amtB glnK operon, GlnR served as both a negative regulator in the presence of ammonium and a positive regulator in the glutamine medium. The gdhA gene was subject to a third mode of regulation that apparently required an elevated level of GlnR for repression. Finally, activity of glutamate dehydrogenase encoded by the gdhA gene appeared to correlate inversely with expression of gltAB, the operon that encodes the other major glutamate-synthesizing enzyme, glutamate synthase. Both gdhA and amtB were also regulated, in a negative manner, by the global transcriptional regulator CodY. IMPORTANCEL. monocytogenes is a widespread foodborne pathogen. Nitrogen-containing compounds, such as the glutamate-containing tripeptide, glutathione, and glutamine, have been shown to be important for expression of L. monocytogenes virulence genes. In this work, we showed that a transcriptional regulator, GlnR, controls expression of critical listerial genes of nitrogen metabolism that are involved in ammonium uptake and biosynthesis of glutamine and glutamate. A different mode of GlnR-mediated regulation was found for each of these three pathways.

The objective of this study was to evaluate a novel automated random-access test, mariPOC CDI (ArcDia Ltd., Finland), for the detection of Clostridioides difficile glutamate dehydrogenase (GDH) and toxins A and B directly from fecal specimens. The mariPOC test was compared with both the GenomEra C. difficile PCR assay (Abacus Diagnostica Oy, Finland) and the TechLab C. diff Quik Chek Complete (Alere Inc.; now Abbot) membrane enzyme immunoassay (MEIA). Culture and the Xpert C. difficile assay (Cepheid Inc., USA) were used to resolve discrepant results. In total, 337 specimens were tested with the mariPOC CDI test and GenomEra PCR. Of these specimens, 157 were also tested with the TechLab MEIA. The sensitivity of the mariPOC test for GDH was slightly lower (95.2%) than that obtained with the TechLab assay (100.0%), but no toxin-positive cases were missed. The sensitivity of the mariPOC test for the detection of toxigenic C. difficile by analyzing toxin expression was better (81.6%) than that of the TechLab assay (71.1%). The analytical specificities for the mariPOC and the TechLab tests were 98.3% and 100.0% for GDH and 100.0% and 99.2% for toxin A/B, respectively. The analytical specificity of the GenomEra method was 100.0%. The mariPOC and TechLab GDH tests and GenomEra PCR had high negative predictive values of 99.3%, 98.3%, and 99.7%, respectively, in excluding infection with toxigenic C. difficile. The mariPOC toxin A/B test and GenomEra PCR had an identical analytical positive predictive value of 100%, providing highly reliable information about toxin expression and the presence of toxin genes, respectively.

The Singulex Clarity C. diff toxins A/B (Clarity) assay is an automated, ultrasensitive immunoassay for the detection of Clostridioides difficile toxins in stool. In this study, the performance of the Clarity assay was compared to that of a multistep algorithm using an enzyme immunoassay (EIA) for detection of glutamate dehydrogenase (GDH) and toxins A and B arbitrated by a semiquantitative cell cytotoxicity neutralization assay (CCNA). The performance of the assay was evaluated using 211 residual deidentified stool samples tested with a GDH-and-toxin EIA (C. Diff Quik Chek Complete; Techlab), with GDH-and-toxin discordant samples tested with CCNA. The stool samples were stored at –80°C before being tested with the Clarity assay. For samples discordant between Clarity and the standard-of-care algorithm, the samples were tested with PCR (Xpert C. difficile; Cepheid), and chart review was performed. The testing algorithm resulted in 34 GDH(+)/toxin(+), 53 GDH(−)/toxin(−), and 124 GDH(+)/toxin(−) samples, of which 39 were CCNA(+) and 85 were CCNA(−). Clarity had 96.2% negative agreement with GDH(−)/toxin(−) samples, 100% positive agreement with GDH(+)/toxin(+) samples, and 95.3% agreement with GDH(+)/toxin(−)/CCNA(−) samples. The Clarity result was invalid for one sample. Clarity agreed with 61.5% of GDH(+)/toxin(−)/CCNA(+) samples, 90.0% of GDH(+)/toxin(−)/CCNA(+) (high-positive) samples, and 31.6% of GDH(+)/toxin(−)/CCNA(+) (low-positive) samples. The Singulex Clarity C. diff toxins A/B assay demonstrated high agreement with a testing algorithm utilizing a GDH-and-toxin EIA and CCNA. This novel automated assay may offer an accurate, stand-alone solution for C. difficile infection (CDI) diagnostics, and further prospective clinical studies are merited.

Clostridioides (Clostridium) difficile infection (CDI) is the most common causative pathogen of health care-associated gastrointestinal infections; however, due to the overlap of clinical symptoms with those of other causes of acute gastroenteritis, the selection of the most appropriate laboratory test is difficult. From April to October 2018, 640 stool samples requested for CDI testing were examined using the mariPOC CDI and Gastro test (ArcDia), which allows the detection of C. difficile glutamate dehydrogenase (GDH) and toxin A/B, norovirus genogroups GI and GII.4, rotavirus, adenovirus, and Campylobacter spp. In parallel, the C. Diff Quik Chek Complete test (Alere) was used as a routine diagnostic assay, and C. difficile toxigenic culture was used as a reference method. The sensitivity of the mariPOC CDI and Gastro test was comparable to that of C. Diff Quik Chek Complete for the detection of GDH (96.40% [95% confidence interval {CI}, 91.81% to 98.82%] versus 95.68% [95% CI, 90.84 to 98.40%]; P = 1.00) and was higher for the detection of toxin A/B (66.67% [95% CI, 57.36 to 75.11%] versus 55.56% [95% CI, 46.08 to 64.74%]; P = 0.00). The specificity of the mariPOC CDI and Gastro test was lower than that of C. Diff Quik Chek Complete for GDH detection (95.21% [95% CI, 92.96% to 96.91%] versus 97.60% [95% CI, 95.85% to 98.76%]; P = 0.04) and comparable to that of C. Diff Quik Chek Complete for toxin A/B detection (99.24 [95% CI, 98.05% to 99.79%] versus 99.81% [95% CI, 98.94% to 100.0%]; P = 0.37). In 29 cases (4.53%), other causative agents of diarrhea were detected (Campylobacter spp. [n = 17], rotavirus [n = 7], and norovirus genogroup GII.4 [n = 5]).

Persister cells are rare phenotypic variants that are able to survive antibiotic treatment. Unlike resistant bacteria, which have specific mechanisms to prevent antibiotics from binding to their targets, persisters evade antibiotic killing by entering a tolerant nongrowing state. Persisters have been implicated in chronic infections in multiple species, and growing evidence suggests that persister cells are responsible for many cases of antibiotic treatment failure. New antibiotic treatment strategies aim to kill tolerant persister cells more effectively, but the mechanism of tolerance has remained unclear until now., Chronic bacterial infections are difficult to eradicate, though they are caused primarily by drug-susceptible pathogens. Antibiotic-tolerant persisters largely account for this paradox. In spite of their significance in the recalcitrance of chronic infections, the mechanism of persister formation is poorly understood. We previously reported that a decrease in ATP levels leads to drug tolerance in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. We reasoned that stochastic fluctuation in the expression of tricarboxylic acid (TCA) cycle enzymes can produce cells with low energy levels. S. aureus knockouts in glutamate dehydrogenase, 2-oxoketoglutarate dehydrogenase, succinyl coenzyme A (CoA) synthetase, and fumarase have low ATP levels and exhibit increased tolerance of fluoroquinolone, aminoglycoside, and β-lactam antibiotics. Fluorescence-activated cell sorter (FACS) analysis of TCA genes shows a broad Gaussian distribution in a population, with differences of over 3 orders of magnitude in the levels of expression between individual cells. Sorted cells with low levels of TCA enzyme expression have an increased tolerance of antibiotic treatment. These findings suggest that fluctuations in the levels of expression of energy-generating components serve as a mechanism of persister formation.

Glutamate dehydrogenase (GDH) from a thermophilic bacterium, Thermus thermophilus , is composed of two heterologous subunits, GdhA and GdhB. In the hetero-complex, GdhB acts as the catalytic subunit, whereas GdhA lacks enzymatic activity and acts as the regulatory subunit for activation by leucine. In the present study, we performed a pull-down assay using recombinant T. thermophilus , producing GdhA fused with a His-tag at the N terminus and found that TTC1249 (APRTh), which is annotated as adenine phosphoribosyltransferase but lacks the enzymatic activity, was co-purified with GdhA. When GdhA, GdhB, and APRTh were co-produced in E. coli cells, they were purified as a ternary complex. The ternary complex exhibited the GDH activity that was activated by leucine as observed for the GdhA⋅GdhB binary complex. Furthermore, AMP activated the GDH activity of the ternary complex, whereas such activation was not observed for the GdhA⋅GdhB binary complex. This suggests that APRTh mediates the allosteric activation of GDH by AMP. The present study demonstrates the presence of complicated regulatory mechanisms of GDH mediated by multiple compounds to control the carbon-nitrogen balance in bacterial cells. IMPORTANCE Glutamate dehydrogenase (GDH), which catalyzes the synthesis and degradation of glutamate using NAD(P)(H), is a widely distributed enzyme among all domains of life. Mammalian GDH is regulated allosterically by multiple metabolites, in which the ‘antenna helix’ plays a key role to transmit the allosteric signals. In contrast, bacterial GDH was believed not to be regulated allosterically because it lacks the antenna helix. We previously reported that GDH from Thermus thermophilus (TtGDH), which is composed of two heterologous subunits, is activated by leucine. In the present study, we found that AMP activates TtGDH using a catalytically-inactive adenine phosphoribosyltransferase homolog (APRTh) as the sensory subunit. This suggests that T. thermophilus possess a complicated regulatory mechanism of GDH to control carbon and nitrogen metabolism.

Staphylococcus aureus has the ability to cause infections in multiple organ systems, suggesting an ability to rapidly adapt to changing carbon and nitrogen sources. Although there is little information about the nutrients available at specific sites of infection, a mature skin abscess has been characterized as glucose depleted, indicating that peptides and free amino acids are an important source of nutrients for the bacteria. Our studies have found that mutations in enzymes necessary for growth on amino acids, including pyruvate carboxykinase (ΔpckA) and glutamate dehydrogenase (ΔgudB), reduced the ability of the bacteria to proliferate within a skin abscess, suggesting that peptides and free amino acids are important for S. aureus growth. Furthermore, we found that collagen, an abundant host protein that is present throughout a skin abscess, serves as a reservoir of peptides. To liberate peptides from the collagen, we identified that the host protease, MMP-9, as well as the staphylococcal proteases aureolysin and staphopain B function to cleave collagen into peptide fragments that can support S. aureus growth under nutrient-limited conditions. Moreover, the oligopeptide transporter Opp3 is the primary staphylococcal transporter responsible for peptide acquisition. Lastly, we observed that the presence of peptides (3-mer to 7-mer) induces the expression of aureolysin, suggesting that S. aureus has the ability to detect peptides in the environment. IMPORTANCEStaphylococcus aureus has the ability to cause infections in a variety of niches, suggesting a robust metabolic capacity facilitating proliferation under various nutrient conditions. The mature skin abscess is glucose depleted, indicating that peptides and free amino acids are important sources of nutrients for S. aureus. Our studies have found that mutations in both pyruvate carboxykinase and glutamate dehydrogenase, enzymes that function in essential gluconeogenesis reactions when amino acids serve as the major carbon source, reduce bacterial burden in a murine skin abscess model. Moreover, peptides liberated from collagen by host protease MMP-9 as well as the staphylococcal protease aureolysin support S. aureus growth in an Opp3-dependent manner under nutrient-limited conditions. Additionally, the presence of peptides induces aureolysin expression. Overall, these studies define one pathway by which S. aureus senses a nutrient-limiting environment and induces factors that function to acquire and utilize carbon from host-derived sources.

Accumulating evidence shows a high prevalence of Clostridium difficile in Southeast Asia associated with a range of clinical presentations. However, severe infections are rarely reported. We investigated C. difficile infection (CDI) across four hospitals in Kuala Lumpur and Kota Bharu, Malaysia. Enzyme immunoassays for glutamate dehydrogenase (GDH) and toxin A or B were performed on diarrheal stool specimens collected from patients in 2015 and 2016. Specimens were also cultured and isolates of C. difficile characterized by PCR ribotyping and detection of toxin genes. In total, 437 specimens were collected and fecal toxin was detected in 3.0%. A further 16.2% of specimens were GDH positive and toxin negative. After culture, toxigenic strains were isolated from 10.3% and nontoxigenic strains from 12.4% of specimens. The most prevalent PCR ribotypes (RTs) were RT 017 (20.0%) and RT 043 (10.0%). The high prevalence of RT 017 and nontoxigenic strains in Malaysia and in neighboring Thailand and Indonesia suggests that they localize to the region of Southeast Asia, with an implication that they may mediate the burden of CDI in the region.

While glutamine is a nonessential amino acid that can be synthesized from glucose, some cancer cells primarily depend on glutamine for their growth, proliferation, and survival. Numerous types of cancer also depend on asparagine for cell proliferation. The underlying mechanisms of the glutamine and asparagine requirement in cancer cells in different contexts remain unclear. In this study, we show that the oncogenic virus Kaposi’s sarcoma-associated herpesvirus (KSHV) accelerates the glutamine metabolism of glucose-independent proliferation of cancer cells by upregulating the expression of numerous critical enzymes, including glutaminase 2 (GLS2), glutamate dehydrogenase 1 (GLUD1), and glutamic-oxaloacetic transaminase 2 (GOT2), to support cell proliferation. Surprisingly, cell crisis is rescued only completely by supplementation with asparagine but minimally by supplementation with α-ketoglutarate, aspartate, or glutamate upon glutamine deprivation, implying an essential role of γ-nitrogen in glutamine and asparagine for cell proliferation. Specifically, glutamine and asparagine provide the critical γ-nitrogen for purine and pyrimidine biosynthesis, as knockdown of four rate-limiting enzymes in the pathways, including carbamoylphosphate synthetase 2 (CAD), phosphoribosyl pyrophosphate amidotransferase (PPAT), and phosphoribosyl pyrophosphate synthetases 1 and 2 (PRPS1 and PRPS2, respectively), suppresses cell proliferation. These findings indicate that glutamine and asparagine are shunted to the biosynthesis of nucleotides and nonessential amino acids from the tricarboxylic acid (TCA) cycle to support the anabolic proliferation of KSHV-transformed cells. Our results illustrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway., IMPORTANCE We have previously found that Kaposi’s sarcoma-associated herpesvirus (KSHV) can efficiently infect and transform primary mesenchymal stem cells; however, the metabolic pathways supporting the anabolic proliferation of KSHV-transformed cells remain unknown. Glutamine and asparagine are essential for supporting the growth, proliferation, and survival of some cancer cells. In this study, we have found that KSHV accelerates glutamine metabolism by upregulating numerous critical metabolic enzymes. Unlike most cancer cells that primarily utilize glutamine and asparagine to replenish the TCA cycle, KSHV-transformed cells depend on glutamine and asparagine for providing γ-nitrogen for purine and pyrimidine biosynthesis. We identified four rate-limiting enzymes in this pathway that are essential for the proliferation of KSHV-transformed cells. Our results demonstrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway.

Staphylococcus aureus must rapidly adapt to a variety of carbon and nitrogen sources during invasion of a host. Within a staphylococcal abscess, preferred carbon sources such as glucose are limiting, suggesting that S. aureus survives through the catabolism of secondary carbon sources. S. aureus encodes pathways to catabolize multiple amino acids, including those that generate pyruvate, 2-oxoglutarate, and oxaloacetate. To assess amino acid catabolism, S. aureus JE2 and mutants were grown in complete defined medium containing 18 amino acids but lacking glucose (CDM). A mutation in the gudB gene, coding for glutamate dehydrogenase, which generates 2-oxoglutarate from glutamate, significantly reduced growth in CDM, suggesting that glutamate and those amino acids generating glutamate, particularly proline, serve as the major carbon source in this medium. Nuclear magnetic resonance (NMR) studies confirmed this supposition. Furthermore, a mutation in the ackA gene, coding for acetate kinase, also abrogated growth of JE2 in CDM, suggesting that ATP production from pyruvate-producing amino acids is also critical for growth. In addition, although a functional respiratory chain was absolutely required for growth, the oxygen consumption rate and intracellular ATP concentration were significantly lower during growth in CDM than during growth in glucose-containing media. Finally, transcriptional analyses demonstrated that expression levels of genes coding for the enzymes that synthesize glutamate from proline, arginine, and histidine are repressed by CcpA and carbon catabolite repression. These data show that pathways important for glutamate catabolism or ATP generation via Pta/AckA are important for growth in niches where glucose is not abundant, such as abscesses within skin and soft tissue infections., IMPORTANCE S. aureus is a significant cause of both morbidity and mortality worldwide. This bacterium causes infections in a wide variety of organ systems, the most common being skin and soft tissue. Within a staphylococcal abscess, levels of glucose, a preferred carbon source, are limited due to the host immune response. Therefore, S. aureus must utilize other available carbon sources such as amino acids or peptides to proliferate. Our results show that glutamate and amino acids that serve as substrates for glutamate synthesis, particularly proline, function as major carbon sources during growth, whereas other amino acids that generate pyruvate are important for ATP synthesis via substrate-level phosphorylation in the Pta-AckA pathway. Our data support a model whereby certain amino acid catabolic pathways, and acquisition of those particular amino acids, are crucial for growth in niches where glucose is not abundant.

Clostridium difficile is a well-known enteric pathogen of humans and the causative agent of high-morbidity enteritis in piglets aged 1 to 7 days. C. difficile prevalence in Australian piglets is as high as 70%. The current diagnostic assays have been validated only for human infections, and there are no published studies assessing their performance in Australian piglets. We evaluated the suitability of five assays for detecting C. difficile in 157 specimens of piglet feces. The assays included a loop-mediated isothermal amplification (LMIA)-PCR for tcdA (illumigene C. difficile ; Meridian), a real-time PCR for tcdB (GeneOhm Cdiff; Becton Dickinson), two-component enzyme immunoassays (EIA) for C. difficile glutamate dehydrogenase (GDH) (EIA-GDH) and TcdA/TcdB (EIA-TcdA/TcdB) (C. diff Quik Chek; Alere), and direct culture (DC) ( C. difficile chromID agar; bioMérieux). The assays for detection of the organism were compared against enrichment culture (EC), and assays for detection of toxins/toxin genes were compared against EC followed by PCR for toxin genes (toxigenic EC [TEC]). The recovery of C. difficile by EC was 39.5% ( n = 62/157), and TEC revealed that 58.1% ( n = 36/62) of isolates were positive for at least one toxin gene ( tcdA / tcdB ). Compared with those for EC/TEC, the sensitivities, specificities, positive predictive values, and negative predictive values were, respectively, as follows: DC, 91.9, 100.0, 100.0, and 95.0%; EIA-GDH, 41.9, 92.6, 78.8, and 71.0%; EIA-TcdA/TcdB, 5.6, 99.2, 66.7, and 77.9%; real-time PCR, 42.9, 96.7, 78.9, and 85.4% and LMIA-PCR, 25.0, 95.9, 64.3, and 81.1%. The performance of the molecular methods was poor, suggesting that the current commercially available assays for diagnosis of C. difficile in humans are not suitable for use in piglets. C. difficile recovery by the DC provides a cost-effective alternative.

Effective and easy-to-use methods for detecting Clostridium difficile spore contamination would be useful for identifying environmental reservoirs and monitoring the effectiveness of room disinfection. Culture-based detection methods are sensitive for detecting C. difficile , but their utility is limited due to the requirement of anaerobic culture conditions and microbiological expertise. We developed a low-cost selective broth medium containing thioglycolic acid and l -cystine, termed C. difficile brucella broth with thioglycolic acid and l -cystine (CDBB-TC), for the detection of C. difficile from environmental specimens under aerobic culture conditions. The sensitivity and specificity of CDBB-TC (under aerobic culture conditions) were compared to those of CDBB (under anaerobic culture conditions) for the recovery of C. difficile from swabs collected from hospital room surfaces. CDBB-TC was significantly more sensitive than CDBB for recovering environmental C. difficile (36/41 [88%] versus 21/41 [51%], respectively; P = 0.006). C. difficile latex agglutination, an enzyme immunoassay for toxins A and B or glutamate dehydrogenase, and a PCR for toxin B genes were all effective as confirmatory tests. For 477 total environmental cultures, the specificity of CDBB-TC versus that of CDBB based upon false-positive yellow-color development of the medium without recovery of C. difficile was 100% (0 false-positive results) versus 96% (18 false-positive results), respectively. False-positive cultures for CDBB were attributable to the growth of anaerobic non- C. difficile organisms that did not grow in CDBB-TC. Our results suggest that CDBB-TC provides a sensitive and selective medium for the recovery of C. difficile organisms from environmental samples, without the need for anaerobic culture conditions.

The gastric pathogen Helicobacter pylori possesses a highly active urease to support acid tolerance. Urea hydrolysis occurs inside the cytoplasm, resulting in the production of NH 3 that is immediately protonated to form NH 4 + . This ammonium must be metabolized or effluxed because its presence within the cell is counterproductive to the goal of raising pH while maintaining a viable proton motive force (PMF). Two compatible hypotheses for mitigating intracellular ammonium toxicity include (i) the exit of protonated ammonium outward via the UreI permease, which was shown to facilitate diffusion of both urea and ammonium, and/or (ii) the assimilation of this ammonium, which is supported by evidence that H. pylori assimilates urea nitrogen into its amino acid pools. We investigated the second hypothesis by constructing strains with altered expression of the ammonium-assimilating enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) and the ammonium-evolving periplasmic enzymes glutaminase (Ggt) and asparaginase (AsnB). H. pylori strains expressing elevated levels of either GS or GDH are more acid tolerant than the wild type, exhibit enhanced ammonium production, and are able to alkalize the medium faster than the wild type. Strains lacking the genes for either Ggt or AsnB are acid sensitive, have 8-fold-lower urea-dependent ammonium production, and are more acid sensitive than the parent. Additionally, we found that purified H. pylori GS produces glutamine in the presence of Mg 2+ at a rate similar to that of unadenylated Escherichia coli GS. These data reveal that all four enzymes contribute to whole-cell acid resistance in H. pylori and are likely important for assimilation and/or efflux of urea-derived ammonium.

Simplexa Clostridium difficile universal direct PCR, a real-time PCR assay for the detection of the C. difficile toxin B ( tcdB ) gene using the 3M integrated cycler, was compared with a two-step algorithm which includes the C. Diff Chek-60 glutamate dehydrogenase (GDH) antigen assay followed by cytotoxin neutralization. Three hundred forty-two liquid or semisolid stools submitted for diagnostic C. difficile testing, 171 GDH antigen positive and 171 GDH antigen negative, were selected for the study. All samples were tested by the C. Diff Chek-60 GDH antigen assay, cytotoxin neutralization, and Simplexa direct PCR. Of 171 GDH-positive samples, 4 were excluded (from patients on therapy or from whom duplicate samples were obtained) and 88 were determined to be true positives for toxigenic C. difficile . Of the 88, 67 (76.1%) were positive by the two-step method and 86 (97.7%) were positive by PCR. Seventy-nine were positive by the GDH antigen assay only. Of 171 GDH antigen-negative samples, none were positive by PCR. One antigen-negative sample positive by the cytotoxin assay only was deemed a false positive based on chart review. Simplexa C. difficile universal direct PCR was significantly more sensitive for detecting toxigenic C. difficile bacteria than cytotoxin neutralization ( P = 0.0002). However, most PCR-positive/cytotoxin-negative patients did not have clear C. difficile disease. The estimated cost avoidance provided by a more rapid molecular diagnosis was outweighed by the cost of isolating and treating PCR-positive/cytotoxin-negative patients. The costs, clinical consequences, and impact on nosocomial transmission of treating and/or isolating patients positive for toxigenic C. difficile by PCR but negative for in vivo toxin production merit further study.

Coaggregation of Porphyromonas gingivalis and oral streptococci is thought to play an important role in P. gingivalis colonization. Previously, we reported that P. gingivalis major fimbriae interacted with Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and that amino acid residues 166 to 183 of GAPDH exhibited strong binding activity toward P. gingivalis fimbriae (H. Nagata, M. Iwasaki, K. Maeda, M. Kuboniwa, E. Hashino, M. Toe, N. Minamino, H. Kuwahara, and S. Shizukuishi, Infect. Immun. 77: 5130–5138, 2009). The present study aimed to identify and characterize P. gingivalis components other than fimbriae that interact with S. oralis GAPDH. A pulldown assay was performed to detect potential interactions between P. gingivalis client proteins and S. oralis recombinant GAPDH with amino acid residues 166 to 183 deleted by site-directed mutagenesis. Seven proteins, namely, tonB -dependent receptor protein (RagA4), arginine-specific proteinase B, 4-hydroxybutyryl-coenzyme A dehydratase (AbfD), lysine-specific proteinase, GAPDH, NAD-dependent glutamate dehydrogenase (GDH), and malate dehydrogenase (MDH), were identified by two-dimensional gel electrophoresis followed by proteomic analysis using tandem mass spectrometry. Interactions between these client proteins and S. oralis GAPDH were analyzed with a biomolecular interaction analysis system. S. oralis GAPDH showed high affinity for five of the seven client proteins (RagA4, AbfD, GAPDH, GDH, and MDH). Interactions between P. gingivalis and S. oralis were measured by a turbidimetric method and fluorescence microscopy. RagA4, AbfD, and GDH enhanced coaggregation, whereas GAPDH and MDH inhibited coaggregation. Furthermore, the expression of luxS in P. gingivalis was upregulated by RagA4, AbfD, and GDH but was downregulated by MDH. These results indicate that the five P. gingivalis client proteins function as regulators in P. gingivalis biofilm formation with oral streptococci.

We present the evolution of testing algorithms at our institution in which the C. Diff Quik Chek Complete immunochromatographic cartridge assay determines the presence of both glutamate dehydrogenase and Clostridium difficile toxins A and B as a primary screen for C. difficile infection and indeterminate results (glutamate dehydrogenase positive, toxin A and B negative) are confirmed by the GeneXpert C. difficile PCR assay. This two-step algorithm is a cost-effective method for highly sensitive detection of toxigenic C. difficile .

We evaluated the performance of the rapid C. diff Quik Chek Complete's glutamate dehydrogenase antigen (GDH) and toxin A/B (CDT) tests in two algorithmic approaches for a tertiary pediatric population: algorithm 1 entailed initial testing with GDH/CDT followed by loop-mediated isothermal amplification (LAMP), and algorithm 2 entailed GDH/CDT followed by cytotoxicity neutralization assay (CCNA) for adjudication of discrepant GDH-positive/CDT-negative results. A true positive (TP) was defined as positivity by CCNA or positivity by LAMP plus another test (GDH, CDT, or the Premier C. difficile toxin A and B enzyme immunoassay [P-EIA]). A total of 141 specimens from 141 patients yielded 27 TPs and 19% prevalence. Sensitivity, specificity, positive predictive value, and negative predictive value were 56%, 100%, 100%, and 90% for P-EIA and 81%, 100%, 100%, and 96% for both algorithm 1 and algorithm 2. In summary, GDH-based algorithms detected C. difficile infections with superior sensitivity compared to P-EIA. The algorithms allowed immediate reporting of half of all TPs, but LAMP or CCNA was required to confirm the presence or absence of toxigenic C. difficile in GDH-positive/CDT-negative specimens.

Common laboratory strains of Bacillus subtilis encode two glutamate dehydrogenases: the enzymatically active protein RocG and the cryptic enzyme GudB that is inactive due to a duplication of three amino acids in its active center. The inactivation of the rocG gene results in poor growth of the bacteria on complex media due to the accumulation of toxic intermediates. Therefore, rocG mutants readily acquire suppressor mutations that decryptify the gudB gene. This decryptification occurs by a precise deletion of one part of the 9-bp direct repeat that causes the amino acid duplication. This mutation occurs at the extremely high frequency of 10(-4). Mutations affecting the integrity of the direct repeat result in a strong reduction of the mutation frequency; however, the actual sequence of the repeat is not essential. The mutation frequency of gudB was not affected by the position of the gene on the chromosome. When the direct repeat was placed in the completely different context of an artificial promoter, the precise deletion of one part of the repeat was also observed, but the mutation frequency was reduced by 3 orders of magnitude. Thus, transcription of the gudB gene seems to be essential for the high frequency of the appearance of the gudB1 mutation. This idea is supported by the finding that the transcription-repair coupling factor Mfd is required for the decryptification of gudB. The Mfd-mediated coupling of transcription to mutagenesis might be a built-in precaution that facilitates the accumulation of mutations preferentially in transcribed genes.

Iron is vital for all eukaryotes. The ability of this metal to exist in two redox states confers properties that make it an essential participant at the active center of several enzymes that are involved in critical cellular processes, such as amino acid biosynthesis, energy production, and lipid metabolism. Paradoxically, the properties that make iron essential can also make it toxic under certain conditions. Excess iron has the ability to generate toxic reactive oxygen species that can damage cellular components (15). Consequently, cells have developed tightly regulated homeostatic mechanisms in order to optimize iron uptake while keeping its reactivity under tight control. Despite the fact that iron is one of the most abundant elements on earth, its bioavailability remains highly limited at physiological pH owing to its oxidation into insoluble ferric hydroxides under atmospheric oxygen conditions (4). To overcome this life-threatening issue, the fission yeast Schizosaccharomyces pombe, as well as most fungi, uses two high-affinity iron acquisition systems that involve either a reductive or a nonreductive mechanism (20). In S. pombe, the reductive iron acquisition system relies on the ferrireductase Frp1, whose role is to solubilize extracellular iron by reducing its ferric form (Fe3+) to its ferrous form (Fe2+) (42). The subsequent transport of Fe2+ into cells is predominantly mediated by an oxidase-permease complex consisting of Fio1 and Fip1 (2). The nonreductive iron uptake system involves the acquisition of iron-loaded siderophores. Produced by most microorganisms, siderophores are small organic molecules that bind ferric iron with very high affinity and specificity (32). Fission yeast biosynthesizes, accumulates, and excretes only one type of siderophore, which is designated ferrichrome (45). Once loaded with iron, Fe3+-ferrichrome is captured and internalized by S. pombe via the cell surface transporters Str1 and Str2 (36). According to the general biosynthetic pathway of fungal hydroxamate siderophores proposed by Plattner and Diekmann (39), the first step in the biosynthesis of ferrichrome resides in the N5 hydroxylation of ornithine by ornithine-N5-oxygenase. The homology of sequences of the ornithine-N5-oxygenases characterized to date, such as Sid1 (from Ustilago maydis) and SidA (from Aspergillus nidulans), suggest that a similar enzymatic reaction is catalyzed by the product of the sib2+ gene in fission yeast (13). The subsequent step in the biosynthesis of ferrichrome is the formation of the hydroxamate group. Newly synthesized molecules of N5-hydroxyornithine are acylated by an N5-transacetylase, which is predicted to be encoded by the SPBC17G9.06c locus in S. pombe. Ultimately, the hydroxamate group is processed by nonribosomal peptide synthetases (NRPSs) (11, 53). In S. pombe, the NRPS Sib1 catalyzes the last step of ferrichrome biosynthesis (40, 46). Iron transport in fission yeast is primarily regulated at the transcriptional level by the iron-sensing GATA-type repressor Fep1 (20). When iron is abundant, Fep1 binds to GATA-type cis-acting elements (A/T)GATA(A/T) and downregulates frp1+, fio1+, fip1+, str1+, str2+, str3+, and abc3+ gene expression (18, 35, 36, 41). The strength of the Fep1-mediated transcriptional repression is maximized when target gene promoters harbor the modified GATA-type sequence ATC(A/T)GATA(A/T) (41). Fep1 orthologs in U. maydis (Urbs1) and Histoplasma capsulatum (Sre1) are also known to bind to this motif (1, 7). Another member of the Fep1 regulon is php4+, a gene encoding the iron-responsive CCAAT-binding subunit Php4 (29). During iron starvation, Php4 coordinates the S. pombe iron-sparing response by repressing genes that encode components of iron-requiring metabolic pathways, such as the tricarboxylic acid (TCA) cycle, the electron transport chain, and the iron-sulfur cluster biogenesis machinery (30). Similarly, the Php4 ortholog in A. nidulans, denoted HapX, regulates the expression of genes encoding iron-using proteins (17). At the molecular level, Php4 associates with its target genes by recognition of the CCAAT-binding complex, which is composed of Php2, Php3, and Php5 (28, 29). The Php2/3/5 heterotrimer binds CCAAT cis-acting elements, whereas Php4 lacks DNA-binding activity. It has been demonstrated that the gene encoding the transcriptional repressor Fep1 is regulated by Php4, creating a reciprocal regulatory loop between both iron sensors (30). Therefore, Php4 and Fep1 act as key regulators of iron homeostasis in fission yeast by controlling iron utilization and iron acquisition. The assembly of ferrichrome requires ornithine, a nonproteinogenic amino acid whose synthesis is tightly associated with nitrogen metabolism. In yeast, more than 80% of cellular nitrogen derives from the incorporation of ammonia into glutamate, a precursor of ornithine (24). Two metabolic branches mediate assimilation of ammonia into glutamate in fission yeast (Fig. ​(Fig.1).1). One is mediated by the glutamate dehydrogenase Gdh1, while the other consists of a coordinated reaction involving glutamine synthetase (Gln1) and glutamate synthase (Glt1) (37, 38). In the Gln1/Glt1 branch, Gln1 first catalyzes the ATP-dependent incorporation of ammonia into glutamine. The newly acquired amino group of glutamine is then transferred to a 2-ketoglutarate acceptor by Glt1. This reaction ultimately generates two molecules of glutamate. This ammonia assimilation pathway is iron dependent, since Glt1 requires the incorporation of an iron-sulfur cluster for activity (9). Glutamate generated by ammonia assimilation pathways serves in numerous biological processes, including the biosynthesis of arginine, polyamines, and hydroxamate siderophores. An alternative metabolic pathway that produces ornithine originates from the urea cycle. In this pathway, arginase catalyzes the degradation of arginine into urea and ornithine. According to complementation studies using a Saccharomyces cerevisiae arginase null strain (car1Δ), a similar reaction is mediated by the product of the car1+ gene in S. pombe (49). FIG. 1. Outlines of ferrichrome biosynthesis in S. pombe grown under conditions of low iron concentrations. The metabolic relationships between glutamate, ornithine, and ferrichrome in fission yeast are depicted. Transcription of genes that are induced (+) ... Here we report that ornithine and ferrichrome are critical to S. pombe survival under conditions of iron deficiency. We further show that the metabolic pathways related to ornithine and ferrichrome synthesis are regulated at the transcriptional level by Fep1 and Php4 as a function of iron availability. Our findings for S. pombe also constitute the first reported case of a microbial organism expressing two genetically distinct arginases, specifically Car1 and Car3. In addition, these arginases were found to be differentially modulated by the cellular iron status.

Human fibroblasts infected with human cytomegalovirus (HCMV) were more viable than uninfected cells during glucose starvation, suggesting that an alternate carbon source was used. We have determined that infected cells require glutamine for ATP production, whereas uninfected cells do not. This suggested that during infection, glutamine is used to fill the tricarboxylic acid (TCA) cycle (anaplerosis). In agreement with this, levels of glutamine uptake and ammonia production increased in infected cells, as did the activities of glutaminase and glutamate dehydrogenase, the enzymes needed to convert glutamine to α-ketoglutarate to enter the TCA cycle. Infected cells starved for glutamine beginning 24 h postinfection failed to produce infectious virions. Both ATP and viral production could be rescued in glutamine-starved cells by the TCA intermediates α-ketoglutarate, oxaloacetate, and pyruvate, confirming that in infected cells, a program allowing glutamine to be used anaplerotically is induced. Thus, HCMV infection activates the mechanisms needed to switch the anaplerotic substrate from glucose to glutamine to accommodate the biosynthetic and energetic needs of the viral infection and to allow glucose to be used biosynthetically.

Pseudomonas aeruginosa strain PP4 and Acinetobacter lwoffii strain ISP4 metabolize isophthalate as a sole source of carbon and energy. Isophthalate is known to be a competitive inhibitor of glutamate dehydrogenase (GDH), which is involved in C and N metabolism. Strain PP4 showed carbon source-dependent modulation of NADP-GDH; GDH I was produced when cells were grown on isophthalate, while GDH II was produced when cells were grown on glucose. Strain ISP4 produced a single form of NADP-GDH, GDH P , when it was grown on either isophthalate or rich medium (2YT). All of the forms of GDH were purified to homogeneity and characterized. GDH I and GDH II were found to be homotetramers, while GDH P was found to be a homohexamer. GDH II was more sensitive to inhibition by isophthalate (2.5- and 5.5-fold more sensitive for amination and deamination reactions, respectively) than GDH I . Differences in the N-terminal sequences and electrophoretic mobilities in an activity-staining gel confirmed the presence of two forms of GDH, GDH I and GDH II , in strain PP4. In strain ISP4, irrespective of the carbon source, the GDH P produced showed similar levels of inhibition with isophthalate. However, the specific activity of GDH P from isophthalate-grown cells was 2.5- to 3-fold higher than that of GDH P from 2YT-grown cells. Identical N-terminal sequences and electrophoretic mobilities in the activity-staining gel suggested the presence of a single form of GDH P in strain ISP4. These results demonstrate the ability of organisms to modulate GDH either by producing an entirely different form or by increasing the level of the enzyme, thus enabling strains to utilize isophthalate more efficiently as a sole source of carbon and energy.

The BD GeneOhm Cdiff assay, a real-time PCR assay for the detection of the Clostridium difficile toxin B ( tcdB ) gene, was compared with the toxin A/B (Tox A/B) II enzyme-linked immunosorbent assay (ELISA) and a two-step algorithm which includes a C. Diff Chek-60 glutamate dehydrogenase (GDH) antigen assay followed by cytotoxin neutralization. Four hundred liquid or semisolid stool samples submitted for diagnostic C. difficile testing, 200 GDH antigen positive and 200 GDH antigen negative, were selected for analysis. All samples were tested by the C. Diff Chek-60 GDH antigen and cytotoxin neutralization assays, the Tox A/B II ELISA, and the BD GeneOhm Cdiff assay. Specimens with discrepant results were tested by toxigenic culture as an independent “gold standard.” Of 200 GDH-positive samples, 71 were positive by the Tox A/B II ELISA, 88 were positive by the two-step method, 93 were positive by PCR, and 96 were positive by the GDH antigen assay only. Of 200 GDH-negative samples, 3 were positive by PCR only. Toxigenic culture was performed for 41 samples with discrepant results, and 39 were culture positive. Culture resolution of discrepant results showed the Tox A/B II assay to have detected 70 (66.7%), the two-step method to have detected 87 (82.9%), and PCR to have detected 96 (91.4%) of 105 true positives. The BD GeneOhm Cdiff assay was more sensitive in detecting toxigenic C. difficile than the Tox A/B II assay ( P < 0.0001); however, the difference between PCR and the two-step method was not significant ( P = 0.1237). Enhanced sensitivity and rapid turnaround time make the BD GeneOhm Cdiff assay an important advance in the diagnosis of toxigenic C. difficile infection.

The continuing rise in the incidence of Clostridium difficile infection is a cause for concern, with implications for patients and health care systems. Laboratory diagnosis largely relies on rapid toxin detection kits, although assays detecting alternative targets, including glutamate dehydrogenase (GDH) and toxin genes, are now available. Six hundred routine diagnostic diarrheal samples were tested prospectively using nine commercial toxin detection assays, cytotoxin assay (CYT), and cytotoxigenic culture (CYTGC) and retrospectively using a GDH detection assay and PCR for the toxin B gene. The mean sensitivity and specificity for toxin detection assays were 82.8% (range, 66.7 to 91.7%) and 95.4% (range, 90.9 to 98.8%), respectively, in comparison with CYT and 75.0% (range, 60.0 to 86.4%) and 96.1% (91.4 to 99.4%), respectively, in comparison with CYTGC. The sensitivity and specificity of the GDH assay were 90.1% and 92.9%, respectively, compared to CYT and 87.6% and 94.3%, respectively, compared to CYTGC. The PCR assay had the highest sensitivity of all the tests in comparison with CYT (92.2%) and CYTGC (88.5%), and the specificities of the PCR assay were 94.0% and 95.4% compared to CYT and CYTGC, respectively. All kits had low positive predictive values (range, 48.6 to 86.8%) compared with CYT, assuming a positive sample prevalence of 10% (representing the hospital setting), which compromises the clinical utility of single tests for the laboratory diagnosis of C . difficile infection. The optimum rapid single test was PCR for toxin B gene, as this had the highest negative predictive value. Diagnostic algorithms that optimize test combinations for the laboratory diagnosis of C . difficile infection need to be defined.

α-Ketoglutarate (KG) is a crucial metabolite in all living organisms, as it participates in a variety of biochemical processes. We have previously shown that this keto acid is an antioxidant and plays a key role in the detoxification of reactive oxygen species (ROS). In an effort to further confirm this intriguing phenomenon, Pseudomonas fluorescens was exposed to menadione-containing media, with various amino acids as the sources of nitrogen. Here, we demonstrate that KG dehydrogenase (KGDH) and NAD-dependent glutamate dehydrogenase (GDH) work in tandem to modulate KG homeostasis. While KGDH was sharply decreased in cells challenged with menadione, GDH was markedly increased in cultures containing arginine (Arg), glutamate (Glu), and proline (Pro). When ammonium (NH 4 ) was utilized as the nitrogen source, both KGDH and GDH levels were diminished. These enzymatic profiles were reversed when control cells were incubated in menadione media. 13 C nuclear magnetic resonance and high-performance liquid chromatography studies revealed how KG was utilized to eliminate ROS with the concomitant formation of succinate. The accumulation of KG in the menadione-treated cells was dependent on the redox status of the lipoic acid residue in KGDH. Indeed, the treatment of cellular extracts from the menadione-exposed cells with dithiothreitol, a reducing agent, partially restored the activity of KGDH. Taken together, these data reveal that KG is pivotal to the antioxidative defense strategy of P. fluorescens and also point to the ROS-sensing role for KGDH.

The glutamate dehydrogenase (GDH) enzymes of 19 Streptococcus suis serotype 2 strains, consisting of 18 swine isolates and 1 human clinical isolate from a geographically varied collection, were analyzed by activity staining on a nondenaturing gel. All seven (100%) of the highly virulent strains tested produced an electrophoretic type (ET) distinct from those of moderately virulent and nonvirulent strains. By PCR and nucleotide sequence determination, the gdh genes of the 19 strains and of 2 highly virulent strains involved in recent Chinese outbreaks yielded a 1,820-bp fragment containing an open reading frame of 1,344 nucleotides, which encodes a protein of 448 amino acid residues with a calculated molecular mass of approximately 49 kDa. The nucleotide sequences contained base pair differences, but most were silent. Cluster analysis of the deduced amino acid sequences separated the isolates into three groups. Group I (ETI) consisted of the seven highly virulent isolates and the two Chinese outbreak strains, containing Ala 299 -to-Ser, Glu 305 -to-Lys, and Glu 330 -to-Lys amino acid substitutions compared with groups II and III (ETII). Groups II and III consisted of moderately virulent and nonvirulent strains, which are separated from each other by Tyr 72 -to-Asp and Thr 296 -to-Ala substitutions. Gene exchange studies resulted in the change of ETI to ETII and vice versa. A spectrophotometric activity assay for GDH did not show significant differences between the groups. These results suggest that the GDH ETs and sequence types may serve as useful markers in predicting the pathogenic behavior of strains of this serotype and that the molecular basis for the observed differences in the ETs was amino acid substitutions and not deletion, insertion, or processing uniqueness.

We examined the incremental yield of stool culture (with toxin testing on isolates) versus our two-step algorithm for optimal detection of toxigenic Clostridium difficile . Per the two-step algorithm, stools were screened for C. difficile -associated glutamate dehydrogenase (GDH) antigen and, if positive, tested for toxin by a direct (stool) cell culture cytotoxicity neutralization assay (CCNA). In parallel, stools were cultured for C. difficile and tested for toxin by both indirect (isolate) CCNA and conventional PCR if the direct CCNA was negative. The “gold standard” for toxigenic C. difficile was detection of C. difficile by the GDH screen or by culture and toxin production by direct or indirect CCNA. We tested 439 specimens from 439 patients. GDH screening detected all culture-positive specimens. The sensitivity of the two-step algorithm was 77% (95% confidence interval [CI], 70 to 84%), and that of culture was 87% (95% CI, 80 to 92%). PCR results correlated completely with those of CCNA testing on isolates (29/29 positive and 32/32 negative, respectively). We conclude that GDH is an excellent screening test and that culture with isolate CCNA testing detects an additional 23% of toxigenic C. difficile missed by direct CCNA. Since culture is tedious and also detects nontoxigenic C. difficile , we conclude that culture is most useful (i) when the direct CCNA is negative but a high clinical suspicion of toxigenic C. difficile remains, (ii) in the evaluation of new diagnostic tests for toxigenic C. difficile (where the best reference standard is essential), and (iii) in epidemiologic studies (where the availability of an isolate allows for strain typing and antimicrobial susceptibility testing).

The global effect of the CbrAB and NtrBC two-component systems on the control of carbon and nitrogen utilization in Pseudomonas aeruginosa was characterized by phenotype microarray analyses with single and double mutants and the isogenic parent strain. The tested compounds were clustered based on the growth phenotypes of these strains, and the results clearly demonstrated the pivotal roles of CbrAB and NtrBC in carbon and nitrogen utilization, respectively. Growth of the cbrAB deletion mutant on arginine, histidine, and polyamines used as the sole carbon source was abolished, while growth on the tricarboxylic acid (TCA) cycle intermediates was sustained. In this study, suppressors of the cbr mutant were selected from minimal medium containing l -arginine as the sole carbon and nitrogen source. These mutants fell into two groups according to the ability to utilize histidine. The genomic library of a histidine-positive suppressor mutant was constructed, and the corresponding suppressor gene was identified by complementation as an ntrB allele. Similar results were obtained from four additional suppressor mutants, and point mutations of these ntrB alleles resulting in the following changes in residues were identified, with implications for reduced phosphatase activities: L126W, D227A, P228L, and S229I. The Ntr systems of these ntrB mutants became constitutively active, as revealed by the activity profiles of glutamate dehydrogenase, glutamate synthase, and glutamine synthetase. As a result, these mutants not only regain the substrate-specific induction on catabolic arginine and histidine operons but are also expressed to higher levels than the wild type. While the Δ cbrAB ntrB (Con) mutant restored growth on many N-containing compounds used as the carbon sources, its capability to grow on TCA cycle intermediates and glucose was compromised when ammonium served as the sole nitrogen source, mostly due to an extreme imbalance of carbon and nitrogen regulatory systems. In summary, this study supports the notion that CbrAB and NtrBC form a network to control the C/N balance in P. aeruginosa . Possible molecular mechanisms of these two regulatory elements in the control of arginine and histidine operons used as the model systems are discussed.

CodY is a transcriptional regulator conserved in the low-GC group of Gram-positive bacteria. In this work, we demonstrated the presence in Streptococcus thermophilus ST2017 of a functional member of the CodY family of global regulatory proteins, S. thermophilus CodY (CodY St ). The CodY St regulon was identified by transcriptome analysis; it consisted predominantly of genes involved in amino acid metabolism but also included genes involved in several other cellular processes, including carbon metabolism, nutrient transport, and stress response. It was revealed that CodY St repressed the transformation of the central metabolic pathway to amino acid metabolism and improved lactose utilization. Furthermore, the glutamate dehydrogenase gene ( gdhA ), repressed by CodY St , was suggested to coordinate the interconversion between carbon metabolism and amino acid metabolism and to play an important role on the optimal growth of S. thermophilus ST2017 in milk. A conserved CodY St box [AA(T/A)(A/T)TTCTGA(A/C)AATT] was indeed required for in vitro binding of CodY St to the target regions of DNA. These results provided evidence for the function of CodY St , by which this strain coordinately regulates its various metabolic pathways so as to adapt to the milk environment.

We evaluated a two-step algorithm for detecting toxigenic Clostridium difficile : an enzyme immunoassay for glutamate dehydrogenase antigen (Ag-EIA) and then, for antigen-positive specimens, a concurrent cell culture cytotoxicity neutralization assay (CCNA). Antigen-negative results were ≥99% predictive of CCNA negativity. Because the Ag-EIA reduced cell culture workload by ≈75 to 80% and two-step testing was complete in ≤3 days, we decided that this algorithm would be effective. Over 6 months, our laboratories' expenses were US$143,000 less than if CCNA alone had been performed on all 5,887 specimens.

Clostridium difficile is the primary causative agent of antibiotic-associated diarrheal disease. To facilitate molecular genetic analysis of gene expression in this organism, methods were developed to study transcriptional regulation in vitro and in vivo. That is, C. difficile RNA polymerase was partially purified and shown to bind to and initiate transcription in vitro from bona fide C. difficile promoters for rRNA and glutamate dehydrogenase genes. In addition, primer extension analyses and a β-glucuronidase reporter system were used to quantitate transcription from these promoters in vivo. With these tools in hand, it is now possible to characterize the behavior of any C. difficile gene in vivo and to study the regulation of its expression in detail.