Microarray expression profile analysis of circular RNAs and their potential regulatory role in bladder carcinoma.

Dysregulated circular RNAs (circRNAs) often contribute to the occurrence and development of various tumors; however, the function and mechanism of circRNAs are largely unknown in human bladder cancer (BC). In the present study, dysregulated circRNAs between BC and adjacent non­neoplastic bladder tissues were analyzed by circRNA microarray. We randomly selected 10 upregulated and five downregulated circRNAs for validation by quantitative real­time PCR. Bioinformatics analysis was further conducted to investigate the potential function of these differentially expressed circRNAs, with the differential expression of hsa_circRNA_100876, mir­136­5p, and mRNA­chromobox 4 (CBX4) subsequently verified. A total of 512 differentially expressed circRNAs were identified after scanning and normalization (340 upregulated and 172 downregulated circRNAs), with pathway and Gene Ontology analyses revealing their association with multiple significant cancer pathways. Construction of a circRNA­microRNA­mRNA network suggested additional potential roles of these circRNAs. The expression of hsa_circRNA_100876 and CBX4 was significantly negatively correlated with the expression of miR­136­5p. Additionally, hsa_circRNA_100876 was highly positively correlated with CBX4 expression. The results revealed that hsa_circRNA_100876 inhibition suppressed BC cell proliferation and it was associated with advanced T stage and lymphatic metastasis, and poor overall survival of BC patients. In conclusion, these differentially expressed circRNAs offer novel insights into potential biological markers or new therapeutic targets for the treatment of BC. Furthermore, hsa_circRNA_100876 may increase the expression of CBX4 by competing with miR­136­5p, ultimately promoting the malignant biological behavior of BC. Aberrantly expressed hsa_circRNA_100876 could be used as a potential non­invasive biomarker for the early detection and screening of BC.

A nucleotide-switch mechanism mediates opposing catalytic activities of Rel enzymes.

Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3' of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3' pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (RelTt). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of RelTt (RelTtNTD) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation.

Maintenance of Nucleolar Homeostasis by CBX4 Alleviates Senescence and Osteoarthritis.

CBX4, a component of polycomb repressive complex 1 (PRC1), plays important roles in the maintenance of cell identity and organ development through gene silencing. However, whether CBX4 regulates human stem cell homeostasis remains unclear. Here, we demonstrate that CBX4 counteracts human mesenchymal stem cell (hMSC) aging via the maintenance of nucleolar homeostasis. CBX4 protein is downregulated in aged hMSCs, whereas CBX4 knockout in hMSCs results in destabilized nucleolar heterochromatin, enhanced ribosome biogenesis, increased protein translation, and accelerated cellular senescence. CBX4 maintains nucleolar homeostasis by recruiting nucleolar protein fibrillarin (FBL) and heterochromatin protein KRAB-associated protein 1 (KAP1) at nucleolar rDNA, limiting the excessive expression of rRNAs. Overexpression of CBX4 alleviates physiological hMSC aging and attenuates the development of osteoarthritis in mice. Altogether, our findings reveal a critical role of CBX4 in counteracting cellular senescence by maintaining nucleolar homeostasis, providing a potential therapeutic target for aging-associated disorders.

Trivial role for NSMCE2 during in vitro proliferation and differentiation of male germline stem cells.

Spermatogenesis, starting with spermatogonial differentiation, is characterized by ongoing and dramatic alterations in composition and function of chromatin. Failure to maintain proper chromatin dynamics during spermatogenesis may lead to mutations, chromosomal aberrations or aneuploidies. When transmitted to the offspring, these can cause infertility or congenital malformations. The structural maintenance of chromosomes (SMC) 5/6 protein complex has recently been described to function in chromatin modeling and genomic integrity maintenance during spermatogonial differentiation and meiosis. Among the subunits of the SMC5/6 complex, non-SMC element 2 (NSMCE2) is an important small ubiquitin-related modifier (SUMO) ligase. NSMCE2 has been reported to be essential for mouse development, prevention of cancer and aging in adult mice and topological stress relief in human somatic cells. By using in vitro cultured primary mouse spermatogonial stem cells (SSCs), referred to as male germline stem (GS) cells, we investigated the function of NSMCE2 during spermatogonial proliferation and differentiation. We first optimized a protocol to generate genetically modified GS cell lines using CRISPR-Cas9 and generated an Nsmce2-/- GS cell line. Using this Nsmce2-/- GS cell line, we found that NSMCE2 was dispensable for proliferation, differentiation and topological stress relief in mouse GS cells. Moreover, RNA sequencing analysis demonstrated that the transcriptome was only minimally affected by the absence of NSMCE2. Only differential expression of Sgsm1 appeared highly significant, but with SGSM1 protein levels being unaffected without NSMCE2. Hence, despite the essential roles of NSMCE2 in somatic cells, chromatin integrity maintenance seems differentially regulated in the germline.

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone.

Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named "(p)ppGpp"] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) from Bacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (SN2) reaction. We show that pppGpp-but not ppGpp-positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles.

An ENU-induced mutation in the mouse Rnf212 gene is associated with male meiotic failure and infertility.

The ENU-induced repro57 mutation was identified in an unbiased screen for the discovery of novel genes for fertility. Male repro57 homozygous mice are infertile and exhibit significantly reduced testis weight compared with WT mice. Histological examination of mutant testes revealed that spermatocytes degenerated during late prophase, and no mature spermatozoa were found in the seminiferous epithelium, suggesting that infertility is caused by the arrest of spermatogenesis at late meiotic prophase. Consistent with this hypothesis, the number of foci with MLH1, a protein essential for crossing over, is greatly reduced in repro57 mutant spermatocytes, which also lack chiasmata between homologs and exhibit premature dissociation of XY chromosomes. In repro57 mutant mice, we identified a mutation in the Rnf212 gene, encoding Ring finger protein 212. The overall phenotype of repro57 mice is consistent with the recently reported phenotype of the Rnf212 knockout mice; slight differences may be due to genetic background effects. Thus, the repro57 nonsense mutation provides a new allele of the mouse Rnf212 gene.

RelA protein stimulates the activity of RyhB small RNA by acting on RNA-binding protein Hfq.

The conserved RNA-binding protein Hfq and its associated small regulatory RNAs (sRNAs) are increasingly recognized as the players of a large network of posttranscriptional control of gene expression in Gram-negative bacteria. The role of Hfq in this network is to facilitate base pairing between sRNAs and their trans-encoded target mRNAs. Although the number of known sRNA-mRNA interactions has grown steadily, cellular factors that influence Hfq, the mediator of these interactions, have remained unknown. We report that RelA, a protein long known as the central regulator of the bacterial-stringent response, acts on Hfq and thereby affects the physiological activity of RyhB sRNA as a regulator of iron homeostasis. RyhB requires RelA in vivo to arrest growth during iron depletion and to down-regulate a subset of its target mRNAs (fdoG, nuoA, and sodA), whereas the sodB and sdhC targets are barely affected by RelA. In vitro studies with recombinant proteins show that RelA enhances multimerization of Hfq monomers and stimulates Hfq binding of RyhB and other sRNAs. Hfq from polysomes extracted from wild-type cells binds RyhB in vitro, whereas Hfq from polysomes of a relA mutant strain shows no binding. We propose that, by increasing the level of the hexameric form of Hfq, RelA enables binding of RNAs whose affinity for Hfq is low. Our results suggest that, under specific conditions and/or environments, Hfq concentrations are limiting for RNA binding, which thereby provides an opportunity for cellular proteins such as RelA to impact sRNA-mediated responses by modulating the activity of Hfq.

The (p)ppGpp synthetase RelA contributes to stress adaptation and virulence in Enterococcus faecalis V583.

Guanosine penta- and tetraphosphate [(p)ppGpp] are two unusual nucleotides implied in the bacterial stringent response. In many pathogenic bacteria, mutants unable to synthesize these molecules lose their virulence. In Gram-positive bacteria such as Enterococcus faecalis, the synthesis and degradation of (p)ppGpp mainly depend on the activity of a bifunctional enzyme, encoded by the relA gene. By analysing DeltarelA and DeltarelQ (which encodes a protein harbouring a ppGpp synthetase activity) deletion mutants, we showed that RelA is by far the main system leading to (p)ppGpp production under our experimental conditions, and during the development of a stringent response induced by mupirocin. We also constructed a mutant (DeltarelAsp) in which a small part of the relA gene (about 0.7 kbp) encoding the carboxy-terminal domain of the RelA protein was deleted. Both relA mutants were more resistant than the wild-type strain to 0.3 % bile salts, 25 % ethanol and acid (pH 2.3) challenges. Interestingly, the DeltarelAsp mutant grew better than the two other strains in the presence of 1 mM H(2)O(2), but did not display increased tolerance when subjected to lethal doses of H(2)O(2) (45 mM). By contrast, the DeltarelA mutant was highly sensitive to 45 mM H(2)O(2) and displayed reduced growth in a medium containing 1 M NaCl. The two mutants also displayed contrasting virulence phenotypes towards larvae of the Greater Wax Moth infection model Galleria mellonella. Indeed, although the DeltarelA mutant did not display any phenotype, the DeltarelAsp mutant was more virulent than the wild-type strain. This virulent phenotype should stem from its increased ability to proliferate under oxidative environments.

Ubiquitin proteolytic system: focus on SUMO.

The small ubiquitin-like modifier proteins (Smt3 in yeast and SUMOs 1-4 in vertebrates) are members of the ubiquitin super family. Like ubiquitin, the SUMOs are protein modifiers that are covalently attached to the epsilon-amino group of lysine residues in the substrates. The application of proteomics to the SUMO field has greatly expanded both the number of known targets and the number of identified target lysines. As new refinements of proteomic techniques are developed and applied to sumoylation, an explosion of novel data is likely in the next 5 years. This ability to examine sumoylated proteins globally, rather than individually, will lead to new insights into both the functions of the individual SUMO types, and how dynamic changes in overall sumoylation occur in response to alterations in cellular environment. In addition, there is a growing appreciation for the existence of cross-talk mechanisms between the sumoylation and ubiquitinylation processes. Rather than being strictly parallel, these two systems have many points of intersection, and it is likely that the coordination of these two systems is a critical contributor to the regulation of many fundamental cellular events.

Histone H2A monoubiquitination represses transcription by inhibiting RNA polymerase II transcriptional elongation.

Solving the biological roles of covalent histone modifications, including monoubiquitination of histone H2A, and the molecular mechanisms by which these modifications regulate specific transcriptional programs remains a central question for all eukaryotes. Here we report that the N-CoR/HDAC1/3 complex specifically recruits a specific histone H2A ubiquitin ligase, 2A-HUB/hRUL138, to a subset of regulated gene promoters. 2A-HUB catalyzes monoubiquitination of H2A at lysine 119, functioning as a combinatoric component of the repression machinery required for specific gene regulation programs. Thus, 2A-HUB mediates a selective repression of a specific set of chemokine genes in macrophages, critically modulating migratory responses to TLR activation. H2A monoubiquitination acts to prevent FACT recruitment at the transcriptional promoter region, blocking RNA polymerase II release at the early stage of elongation. We suggest that distinct H2A ubiquitinases, each recruited based on interactions with different corepressor complexes, contribute to distinct transcriptional repression programs.

Identification and functional analysis of novel (p)ppGpp synthetase genes in Bacillus subtilis.

Bacterial alarmone (p)ppGpp, is a global regulator responsible for the stringent control. Two homologous (p)ppGpp synthetases, RelA and SpoT, have been identified and characterized in Escherichia coli, whereas Gram-positive bacteria such as Bacillus subtilis have been thought to possess only a single RelA-SpoT enzyme. We have now identified two genes, yjbM and ywaC, in B. subtilis that encode a novel type of alarmone synthetase. The predicted products of these genes are relatively small proteins ( approximately 25 kDa) that correspond to the (p)ppGpp synthetase domain of RelA-SpoT family members. A database survey revealed that genes homologous to yjbM and ywaC are conserved in certain bacteria belonging to Firmicutes or Actinobacteria phyla but not in other phyla such as Proteobacteria. We designated the proteins as small alarmone synthetases (SASs) to distinguish them from RelA-SpoT proteins. The (p)ppGpp synthetase function of YjbM and YwaC was confirmed by genetic complementation analysis and by in vitro assay of enzyme activity. Molecular genetic analysis also revealed that ywaC is induced by alkaline shock, resulting in the transient accumulation of ppGpp. The SAS proteins thus likely function in the biosynthesis of alarmone with a mode of action distinct from that of RelA-SpoT homologues.

Differential stringent responses of Streptomyces coelicolor M600 to starvation of specific nutrients.

This study focused on the involvement of the unusual nucleotide (p)ppGpp, a stringent factor, during the morphological and physiological differentiation of Streptomyces coelicolor. Two genes, relA and rshA, were disrupted to demonstrate the roles of the stringent factor in the differentiation. The intracellular concentration of (p)ppGpp in the wild-type (M600) and disrupted mutants was measured in relation to the intentional starvation of a specific nutrient such as carbon, nitrogen, and phosphate or the in situ depletion of nutrients in a batch culture. As a result, it was found that the morphological characteristic of the deltarelA mutant was a bld phenotype forming condensed mycelia, whereas the deltarshA mutant grew fast-forming spores and straightforward mycelia. In both mutants, the production of actinorhodin (Act) was completely abolished, yet the undecylprodigiosin (Red) production was increased. Intracellular (p)ppGpp was detected in the deltarelA mutant in the case of limited phosphate, yet not with limited carbon or nitrogen sources. In contrast, (p)ppGpp was produced in the deltarshA mutant under limited carbon and nitrogen conditions. Therefore, (p)ppGpp in S. coelicolor was found to be selectively regulated by either the RelA or RshA protein, which was differentially expressed in response to the specific nutrient limitation. These results were also supported by the in situ ppGpp production during a batch culture. Furthermore, it is suggested that RelA and RshA are bifunctional proteins that possess the ability to both synthesize and hydrolyze (p)ppGpp.

A charge reversal differentiates (p)ppGpp synthesis by monofunctional and bifunctional Rel proteins.

A major regulatory mechanism evolved by microorganisms to combat stress is the regulation mediated by (p)ppGpp (the stringent response molecule), synthesized and hydrolyzed by Rel proteins. These are divided into bifunctional and monofunctional proteins based on the presence or absence of the hydrolysis activity. Although these proteins require Mg(2+) for (p)ppGpp synthesis, high Mg(2+) was shown to inhibit this reaction in bifunctional Rel proteins from Mycobacterium tuberculosis and Streptococcus equisimilis. This is not a characteristic feature in enzymes that use a dual metal ion mechanism, such as DNA polymerases that are known to carry out a similar pyrophosphate transfer reaction. Comparison of polymerase Polbeta and Rel(Seq) structures that share a common fold led to the proposal that the latter would follow a single metal ion mechanism. Surprisingly, in contrast to bifunctional Rel, we did not find inhibition of guanosine 5'-triphosphate, 3'-diphosphate (pppGpp) synthesis at higher Mg(2+) in the monofunctional RelA from Escherichia coli. We show that a charge reversal in a conserved motif in the synthesis domains explains this contrast; an RXKD motif in the bifunctional proteins is reversed to an EXDD motif. The differential response of these proteins to Mg(2+) could also be noticed in fluorescent nucleotide binding and circular dichroism experiments. In mutants where the motifs were reversed, the differential effect could also be reversed. We infer that although a catalytic Mg(2+) is common to both bifunctional and monofunctional proteins, the latter would utilize an additional metal binding site formed by EXDD. This work, for the first time, brings out differences in (p)ppGpp synthesis by the two classes of Rel proteins.

The global role of ppGpp synthesis in morphological differentiation and antibiotic production in Streptomyces coelicolor A3(2).

BACKGROUND: Regulation of production of the translational apparatus via the stringent factor ppGpp in response to amino acid starvation is conserved in many bacteria. However, in addition to this core function, it is clear that ppGpp also exhibits genus-specific regulatory effects. In this study we used Affymetrix GeneChips to more fully characterize the regulatory influence of ppGpp synthesis on the biology of Streptomyces coelicolor A3(2), with emphasis on the control of antibiotic biosynthesis and morphological differentiation. RESULTS: Induction of ppGpp synthesis repressed transcription of the major sigma factor hrdB, genes with functions associated with active growth, and six of the thirteen conservons present in the S. coelicolor genome. Genes induced following ppGpp synthesis included the alternative sigma factor SCO4005, many for production of the antibiotics CDA and actinorhodin, the regulatory genes SCO4198 and SCO4336, and two alternative ribosomal proteins. Induction of the CDA and actinorhodin clusters was accompanied by an increase in transcription of the pathway regulators cdaR and actII-ORF4, respectively. Comparison of transcriptome profiles of a relA null strain, M570, incapable of ppGpp synthesis with its parent M600 suggested the occurrence of metabolic stress in the mutant. The failure of M570 to sporulate was associated with a stalling between production of the surfactant peptide SapB, and of the hydrophobins: it overproduced SapB but failed to express the chaplin and rodlin genes. CONCLUSION: In S. coelicolor, ppGpp synthesis influences the expression of several genomic elements that are particularly characteristic of streptomycete biology, notably antibiotic gene clusters, conservons, and morphogenetic proteins.

Multicellular development in Myxococcus xanthus is stimulated by predator-prey interactions.

Myxococcus xanthus is a predatory bacterium that exhibits complex social behavior. The most pronounced behavior is the aggregation of cells into raised fruiting body structures in which cells differentiate into stress-resistant spores. In the laboratory, monocultures of M. xanthus at a very high density will reproducibly induce hundreds of randomly localized fruiting bodies when exposed to low nutrient availability and a solid surface. In this report, we analyze how M. xanthus fruiting body development proceeds in a coculture with suitable prey. Our analysis indicates that when prey bacteria are provided as a nutrient source, fruiting body aggregation is more organized, such that fruiting bodies form specifically after a step-down or loss of prey availability, whereas a step-up in prey availability inhibits fruiting body formation. This localization of aggregates occurs independently of the basal nutrient levels tested, indicating that starvation is not required for this process. Analysis of early developmental signaling relA and asgD mutants indicates that they are capable of forming fruiting body aggregates in the presence of prey, demonstrating that the stringent response and A-signal production are surprisingly not required for the initiation of fruiting behavior. However, these strains are still defective in differentiating to spores. We conclude that fruiting body formation does not occur exclusively in response to starvation and propose an alternative model in which multicellular development is driven by the interactions between M. xanthus cells and their cognate prey.

Genome-wide expression profiling in Geobacter sulfurreducens: identification of Fur and RpoS transcription regulatory sites in a relGsu mutant.

Rel(Gsu) is the single Geobacter sulfurreducens homolog of RelA and SpoT proteins found in many organisms. These proteins are involved in the regulation of levels of guanosine 3', 5' bispyrophosphate, ppGpp, a molecule that signals slow growth and stress response under nutrient limitation in bacteria. We used information obtained from genome-wide expression profiling of the rel(Gsu) deletion mutant to identify putative regulatory sites involved in transcription networks modulated by Rel(Gsu) or ppGpp. Differential gene expression in the rel(Gsu) deletion mutant, as compared to the wild type, was available from two growth conditions, steady state chemostat cultures and stationary phase batch cultures. Hierarchical clustering analysis of these two datasets identified several groups of operons that are likely co-regulated. Using a search for conserved motifs in the upstream regions of these co-regulated operons, we identified sequences similar to Fur- and RpoS-regulated sites. These findings suggest that Fur- and RpoS-dependent gene expression in G. sulfurreducens is affected by Rel(Gsu)-mediated signaling.

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase.

The apicomplexan Cryptosporidium parvum possesses a unique 1500-kDa polyketide synthase (CpPKS1) comprised of 29 enzymes for synthesising a yet undetermined polyketide. This study focuses on the biochemical characterization of the 845-amino acid loading unit containing acyl-[ACP] ligase (AL) and acyl carrier protein (ACP). The CpPKS1-AL domain has a substrate preference for long chain fatty acids, particularly for the C20:0 arachidic acid. When using [3H]palmitic acid and CoA as co-substrates, the AL domain displayed allosteric kinetics towards palmitic acid (Hill coefficient, h=1.46, K50=0.751 microM, Vmax=2.236 micromol mg(-1) min(-1)) and CoA (h=0.704, K50=5.627 microM, Vmax=0.557 micromol mg(-1) min(-1)), and biphasic kinetics towards adenosine 5'-triphosphate (Km1=3.149 microM, Vmax1=373.3 nmol mg(-1) min(-1), Km2=121.0 microM, and Vmax2=563.7 nmol mg(-1) min(-1)). The AL domain is Mg2+-dependent and its activity could be inhibited by triacsin C (IC50=6.64 microM). Furthermore, the ACP domain within the loading unit could be activated by the C. parvum surfactin production element-type phosphopantetheinyl transferase. After attachment of the fatty acid substrate to the AL domain for conversion into the fatty-acyl intermediate, the AL domain is able to transfer palmitic acid to the activated holo-ACP in vitro. These observations ultimately validate the function of the CpPKS1-AL-ACP unit, and make it possible to further dissect the function of this megasynthase using recombinant proteins in a stepwise procedure.

Role for RpoS but not RelA of Legionella pneumophila in modulation of phagosome biogenesis and adaptation to the phagosomal microenvironment.

The induction of virulence traits by Legionella pneumophila at the post-exponential phase has been proposed to be triggered by the stringent response mediated by RelA, which triggers RpoS. We show that L. pneumophila rpoS but not relA is required for early intracellular survival and replication within human monocyte-derived macrophages and Acanthamoeba polyphaga. In addition, L. pneumophila rpoS but not relA is required for expression of the pore-forming activity. We provide evidence that RpoS plays a role in the modulation of phagosome biogenesis and in adaptation to the phagosomal microenvironment. Thus, there is no functional link between the stringent response and RpoS in the pathogenesis of L. pneumophila.

Linking the nutritional status of Streptococcus pyogenes to alteration of transcriptional gene expression: the action of CodY and RelA.

In this investigation, we identify the CodY protein from Streptococcus pyogenes as a pleiotropic transcription regulator with global features. The notion that acquisition of nutrients by this polyauxotrophic organism is the primary event occurring during the establishment of infection and that virulence expression is a result of this quest, led us to study the action of codY and relA genes on transcriptional gene expression under different nutritional conditions using complex and chemically defined media. Real-time reverse transcription PCR was used to determine the extent to which inactivation of codY and relA affects the mRNA levels of selected transcription factors, virulence genes, transporters, and genes encoding metabolic enzymes. The results show that CodY and RelA did not affect the expression of each other but that both exhibited strong negative autoregulatory properties. Genes negatively controlled by the relA-directed stringent response to amino acid starvation included, besides relA itself, transporters, metabolic enzymes, and at least two virulence genes (graB and speH). The expression of many genes of all four groups studied proved to be subject to direct or indirect control by CodY, often in a nutritional status-dependent fashion. One of the most important results implicates CodY in growth phase-dependent positive transcriptional regulation of pel/sagA and mga, loci that themselves positively affect the expression of numerous virulence factors. Increasing the cellular activity of nicotinamidase in both a codY mutant and wild-type background induced extensive transcriptional reprogramming, altering, among others, the growth phase-dependent transcription pattern of the genes for cysteine protease (speB) and several transporters. Inasmuch as CodY influenced the expression of other regulators (pel/sagA, mga, covRS, ropB, pyrR), its action is amplified and expands the complex regulatory network that governs gene expression in S. pyogenes.