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Bacillus sp. TL7-3 has potential as a dietary supplement to promote human and animal health. It produces spores that can survive in harsh environments. Thus, when supplemented with nutrients, these spores can withstand the acidic pH of the stomach and resume vegetative development in the gut when exposed to growth-promoting conditions. Spores are formed as a cellular defense mechanism when a culture experiences stress and process optimization to achieve high spore production in a typical batch process remains challenging. Existing literature on the manipulation of gene expression and enzyme activity during batch cultivation is limited. Studies on the growth patterns, morphological changes, and relevant gene expression have aided in enhancing spore production. The present study used the response surface methodology for medium optimization. The model suggested that yeast extract and NH4Cl were significant factors controlling spore production. A comparison between the high weight ratio of carbon and nitrogen (C:N) substrates (8.57:1) in the optimized and basal media (0.52:1) showed an 8.76-fold increase in the final spore concentration. The expression of major genes, including codY, spo0A, kinA, and spo0F, involved in the sporulation was compared when cultivating Bacillus sp. TL7-3 in media with varying C:N ratios. At high C:N ratios, spo0A, kinA, and spo0F were upregulated, whereas codY was downregulated. This led to decreased guanylate kinase activity, resulting in a low guanosine triphosphate concentration and inactivation of CodY, thereby reducing the repression of spo0A and CodY-repressed genes and stimulating sporulation.

Lactococcus lactis is a key strain in the production of branched-chain aldehydes that contribute to the nutty flavor in cheese. It has been hypothesized that the conversion of branched-chain amino acids (BCAAs) into branched-chain aldehydes is under the regulation of a global transcriptional regulator, CodY; however, the functions remain unclear. This study aimed to elucidate the regulatory effect of CodY on the key genes for the BCAAs conversion into branched-chain aldehydes in L. lactis, namely branched-chain aminotransferase (encoded by bcaT) a-keto acid decarboxylase (encoded by kdcA) and a-keto acid dehydrogenase (encoded by pdhABCD). The deletion of codY was associated with a significant increase in the production of 3-methylbutanal and 2-methylbutanal, indicating the inhibitory effect of CodY on the BCAAs conversion into branched-chain aldehydes. Real-time quantitative reverse transcription-PCR demonstrated the negative regulatory effect of CodY on bcaT and kdcA, whereas pdhABCD was activated by CodY. Potential CodY binding sites in the promoter regions of bcaT, kdcA, and pdhABCD were predicted, and four CodY-boxes within the regulatory regions were verified by electrophoretic mobility shift assay and DNase I footprinting assay. The sites located within the promoter of bcaT and kdcA led to repression by CodY. When one site was located upstream and the other was located within the promoter of pdhABCD, the ultimate effect was activation. Overall, the study revealed that CodY, together with isoleucine, acts as a pleiotropic transcriptional regulator of the conversion of BCAAs into branched-chain aldehydes in L. lactis via binding to various regulatory regions of key genes. IMPORTANCE Branched-chain aldehydes are the primary compounds that contribute to the nutty flavor in cheddar cheese. Lactococcus lactis, which is often applied as primary starter culture, is a significant contributor to the nutty flavor of cheddar cheese due to its ability of conversion of BCAAs into branched-chain aldehydes. In the present study, we found that the regulatory role of CodY is crucial for the conversion. CodY acts as a pleiotropic transcriptional regulator via binding to various regulatory regions of key genes. The results presented valuable knowledge into the role of CodY on the regulation and biosynthetic pathway of branched-chain amino acids and the related aldehydes. Furthermore, it provided new insight for increasing the nutty flavor produced during the manufacture and ripening of cheese. [ABSTRACT FROM AUTHOR]

The research aims to identify the effect of developing the strength and stability of the center muscles on improving the level of performance of the cody skill for trampoline gymnasts, the experimental approach was using the experimental design with pre-post-measurement for one group, the research sample consisted of (۱۱) second-class (junior) players and under years old, for trampoline gymnastics at the Staff Club of the Faculty of Physical Education for Boys in Alexandria and the Sudanese General Union Club in Cairo, season ۲۰۲۲-۲۰۲۳, after applying the program, it was reached: The program of strength and stability of the muscles of the center, which was subjected to the same research, has a positive effect on all physical measurements under study. - The program of strength and stability of the muscles of the center, which the research sample was subjected, has a positive effect on the performance level of the cody skill for the research sampl Through these results, the researcher recommends the following: Be guided by the scientific foundations that were used in designing the proposed training program, which includes strength and stability exercises for the center muscles, for trampoline gymnastics skills. Directing the findings to gymnastics workers to benefit from them in various training programs. Conducting more research to study the feasibility of developing the strength and stability of the core muscles, on the practical performance of gymnastics skills Strengthening the practical performance of all gymnastics skills with strength and stability exercises for the center muscles, because of its impact on improving the performance of the skills. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus (S. aureus), as the main pathogen in milk and dairy products, usually causes intoxication with vomiting and various kinds of inflammation after entering the human body. CodY, an important transcriptional regulator in S. aureus, plays an important role in regulating metabolism, growth, and virulence. However, little is known about the role of CodY on environmental stress tolerance. In this research, we revealed the role of CodY in environmental stress tolerance in foodborne S. aureus RMSA24. codY mutation significantly reduced the tolerance of S. aureus to desiccation and oxidative, salt, and high-temperature stresses. However, S. aureus was more tolerant to low temperature stress due to mutation of codY. We found that the expressions of two important heat shock proteins—GroEL and DanJ—were significantly down-regulated in the mutant codY. This suggests that CodY may indirectly regulate the high- and low-temperature tolerance of S. aureus by regulating the expressions of groEL and danJ. This study reveals a new mechanism of environmental stress tolerance in S. aureus and provides new insights into controlling the contamination and harm caused by S. aureus in the food industry. [ABSTRACT FROM AUTHOR]

ABSTRACT CodY is a conserved broad-acting transcription factor that regulates the expression of genes related to amino acid metabolism and virulence in Gram-positive bacteria. Here, we performed the first in vivo determination of CodY target genes using a novel CodY monoclonal antibody in methicillin-resistant Staphylococcus aureus (MRSA) USA300. Our results showed (i) the same 135 CodY promoter binding sites regulating the 165 target genes identified in two closely related virulent S. aureus USA300 TCH1516 and LAC strains; (ii) the differential binding intensity for the same target genes under the same conditions was due to sequence differences in the same CodY-binding site in the two strains; (iii) a CodY regulon comprising 72 target genes that are differentially regulated relative to a CodY deletion strain, representing genes that are mainly involved in amino acid transport and metabolism, inorganic ion transport and metabolism, transcription and translation, and virulence, all based on transcriptomic data; and (iv) CodY systematically regulated central metabolic flux to generate branched-chain amino acids (BCAAs) by mapping the CodY regulon onto a genome-scale metabolic model of S. aureus. Our study performed the first system-level analysis of CodY in two closely related USA300 TCH1516 and LAC strains, revealing new insights into the similarities and differences of CodY regulatory roles between the closely related strains. IMPORTANCE With the increasing availability of whole-genome sequences for many strains within the same pathogenic species, a comparative analysis of key regulators is needed to understand how the different strains uniquely coordinate metabolism and expression of virulence. To successfully infect the human host, Staphylococcus aureus USA300 relies on the transcription factor CodY to reorganize metabolism and express virulence factors. While CodY is a known key transcription factor, its target genes are not characterized on a genome-wide basis. We performed a comparative analysis to describe the transcriptional regulation of CodY between two dominant USA300 strains. This study motivates the characterization of common pathogenic strains and an evaluation of the possibility of developing specialized treatments for major strains circulating in the population.

ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are widely recognized as bacterial adaptive immune systems against invading viruses and bacteriophages. The oral pathogen Streptococcus mutans encodes two CRISPR-Cas loci (CRISPR1-Cas and CRISPR2-Cas), and their expression under environmental conditions is still under investigation. In this study, we investigated the transcriptional regulation of cas operons by CcpA and CodY, two global regulators that contribute to carbohydrate and (p)ppGpp metabolism. The possible promoter regions for cas operons and the binding sites for CcpA and CodY in the promoter regions of both CRISPR-Cas loci were predicted using computational algorithms. We found that CcpA could directly bind to the upstream region of both cas operons, and detected an allosteric interaction of CodY within the same region. The binding sequences of the two regulators were identified through footprinting analysis. Our results showed that the promoter activity of CRISPR1-Cas was enhanced under fructose-rich conditions, while deletion of the ccpA gene led to reduced activity of the CRISPR2-Cas promoter under the same conditions. Additionally, deletion of the CRISPR systems resulted in a significant decrease in fructose uptake ability compared to the parental strain. Interestingly, the accumulation of guanosine tetraphosphate (ppGpp) was reduced in the presence of mupirocin, which induces a stringent response, in the CRISPR1-Cas-deleted (ΔCR1cas) and both CRISPR-Cas-deleted (ΔCRDcas) mutant strains. Furthermore, the promoter activity of both CRISPRs was enhanced in response to oxidative or membrane stress, while the CRISPR1 promoter activity was reduced under low-pH conditions. Collectively, our findings demonstrate that the transcription of the CRISPR-Cas system is directly regulated by the binding of CcpA and CodY. These regulatory actions play a crucial role in modulating glycolytic processes and exerting effective CRISPR-mediated immunity in response to nutrient availability and environmental cues. IMPORTANCE An effective immune system has evolved not only in eukaryotic organisms but also in microorganisms, enabling them to rapidly detect and neutralize foreign invaders in the environment. Specifically, the CRISPR-Cas system in bacterial cells is established through a complex and sophisticated regulatory mechanism involving specific factors. In this study, we demonstrate that the expression of two CRISPR systems in S. mutans can be controlled by two global regulators, CcpA and CodY, which play critical roles in carbohydrate metabolism and amino acid biosynthesis. Importantly, our results show that the expression of the CRISPR-Cas system in S. mutans influences (p)ppGpp production during the stringent response, which is a gene expression regulatory response that aids in environmental stress adaptation. This transcriptional regulation by these regulators enables a CRISPR-mediated immune response in a host environment with limited availability of carbon sources or amino acids, while ensuring efficient carbon flux and energy expenditure to support multiple metabolic processes.

Cyclic di-AMP is an emerging second messenger that is synthesized by many archaea and bacteria, including the Gram-positive pathogenic bacterium Listeria monocytogenes. Listeria monocytogenes played a crucial role in elucidating the essential function of c-di-AMP, thereby becoming a model system for studying c-di-AMP metabolism and the influence of the nucleotide on cell physiology. c-di-AMP is synthesized by a diadenylate cyclase and degraded by two phosphodiesterases. To date, eight c-di-AMP receptor proteins have been identified in L. monocytogenes , including one that indirectly controls the uptake of osmotically active peptides and thus the cellular turgor. The functions of two c-di-AMP-receptor proteins still need to be elucidated. Here, we provide an overview of c-di-AMP signalling in L. monocytogenes and highlight the main differences compared to the other established model systems in which c-di-AMP metabolism is investigated. Moreover, we discuss the most important questions that need to be answered to fully understand the role of c-di-AMP in osmoregulation and in the control of central metabolism. [ABSTRACT FROM AUTHOR]

Background: Staphylococcus aureus (S. aureus) is the leading cause of various infective diseases, including topical soft tissue infections. The goals of this study were to investigate the roles of YycF and CodY in the regulation of biofilm formation and pathogenicity. Methods: Electrophoretic mobility shift assay (EMSA) was conducted to validate the bound promoter regions of YycF protein. We constructed the codY up-regulated or down-regulated S. aureus mutants. The biofilm biomass was determined by crystal violet microtiter assay and scanning electron microscopy (SEM). Quantitative RT-PCR analysis was used to detect the transcripts of biofilm-related genes. The live and dead cells of S. aureus biofilm were also investigated by confocal laser scanning microscopy (CLSM). We constructed an abscess infection in Sprague Dawley (SD) rat models to determine the effect of CodY on bacterial pathogenicity. We further used the RAW264.7, which were cocultured with S. aureus, to evaluate the effect of CodY on macrophages apoptosis. Result: Quantitative RT-PCR analyses reveled that YycF negatively regulates codY expression. EMSA assays indicated that YycF protein directly binds to the promoter regions of codY gene. Quantitative RT-PCR confirmed the construction of dual-mutant stains codY + ASyycF and codY-ASyycF. The SEM results showed that the biofilm formation in the codY + ASyycF group was sparser than those in the other groups. The crystal violet assays indicated that the codY + ASyycF group formed less biofilms, which was consistent with the immunofluorescence results of the lowest live cell ration in the codY + ASyycF group. The expression levels of biofilm-associated icaA gene were significantly reduced in the codY + strain, indicating codY negatively regulates the biofilm formation. Furthermore, CodY impedes the pathogenicity in a rat-infection model. After cocultured with bacteria or 4-h in vitro, the apoptosis rates of macrophage cells were lowest in the codY + group. Conclusions: YycF negatively regulate the expression of codY. By interaction with codY, YycF could modulate S. aureus biofilm formation via both eDNA-dependent and PIA- dependent pathways, which can be a significant target for antibiofilm. CodY not only impedes the pathogenicity but also has a role on immunoregulation. Thus, the current evidence may provide a supplementary strategy for managing biofilm infections. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a Gram-positive, opportunistic human pathogen that is a leading cause of skin and soft tissue infections and invasive disease worldwide. Virulence in this bacterium is tightly controlled by a network of regulatory factors. One such factor is the global regulatory protein CodY. CodY links branched-chain amino acid sufficiency to the production of surface-associated and secreted factors that facilitate immune evasion and subversion. Our previous work revealed that CodY regulates virulence factor gene expression indirectly in part by controlling the activity of the SaeRS two-component system (TCS). While this is correlated with an increase in membrane anteiso-15:0 and -17:0 branched-chain fatty acids (BCFAs) derived from isoleucine, the true mechanism of control has remained elusive. Herein, we report that CodY-dependent regulation of SaeS sensor kinase activity requires BCFA synthesis. During periods of nutrient sufficiency, BCFA synthesis and Sae TCS activity are kept relatively low by CodY-dependent repression of the ilv-leu operon and the isoleucine-specific permease gene brnQ2 . In a codY null mutant, which simulates extreme nutrient limitation, de-repression of ilv-leu and brnQ2 directs the synthesis of enzymes in redundant de novo and import pathways to upregulate production of BCFA precursors. Overexpression of brnQ2, independent of CodY, is sufficient to increase membrane anteiso BCFAs, Sae-dependent promoter activity, and SaeR ~ P levels. Our results further clarify the molecular mechanisms by which CodY controls virulence in S. aureus .IMPORTANCEExpression of bacterial virulence genes often correlates with the exhaustion of nutrients, but how the signaling of nutrient availability and the resulting physiological responses are coordinated is unclear. In S. aureus, CodY controls the activity of two major regulators of virulence-the Agr and Sae two-component systems (TCSs)-by unknown mechanisms. This work identifies a mechanism by which CodY controls the activity of the sensor kinase SaeS by modulating the levels of anteiso branched-chain amino acids that are incorporated into the membrane. Understanding the mechanism adds to our understanding of how bacterial physiology and metabolism are linked to virulence and underscores the role virulence in maintaining homeostasis. Understanding the mechanism also opens potential avenues for targeted therapeutic strategies against S. aureus infections., Competing Interests: The authors declare no conflict of interest.

Staphylococcus aureus is a leading cause of severe pneumonia. Our recent proteomic investigations into S. aureus invasion of human lung epithelial cells revealed three key adaptive responses: activation of the SigB and CodY regulons and upregulation of the hibernation-promoting factor SaHPF. Therefore, our present study aimed at a functional and proteomic dissection of the contributions of CodY, SigB and SaHPF to host invasion using transposon mutants of the methicillin-resistant S. aureus USA300. Interestingly, disruption of codY resulted in a "small colony variant" phenotype and redirected the bacteria from (phago)lysosomes into the host cell cytoplasm. Furthermore, we show that CodY, SigB and SaHPF contribute differentially to host cell adhesion, invasion, intracellular survival and cytotoxicity. CodY- or SigB-deficient bacteria experienced faster intracellular clearance than the parental strain, underscoring the importance of these regulators for intracellular persistence. We also show an unprecedented role of SaHPF in host cell adhesion and invasion. Proteomic analysis of the different mutants focuses attention on the CodY-perceived metabolic state of the bacteria and the SigB-perceived environmental cues in bacterial decision-making prior and during infection. Additionally, it underscores the impact of the nutritional status and bacterial stress on the initiation and progression of staphylococcal lung infections.

Using the USA300, methicillin-resistant Staphylococcus aureus strain LAC, we previously examined the impact of regulatory mutations implicated in biofilm formation on protease production and virulence in a murine sepsis model. Here we examined the impact of these mutations in the USA200, methicillin-sensitive strain UAMS-1. Mutation of agr, mgrA, rot, sarA and sigB attenuated the virulence of UAMS-1. A common characteristic of codY, rot, sigB, and sarA mutants was increased protease production, with mutation of rot having the least impact followed by mutation of codY, sigB and sarA, respectively. Protein A was undetectable in conditioned medium from all four mutants, while extracellular nuclease was only present in the proteolytically cleaved NucA form. The abundance of high molecular weight proteins was reduced in all four mutants. Biofilm formation was reduced in codY, sarA and sigB mutants, but not in the rot mutant. Eliminating protease production partially reversed these phenotypes and enhanced biofilm formation. This was also true in LAC codY, rot, sarA and sigB mutants. Eliminating protease production enhanced the virulence of LAC and UAMS-1 sarA, sigB and rot mutants in a murine sepsis model but did not significantly impact the virulence of the codY mutant in either strain. Nevertheless, these results demonstrate that repressing protease production plays an important role in defining critical phenotypes in diverse clinical isolates of S. aureus and that Rot, SigB and SarA play critical roles in this regard.

Staphylococcus aureus (S. aureus), as the main pathogen in milk and dairy products, usually causes intoxication with vomiting and various kinds of inflammation after entering the human body. CodY, an important transcriptional regulator in S. aureus, plays an important role in regulating metabolism, growth, and virulence. However, little is known about the role of CodY on environmental stress tolerance. In this research, we revealed the role of CodY in environmental stress tolerance in foodborne S. aureus RMSA24. codY mutation significantly reduced the tolerance of S. aureus to desiccation and oxidative, salt, and high-temperature stresses. However, S. aureus was more tolerant to low temperature stress due to mutation of codY. We found that the expressions of two important heat shock proteins—GroEL and DanJ—were significantly down-regulated in the mutant codY. This suggests that CodY may indirectly regulate the high- and low-temperature tolerance of S. aureus by regulating the expressions of groEL and danJ. This study reveals a new mechanism of environmental stress tolerance in S. aureus and provides new insights into controlling the contamination and harm caused by S. aureus in the food industry.

Group B Streptococcus (GBS) is a Gram-positive bacterium able to switch from a harmless commensal of healthy adults to a pathogen responsible for invasive infections in neonates. The signals and regulatory mechanisms governing this transition are still largely unknown. CodY is a highly conserved global transcriptional regulator that links nutrient availability to the regulation of major metabolic and virulence pathways in low-G+C Gram-positive bacteria. In this work, we investigated the role of CodY in BM110, a GBS strain representative of a hypervirulent lineage associated with the majority of neonatal meningitis. Deletion of codY resulted in a reduced ability of the mutant strain to cause infections in neonatal and adult animal models. The observed decreased in vivo lethality was associated with an impaired ability of the mutant to persist in the blood, spread to distant organs, and cross the blood-brain barrier. Notably, the codY null mutant showed reduced adhesion to monolayers of human epithelial cells in vitro and an increased ability to form biofilms, a phenotype associated with strains able to asymptomatically colonize the host. RNA-seq analysis showed that CodY controls about 13% of the genome of GBS, acting mainly as a repressor of genes involved in amino acid transport and metabolism and encoding surface anchored proteins, including the virulence factor Srr2. CodY activity was shown to be dependent on the availability of branched-chain amino acids, which are the universal cofactors of this regulator. These results highlight a key role for CodY in the control of GBS virulence. [ABSTRACT FROM AUTHOR]

Group B Streptococcus (GBS) is a Gram-positive bacterium able to switch from a harmless commensal of healthy adults to a pathogen responsible for invasive infections in neonates. The signals and regulatory mechanisms governing this transition are still largely unknown. CodY is a highly conserved global transcriptional regulator that links nutrient availability to the regulation of major metabolic and virulence pathways in low-G+C Gram-positive bacteria. In this work, we investigated the role of CodY in BM110, a GBS strain representative of a hypervirulent lineage associated with the majority of neonatal meningitis. Deletion of codY resulted in a reduced ability of the mutant strain to cause infections in neonatal and adult animal models. The observed decreased in vivo lethality was associated with an impaired ability of the mutant to persist in the blood, spread to distant organs, and cross the blood-brain barrier. Notably, the codY null mutant showed reduced adhesion to monolayers of human epithelial cells in vitro and an increased ability to form biofilms, a phenotype associated with strains able to asymptomatically colonize the host. RNA-seq analysis showed that CodY controls about 13% of the genome of GBS, acting mainly as a repressor of genes involved in amino acid transport and metabolism and encoding surface anchored proteins, including the virulence factor Srr2. CodY activity was shown to be dependent on the availability of branched-chain amino acids, which are the universal cofactors of this regulator. These results highlight a key role for CodY in the control of GBS virulence.

Bacteria adapt to the constantly changing environment by regulating their metabolism. The global transcriptional regulator CodY is known to regulate metabolism in low-G1C Gram-positive bacteria. Systems-level identification of its direct targets by proteome and chromatin immunoprecipitation followed by sequencing (ChIP-seq) assays have rarely been reported. Here, we identified that CodY serves as an activator or a repressor of hundreds of genes involved in nitrogen metabolism, carbohydrate metabolism, and transcription through iTRAQ proteome and ChIP-seq. Combined with the electrophoretic mobility shift assay (EMSA), apart from the genes associated with amino acid biosynthesis (ilvD, leuA, optS, ybbD, dtpT, and pepN), genes involved in cell wall synthesis (murD and ftsW) and nisin immunity (nisI) were identified as being regulated by CodY. Moreover, it was demonstrated by nisin resistance assay that CodY activated the transcription of nisI and contributed to nisin immunity. Intriguingly, CodY showed a self-regulation through binding to the motif AAAGGTGTGACAACT in the coding sequence (CDS) region of codY, as verified by DNase I footprinting assay and MEME analysis. In addition, a novel conserved AT-rich motif, AATWTTCTGACAATT, was obtained in L. lactis F44. This study provides new insights into the comprehensive CodY regulation in L. lactis by controlling metabolism, nisin immunity, and self-expression. IMPORTANCE Lactococcus lactis, a species of lactic acid bacteria (LAB) widely used in food fermentation, has been the model strain in genetic engineering, and its application has extended from food to microbial cell factories. CodY is a global regulator in low-G1C Gram-positive bacteria. Its function and direct target genes at the genome-level are little known in L. lactis. In this study, we describe the comprehensive regulation mechanism of CodY. It widely modulated the metabolism of nitrogen and carbohydrate, cell wall synthesis, and nisin immunity in L. lactis F44, and its expression level was regulated by feedback control. [ABSTRACT FROM AUTHOR]

Using the USA300, methicillin-resistant Staphylococcus aureus strain LAC, we previously examined the impact of regulatory mutations implicated in biofilm formation on protease production and virulence in a murine sepsis model. Here we examined the impact of these mutations in the USA200, methicillin-sensitive strain UAMS-1. Mutation of agr, mgrA, rot, sarA and sigB attenuated the virulence of UAMS-1. A common characteristic of codY, rot, sigB, and sarA mutants was increased protease production, with mutation of rot having the least impact followed by mutation of codY, sigB and sarA, respectively. Protein A was undetectable in conditioned medium from all four mutants, while extracellular nuclease was only present in the proteolytically cleaved NucA form. The abundance of high molecular weight proteins was reduced in all four mutants. Biofilm formation was reduced in codY, sarA and sigB mutants, but not in the rot mutant. Eliminating protease production partially reversed these phenotypes and enhanced biofilm formation. This was also true in LAC codY, rot, sarA and sigB mutants. Eliminating protease production enhanced the virulence of LAC and UAMS-1 sarA, sigB and rot mutants in a murine sepsis model but did not significantly impact the virulence of the codY mutant in either strain. Nevertheless, these results demonstrate that repressing protease production plays an important role in defining critical phenotypes in diverse clinical isolates of S. aureus and that Rot, SigB and SarA play critical roles in this regard. [ABSTRACT FROM AUTHOR]

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Bacillus sp. TL7-3 has potential as a dietary supplement to promote human and animal health. It produces spores that can survive in harsh environments. Thus, when supplemented with nutrients, these spores can withstand the acidic pH of the stomach and resume vegetative development in the gut when exposed to growth-promoting conditions. Spores are formed as a cellular defense mechanism when a culture experiences stress and process optimization to achieve high spore production in a typical batch process remains challenging. Existing literature on the manipulation of gene expression and enzyme activity during batch cultivation is limited. Studies on the growth patterns, morphological changes, and relevant gene expression have aided in enhancing spore production. The present study used the response surface methodology for medium optimization. The model suggested that yeast extract and NH 4 Cl were significant factors controlling spore production. A comparison between the high weight ratio of carbon and nitrogen (C:N) substrates (8.57:1) in the optimized and basal media (0.52:1) showed an 8.76-fold increase in the final spore concentration. The expression of major genes, including codY , spo0A , kinA , and spo0F , involved in the sporulation was compared when cultivating Bacillus sp. TL7-3 in media with varying C:N ratios. At high C:N ratios, spo0A , kinA , and spo0F were upregulated, whereas codY was downregulated. This led to decreased guanylate kinase activity, resulting in a low guanosine triphosphate concentration and inactivation of CodY, thereby reducing the repression of spo0A and CodY-repressed genes and stimulating sporulation., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Nuttha Thongchul reports financial support was provided by 10.13039/501100004704National Research Council of Thailand. Nuttha Thongchul reports financial support was provided by 10.13039/501100004192National Science and Technology Development Agency. Suttichai Assabumrungrat reports financial support was provided by Research Chair Professor Grant. Sitanan Thitiprasert reports financial support was provided by Fundamental Research Fund. Benjamas Chiersilp reports financial support was provided by Thailand Research Fund. Phetcharat Jaiaue reports financial support was provided by Development and Promotion of Science and Technology Talents Scholarship Program. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Abstract The ability of Streptococcus mutans to persist in a variety of adverse environments and to emerge as a numerically dominant member of stable oral biofilm communities are essential elements for its cariogenicity. The S. mutans Cid/Lrg system has been studied as a key player in the integration of complex environmental signals into regulatory networks that modulate virulence and cell homeostasis. Cid/Lrg has also been shown to be closely associated with metabolic pathways of this organism, due to distinct patterns of cid and lrg expression in response to growth phase and glucose/oxygen levels. In this study, a comparison of cid and lrg promoter regions with conserved CodY (a regulator which responds to starvation stress)‐binding motifs revealed the presence of a potential CodY‐binding site, which is arranged similarly in both cid and lrg promoters. Electrophoretic mobility shift assays (EMSAs) and promoter reporter assays demonstrated that expression of the cid and lrg operons is directly mediated by the global transcriptional regulator CodY. DNase I footprinting analyses confirmed the predicted binding sequences for CodY in both the cid and the lrg promoter regions. Overexpression of CodY had no obvious effect on lrgAB expression, but deficiency of CodY still affected lrgAB expression in a lytST‐overexpressing strain, suggesting that CodY is required for the full regulation of lrgAB by LytST. We also demonstrated that both CodY and CcpA are involved in regulating pyruvate flux and utilization. Collectively, these data show that CodY directly regulates cid and lrg expression, and together with CcpA (previously shown to directly regulate cid and lrg promoters) contributes to coordinating pyruvate uptake and utilization in response to both the external environment and the cellular metabolic status.

Abstract Background Streptococcus salivarius is an abundant isolate of the human oral microbiota. Since both pH and glucose availability fluctuate frequently in the oral cavity, the goal of this study was to investigate regulation by CodY, a conserved pleiotropic regulator of Gram positive bacteria, in response to these two signals. The chemostat culture system was employed to precisely control the growth parameters, and the transcriptomes of wild-type S. salivarius 57.I and its CodY-null derivative (ΔcodY) grown at pH 7 and 5.5, with limited and excessive glucose supply were determined. Results The transcriptomic analysis revealed that CodY was most active at pH 7 under conditions of glucose limitation. Based on whether a CodY binding consensus could be located in the 5′ flanking region of the identified target, the transcriptomic analysis also found that CodY shaped the transcriptome via both direct and indirect regulation. Inactivation of codY reduced the glycolytic capacity and the viability of S. salivarius at pH 5.5 or in the presence of H2O2. Studies using the Galleria mellonella larva model showed that CodY was essential for the toxicity generated from S. salivarius infection, suggesting that CodY regulation was critical for immune evasion and systemic infections. Furthermore, the CodY-null mutant strain exhibited a clumping phenotype and reduced attachment in biofilm assays, suggesting that CodY also modulates cell wall metabolism. Finally, the expression of genes belonging to the CovR regulon was affected by codY inactivation, but CodY and CovR regulated these genes in opposite directions. Conclusions Metabolic adaptation in response to nutrient availability and growth pH is tightly linked to stress responses and virulence expression in S. salivarius. The regulation of metabolism by CodY allows for the maximal utilization of available nutrients and ATP production. The counteractive regulation of the CovR regulon could fine tune the transcriptomes in response to environmental changes.

Peptides present in growth media are essential for nitrogen nutrition and optimal growth of lactic acid bacteria. In addition, according to their amino acid composition, they can also directly or indirectly play regulatory roles and influence the global metabolism. This is especially relevant during the propagation phase to produce high cell counts of active lactic acid bacteria used as starters in the dairy industry. In the present work, we aimed at investigating how the respective compositions of two different yeast extracts, with a specific focus on peptide content, influenced Streptococcus thermophilus metabolism during growth under pH-controlled conditions. In addition to free amino acids quantification, we used a multi-omics approach (peptidomics, proteomics and transcriptomics) to identify peptide initially present in the two culture media, and to follow S. thermophilus gene expression and bacterial protein production during growth. The free amino acid and peptide composition of the two yeast extracts differed qualitatively and quantitatively. Nevertheless, the two yeast extracts sustained similar growth of S. thermophilus and led to equivalent final biomasses. However, transcriptomics and proteomics showed differential gene expression and protein production in several S. thermophilus metabolic pathways, especially amino acid, citrate, urease, purine and pyrimidine metabolisms. The probable role of the regulator CodY is discussed in this context. Moreover, we observed significant differences in the production of regulators and of a quorum sensing regulatory system. The possible roles of yeast extract peptides on the modulation of the quorum sensing system expression are evaluated. [ABSTRACT FROM AUTHOR]

This paper reports on somewhat puzzling character of the determination of the optical band gap energies of amorphous and polycrystalline semiconductors, where probably the mixed-phase composition and structural and compositional disorder are main reasons for the ambiguities. The analysis is performed using spectra of absorption coefficients of the featured samples of Si:H and Zn-Ti-O extracted by numerical analysis of transmittance measurements. The differences originating from using several procedures to process spectra of absorption coefficients are discussed.

ABSTRACT In Firmicutes, the nutrient-sensing regulators (p)ppGpp, the effector molecule of the stringent response, and CodY work in tandem to maintain bacterial fitness during infection. Here, we tested (p)ppGpp and codY mutant strains of Enterococcus faecalis in a catheter-associated urinary tract infection (CAUTI) mouse model and used global transcriptional analysis to investigate the relationship of (p)ppGpp and CodY. The absence of (p)ppGpp or single inactivation of codY led to lower bacterial loads in catheterized bladders and diminished biofilm formation on fibrinogen-coated surfaces under in vitro and in vivo conditions. Single inactivation of the bifunctional (p)ppGpp synthetase/hydrolase rel did not affect virulence, supporting previous evidence that the association of (p)ppGpp with enterococcal virulence is not dependent on the activation of the stringent response. Inactivation of codY in the (p)ppGpp0 strain restored E. faecalis virulence in the CAUTI model as well as the ability to form biofilms in vitro. Transcriptome analysis revealed that inactivation of codY restores, for the most part, the dysregulated metabolism of (p)ppGpp0 cells. While a clear linkage between (p)ppGpp and CodY with expression of virulence factors could not be established, targeted transcriptional analysis indicates that a possible association between (p)ppGpp and c-di-AMP signaling pathways in response to the conditions found in the bladder may play a role in enterococcal CAUTI. Collectively, data from this study identify the (p)ppGpp-CodY network as an important contributor to enterococcal virulence in catheterized mouse bladder and support that basal (p)ppGpp pools and CodY promote virulence through maintenance of a balanced metabolism under adverse conditions. IMPORTANCE Catheter-associated urinary tract infections (CAUTIs) are one of the most frequent types of infection found in the hospital setting that can develop into serious and potentially fatal bloodstream infections. One of the infectious agents that frequently causes complicated CAUTI is the bacterium Enterococcus faecalis, a leading cause of hospital-acquired infections that are often difficult to treat due to the exceptional multidrug resistance of some isolates. Understanding the mechanisms by which E. faecalis causes CAUTI will aid in the discovery of new druggable targets to treat these infections. In this study, we report the importance of two nutrient-sensing bacterial regulators, named (p)ppGpp and CodY, for the ability of E. faecalis to infect the catheterized bladder of mice.

Antimicrobial drug discovery against drug-resistant bacteria is an urgent need. Beyond agents with direct antibacterial activity, anti-virulent molecules may also be viable compounds to defend against bacterial pathogenesis. Using a high throughput screen (HTS) that utilized Caenorhabditis elegans infected with methicillin-resistant Staphylococcus aureus (MRSA) strain of MW2, we identified 4-(1,3-dimethyl-2,3-dihydro-1H-benzimidazol-2-yl)phenol (BIP). Interestingly, BIP had no in vitro inhibition activity against MW2, at least up to 64 μg/ml. The lack of direct antimicrobial activity suggests that BIP could inhibit bacterial virulence factors. To explore the possible anti-virulence effect of the identified molecule, we first performed real-time PCR to examine changes in virulence expression. BIP was highly active against MRSA virulence factors at sub-lethal concentrations and down-regulated virulence regulator genes, such as agrA and codY. However, the benzimidazole derivatives omeprazole and pantoprazole did not down-regulate virulence genes significantly, compared to BIP. Moreover, the BIP-pretreated MW2 cells were more vulnerable to macrophage-mediated killing, as confirmed by intracellular killing and live/dead staining assays, and less efficient in establishing a lethal infection in the invertebrate host Galleria mellonella (p = 0.0131). We tested the cytotoxicity of BIP against human red blood cells (RBCs), and it did not cause hemolysis at the highest concentration tested (64 μg/ml). Taken together, our findings outline the potential anti-virulence activity of BIP that was identified through a C. elegans-based, whole animal based, screen.

Antimicrobial drug discovery against drug-resistant bacteria is an urgent need. Beyond agents with direct antibacterial activity, anti-virulent molecules may also be viable compounds to defend against bacterial pathogenesis. Using a high throughput screen (HTS) that utilized Caenorhabditis elegans infected with methicillin-resistant Staphylococcus aureus (MRSA) strain of MW2, we identified 4-(1,3-dimethyl-2,3-dihydro-1H-benzimidazol-2-yl)phenol (BIP). Interestingly, BIP had no in vitro inhibition activity against MW2, at least up to 64 μg/ml. The lack of direct antimicrobial activity suggests that BIP could inhibit bacterial virulence factors. To explore the possible anti-virulence effect of the identified molecule, we first performed real-time PCR to examine changes in virulence expression. BIP was highly active against MRSA virulence factors at sub-lethal concentrations and down-regulated virulence regulator genes, such as agrA and codY. However, the benzimidazole derivatives omeprazole and pantoprazole did not down-regulate virulence genes significantly, compared to BIP. Moreover, the BIP-pretreated MW2 cells were more vulnerable to macrophage-mediated killing, as confirmed by intracellular killing and live/dead staining assays, and less efficient in establishing a lethal infection in the invertebrate host Galleria mellonella (p = 0.0131). We tested the cytotoxicity of BIP against human red blood cells (RBCs), and it did not cause hemolysis at the highest concentration tested (64 μg/ml). Taken together, our findings outline the potential anti-virulence activity of BIP that was identified through a C. elegans -based, whole animal based, screen. [ABSTRACT FROM AUTHOR]

• In S. suis, introduction of a codY mutant into ΔrelAΔrelQ strain reduced the expression of virulence factors. • The ΔcodYΔrelAΔrelQ was highly attenuated in S. suis. • GTP is not a ligand of CodY in S. suis. • CodY could interacted with the promoter of relA and positively regulate the expression of relA in S. suis. CodY and (p)ppGpp synthetases are two important global regulators of bacteria. In some pathogens, such as Listeria monocytogenes , the GTP pool links these two regulatory systems, and introducing a codY mutant into the Δ relA strain restored the pathogenicity of the attenuated Δ relA mutant. In previous studies, we identified the (p)ppGpp synthetases (RelA and RelQ) and CodY of Streptococcus suis. To understand the interrelationships between these two regulators in S. suis , a Δ relA Δ relQ Δ codY mutant was constructed, and its growth, morphology, and pathogenicity were evaluated. Compared with Δ relA Δ relQ , Δ codY , its growth was very slow, but its chain length was partly restored to the wild-type length and its capsule became thick and rough. The adherence, invasion ability, and resistance to whole-blood killing in vitro of Δ relA Δ relQ Δ codY and its lethality and colonization ability in mice were clearly reduced, which differs from the effects of these mutations in L. monocytogenes. An analysis of gene expression showed that CodY interacted with the relA promoter in a GTP-independent manner to positively regulate the expression of relA. The introduction of a codY mutant into the Δ relA Δ relQ strain further reduced the expression of virulence factors, which suggests a novel interaction between the (p)ppGpp synthetases and CodY. This study extends our understanding of the relationship between the (p)ppGpp-mediated stringent response and the regulation of CodY in S. suis. [ABSTRACT FROM AUTHOR]

In Lactococcus lactis, the global transcriptional regulatory factor CodY can interact with the promoter DNA to regulate the growth, metabolism, environmental adaptation and other biological activities of the strains. In order to study the mechanism of interaction between CodY and its target DNA, molecular docking and molecular dynamics simulations were used to explore the binding process at molecular level. Through the calculations of the free energy of binding, hydrogen bonding and energy decomposition, nine key residues of CodY were identified, corresponding to SER184, SER186, SER208, THR217, ARG218, SER219, ASN223, LYS242 and GLY243, among which SER186, ARG218 and LYS242 play a vital role in DNA binding. Our research results provide important theoretical guidance for using wet-lab methods to study and optimize the metabolic network regulated by CodY. [ABSTRACT FROM AUTHOR]

Abstract Bacillus subtilis is extensively applied as a microorganism for the high-level production of heterologous proteins. Traditional strategies for increasing the productivity of this microbial cell factory generally focused on the targeted modification of rate-limiting components or steps. However, the longstanding problems of limited productivity of the expression host, metabolic burden and non-optimal nutrient intake, have not yet been completely solved to achieve significant production-strain improvements. To tackle this problem, we systematically rewired the regulatory networks of the global nitrogen and carbon metabolism by random mutagenesis of the pleiotropic transcriptional regulators CodY and CcpA, to allow for optimal nutrient intake, translating into significantly higher heterologous protein production yields. Using a β-galactosidase expression and screening system and consecutive rounds of mutagenesis, we identified mutant variants of both CodY and CcpA that in conjunction increased production levels up to 290%. RNA-Seq and electrophoretic mobility shift assay (EMSA) showed that amino acid substitutions within the DNA-binding domains altered the overall binding specificity and regulatory activity of the two transcription factors. Consequently, fine-tuning of the central metabolic pathways allowed for enhanced protein production levels. The improved cell factory capacity was further demonstrated by the successfully increased overexpression of GFP, xylanase and a peptidase in the double mutant strain. Highlights • gTME was applied to build mutant libraries of CodY and CcpA in Bacillus subtilis. • Specific point mutations altered the binding and expression of major regulators. • Changes in global regulation evoked the reprogrammed central metabolic networks. • Heterologous protein yield was boosted by adjusting nutrient uptake and utilization. [ABSTRACT FROM AUTHOR]

Background: Streptococcus salivarius is an abundant isolate of the human oral microbiota. Since both pH and glucose availability fluctuate frequently in the oral cavity, the goal of this study was to investigate regulation by CodY, a conserved pleiotropic regulator of Gram positive bacteria, in response to these two signals. The chemostat culture system was employed to precisely control the growth parameters, and the transcriptomes of wild-type S. salivarius 57.I and its CodY-null derivative (ΔcodY) grown at pH 7 and 5.5, with limited and excessive glucose supply were determined. Results: The transcriptomic analysis revealed that CodY was most active at pH 7 under conditions of glucose limitation. Based on whether a CodY binding consensus could be located in the 5′ flanking region of the identified target, the transcriptomic analysis also found that CodY shaped the transcriptome via both direct and indirect regulation. Inactivation of codY reduced the glycolytic capacity and the viability of S. salivarius at pH 5.5 or in the presence of H2O2. Studies using the Galleria mellonella larva model showed that CodY was essential for the toxicity generated from S. salivarius infection, suggesting that CodY regulation was critical for immune evasion and systemic infections. Furthermore, the CodY-null mutant strain exhibited a clumping phenotype and reduced attachment in biofilm assays, suggesting that CodY also modulates cell wall metabolism. Finally, the expression of genes belonging to the CovR regulon was affected by codY inactivation, but CodY and CovR regulated these genes in opposite directions. Conclusions: Metabolic adaptation in response to nutrient availability and growth pH is tightly linked to stress responses and virulence expression in S. salivarius. The regulation of metabolism by CodY allows for the maximal utilization of available nutrients and ATP production. The counteractive regulation of the CovR regulon could fine tune the transcriptomes in response to environmental changes. [ABSTRACT FROM AUTHOR]

Beta diversity (β) is used in biogeography, ecology, and conservation to assess the heterogeneity of local communities. Ideally, researchers could include sensitivity to error in the list of reasons to choose a β index. However, only numerical undersampling has been rigorously studied. This study compared multiple β indices to determine which are most robust to geographic undersampling, numerical undersampling, and taxonomic error using simulated landscapes. For these landscapes, eight β indices were chosen to represent families of β and used to measure real and errant data. Six indices used both presence-- absence and abundance data, while two more used only abundance data. Six of the abundance-based indices had adjusted versions for individual undersampling, and these versions were also evaluated (total = 14 indices). Presence--absence- and abundance-based indices were comparable in sensitivity to total method error. Numerical undersampling and taxonomic error generally caused more error in β than randomly distributed geographic undersampling. Among presence--absence-based indices, Jaccard's dissimilarity was the most robust to error overall, while β-3 was the most robust among narrow-sense measures. Among abundance-based indices, Bray-Curtis and BDTOTAL were the most robust to error. Some commonly used β indices (e.g., Sorensen, Simpson) are relatively unreliable given errors of taxonomy or numerical undersampling. Future studies of β should focus on using more robust indices (Jaccard, Bray-Curtis, BDTOTAL), and past studies based on error-sensitive indices should be considered with caution. Studies of β should emphasize adequate numerical sampling and taxonomic accuracy to minimize errors in β. [ABSTRACT FROM AUTHOR]

Bacteria have evolved complex regulatory controls in response to various environmental stresses. Protein toxins of the ζ superfamily, found in prominent human pathogens, are broadly distributed in nature. We show that ζ is a uridine diphosphate-N-acetylglucosamine (UNAG)-dependent ATPase whose activity is inhibited in vitro by stoichiometric concentrations of ε2 antitoxin. In vivo, transient ζ expression promotes a reversible multi-level response by altering the pool of signaling purine nucleotides, which leads to growth arrest (dormancy), although a small cell subpopulation persists rather than tolerating toxin action. High c-di-AMP levels (absence of phosphodiesterase GdpP) decrease, and low c-di-AMP levels (absence of diadenylate cyclase DisA) increase the rate of ζ persistence. The absence of CodY, a transition regulator from exponential to stationary phase, sensitizes cells to toxin action, and suppresses persisters formed in the ΔdisA context. These changes, which do not affect the levels of stochastic ampicillin (Amp) persistence, sensitize cells to toxin and Amp action. Our findings provide an explanation for the connection between ζ-mediated growth arrest (with alterations in the GTP and c-di-AMP pools) and persistence formation.

Streptococcus pneumoniae is a facultative anaerobic pathogen. Although it maintains fermentative metabolism, during aerobic growth pneumococci produce high levels of H2O2, which can have adverse effects on cell viability and DNA, and influence pneumococcal interaction with its host. The pneumococcus is unusual in its dealing with toxic reactive oxygen species (ROS) in that it neither has catalase nor the global regulators of peroxide stress resistance. Previously, we identified pneumococcal thiol peroxidase (TpxD) as the key enzyme for enzymatic removal of H2O2, and showed that TpxD synthesis is up-regulated upon exposure to H2O2. This study aimed to reveal the mechanism controlling TpxD expression under H2O2 stress. We hypothesize that H2O2 activates a transcription factor which in turn up-regulates tpxD expression. Microarray analysis revealed a pneumococcal global transcriptional response to H2O2. Mutation of tpxD abolished H2O2-mediated response to high H2O2 levels, signifying the need for an active TpxD under oxidative stress conditions. Bioinformatic tools, applied to search for a transcription factor modulating tpxD expression, pointed toward CodY as a potential candidate. Indeed, a putative 15-bp consensus CodY binding site was found in the proximal region of tpxD-coding sequence. Binding of CodY to this site was confirmed by EMSA, and genetic engineering techniques demonstrated that this site is essential for TpxD up-regulation under H2O2 stress. Furthermore, tpxD expression was reduced in a ΔcodY mutant. These data indicate that CodY is an activator of tpxD expression, triggering its up-regulation under H2O2 stress. In addition we show that H2O2 specifically oxidizes the 2 CodY cysteines. This oxidation may trigger a conformational change in CodY, resulting in enhanced binding to DNA. A schematic model illustrating the contribution of TpxD and CodY to pneumococcal global transcriptional response to H2O2 is proposed.

Bacteria have evolved complex regulatory controls in response to various environmental stresses. Protein toxins of the ζ superfamily, found in prominent human pathogens, are broadly distributed in nature. We show that ζ is a uridine diphosphate-N-acetylglucosamine (UNAG)-dependent ATPase whose activity is inhibited in vitro by stoichiometric concentrations of +2 antitoxin. In vivo, transient ζ expression promotes a reversible multi-level response by altering the pool of signaling purine nucleotides, which leads to growth arrest (dormancy), although a small cell subpopulation persists rather than tolerating toxin action. High c-di-AMP levels (absence of phosphodiesterase GdpP) decrease, and low c-di-AMP levels (absence of diadenylate cyclase DisA) increase the rate of ζ persistence. The absence of CodY, a transition regulator from exponential to stationary phase, sensitizes cells to toxin action, and suppresses persisters formed in the &3916;disA context. These changes, which do not affect the levels of stochastic ampicillin (Amp) persistence, sensitize cells to toxin and Amp action. Our findings provide an explanation for the connection between ζ-mediated growth arrest (with alterations in the GTP and c-di-AMP pools) and persistence formation. [ABSTRACT FROM AUTHOR]

A growing body of evidence points to CodY, a global regulator in Gram-positive bacteria, as a critical link between microbial physiology and pathogenesis in diverse environments. Recent studies uncovering graded regulation of CodY gene targets reflect the true nature of this transcription factor controlled by ligands and reveal nutrient availability as a potentially critical factor in modulating pathogenesis. This review will serve to update the status of the field and raise new questions to be answered. [ABSTRACT FROM AUTHOR]

The pleiotropism of the GTP-sensing transcriptional regulator CodY is evident by the gamut of processes that it regulates in almost all low G+C Gram-positive bacteria, including general metabolism, biosynthesis of some amino acids and transport systems, nitrogen uptake, sporulation, biofilm formation, motility and virulence. The role of CodY in virulence has been established in Bacillus anthracis, the top rated bioterrorism agent. In this study, we investigated the biochemical attributes of this global regulator. Homology modeling and sequence/structure analysis revealed putative GTP-binding residues in CodY of B. anthracis. CodY exhibited an interaction with the GTP as tested by ultraviolet cross-linking experiments. It could autophosphorylate itself at a conserved Ser215 residue. This was further corroborated by the impairment of autophosphorylation activity in the CodYS215A mutant. Autophosphorylation may be speculated as an additional mechanism regulating CodY activity in the cell. The protein could also hydrolyze GTP, albeit weakly, as indicated by thinlayer chromatography and spectrophotometric quantification of its kinetic parameters. Altogether, these observations provide us an insight into the mechanism of action of this global regulator and a better understanding of its functional regulation. [ABSTRACT FROM AUTHOR]

Aspirin has provided clear benefits to human health. But salicylic acid (SAL) -the main aspirin biometabolite- exerts several effects on eukaryote and prokaryote cells. SAL can affect, for instance, the expression of Staphylococcus aureus virulence factors. SAL can also form complexes with iron cations and it has been shown that different iron chelating molecules diminished the formation of S. aureus biofilm. The aim of this study was to elucidate whether the iron content limitation caused by SAL can modify the S. aureus metabolism and/or metabolic regulators thus changing the expression of the main polysaccharides involved in biofilm formation. The exposure of biofilm to 2mM SAL induced a 27% reduction in the intracellular free Fe2+ concentration compared with the controls. In addition, SAL depleted 23% of the available free Fe2+ cation in culture media. These moderate iron-limited conditions promoted an intensification of biofilms formed by strain Newman and by S. aureus clinical isolates related to the USA300 and USA100 clones. The slight decrease in iron bioavailability generated by SAL was enough to induce the increase of PIA expression in biofilms formed by methicillin-resistant as well as methicillin-sensitive S. aureus strains. S. aureus did not produce capsular polysaccharide (CP) when it was forming biofilms under any of the experimental conditions tested. Furthermore, SAL diminished aconitase activity and stimulated the lactic fermentation pathway in bacteria forming biofilms. The polysaccharide composition of S. aureus biofilms was examined and FTIR spectroscopic analysis revealed a clear impact of SAL in a codY-dependent manner. Moreover, SAL negatively affected codY transcription in mature biofilms thus relieving the CodY repression of the ica operon. Treatment of mice with SAL induced a significant increase of S aureus colonization. It is suggested that the elevated PIA expression induced by SAL might be responsible for the high nasal colonization observed in mice. SAL-induced biofilms may contribute to S. aureus infection persistence in vegetarian individuals as well as in patients that frequently consume aspirin. [ABSTRACT FROM AUTHOR]

CodY is a conserved broad-acting transcription factor that regulates the expression of genes related to amino acid metabolism and virulence in Gram-positive bacteria. Here, we performed the first in vivo determination of CodY target genes using a novel CodY monoclonal antibody in methicillin-resistant Staphylococcus aureus (MRSA) USA300. Our results showed (i) the same 135 CodY promoter binding sites regulating the 165 target genes identified in two closely related virulent S. aureus USA300 TCH1516 and LAC strains; (ii) the differential binding intensity for the same target genes under the same conditions was due to sequence differences in the same CodY-binding site in the two strains; (iii) a CodY regulon comprising 72 target genes that are differentially regulated relative to a CodY deletion strain, representing genes that are mainly involved in amino acid transport and metabolism, inorganic ion transport and metabolism, transcription and translation, and virulence, all based on transcriptomic data; and (iv) CodY systematically regulated central metabolic flux to generate branched-chain amino acids (BCAAs) by mapping the CodY regulon onto a genome-scale metabolic model of S. aureus . Our study performed the first system-level analysis of CodY in two closely related USA300 TCH1516 and LAC strains, revealing new insights into the similarities and differences of CodY regulatory roles between the closely related strains. IMPORTANCE With the increasing availability of whole-genome sequences for many strains within the same pathogenic species, a comparative analysis of key regulators is needed to understand how the different strains uniquely coordinate metabolism and expression of virulence. To successfully infect the human host, Staphylococcus aureus USA300 relies on the transcription factor CodY to reorganize metabolism and express virulence factors. While CodY is a known key transcription factor, its target genes are not characterized on a genome-wide basis. We performed a comparative analysis to describe the transcriptional regulation of CodY between two dominant USA300 strains. This study motivates the characterization of common pathogenic strains and an evaluation of the possibility of developing specialized treatments for major strains circulating in the population., Competing Interests: The authors declare no conflict of interest.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are widely recognized as bacterial adaptive immune systems against invading viruses and bacteriophages. The oral pathogen Streptococcus mutans encodes two CRISPR-Cas loci (CRISPR1-Cas and CRISPR2-Cas), and their expression under environmental conditions is still under investigation. In this study, we investigated the transcriptional regulation of cas operons by CcpA and CodY, two global regulators that contribute to carbohydrate and (p)ppGpp metabolism. The possible promoter regions for cas operons and the binding sites for CcpA and CodY in the promoter regions of both CRISPR-Cas loci were predicted using computational algorithms. We found that CcpA could directly bind to the upstream region of both cas operons, and detected an allosteric interaction of CodY within the same region. The binding sequences of the two regulators were identified through footprinting analysis. Our results showed that the promoter activity of CRISPR1-Cas was enhanced under fructose-rich conditions, while deletion of the ccpA gene led to reduced activity of the CRISPR2-Cas promoter under the same conditions. Additionally, deletion of the CRISPR systems resulted in a significant decrease in fructose uptake ability compared to the parental strain. Interestingly, the accumulation of guanosine tetraphosphate (ppGpp) was reduced in the presence of mupirocin, which induces a stringent response, in the CRISPR1-Cas-deleted (ΔCR1 cas ) and both CRISPR-Cas-deleted (ΔCRD cas ) mutant strains. Furthermore, the promoter activity of both CRISPRs was enhanced in response to oxidative or membrane stress, while the CRISPR1 promoter activity was reduced under low-pH conditions. Collectively, our findings demonstrate that the transcription of the CRISPR-Cas system is directly regulated by the binding of CcpA and CodY. These regulatory actions play a crucial role in modulating glycolytic processes and exerting effective CRISPR-mediated immunity in response to nutrient availability and environmental cues. IMPORTANCE An effective immune system has evolved not only in eukaryotic organisms but also in microorganisms, enabling them to rapidly detect and neutralize foreign invaders in the environment. Specifically, the CRISPR-Cas system in bacterial cells is established through a complex and sophisticated regulatory mechanism involving specific factors. In this study, we demonstrate that the expression of two CRISPR systems in S. mutans can be controlled by two global regulators, CcpA and CodY, which play critical roles in carbohydrate metabolism and amino acid biosynthesis. Importantly, our results show that the expression of the CRISPR-Cas system in S. mutans influences (p)ppGpp production during the stringent response, which is a gene expression regulatory response that aids in environmental stress adaptation. This transcriptional regulation by these regulators enables a CRISPR-mediated immune response in a host environment with limited availability of carbon sources or amino acids, while ensuring efficient carbon flux and energy expenditure to support multiple metabolic processes., Competing Interests: The authors declare no conflict of interest.

Bacillus cereus is a food contaminant with greatly varying enteropathogenic potential. Almost all known strains harbor the genes for at least one of the three enterotoxins Nhe, Hbl and CytK. While some strains show no cytotoxicity, others have caused outbreaks, in rare cases even with lethal outcome. The reason for these differences in cytotoxicity is unknown. To gain insight into the origin of enterotoxin expression heterogeneity in different strains, the architecture and role of 5’ intergenic regions (5’IGRs) upstream of the nhe and hbl operons was investigated. In silico comparison of 142 strains of all seven phylogenetic groups of B. cereus sensu lato proved the presence of long 5’IGRs upstream of the nheABC and hblCDAB operons, which harbor recognition sites for several transcriptional regulators, including the virulence regulator PlcR, redox regulators ResD and Fnr, the nutrient-sensitive regulator CodY as well as the master regulator for biofilm formation SinR. By determining transcription start sites, unusually long 5’ untranslated regions (5’UTRs) upstream of the nhe and hbl start codons were identified, which are not present upstream of cytK-1 and cytK-2. Promoter fusions lacking various parts of the nhe and hbl 5’UTR in B. cereus INRA C3 showed that the entire 331 bp 5’UTR of nhe is necessary for full promoter activity, while the presence of the complete 606 bp hbl 5’UTR lowers promoter activity. Repression was caused by a 268 bp sequence directly upstream of the hbl transcription start. Luciferase activity of reporter strains containing nhe and hbl 5’IGR lux fusions provided evidence that toxin gene transcription is upregulated by the depletion of free amino acids. Electrophoretic mobility shift assays showed that the branched-chain amino acid sensing regulator CodY binds to both nhe and hbl 5’UTR downstream of the promoter, potentially acting as a nutrient-responsive roadblock repressor of toxin gene transcription. PlcR binding sites are highly conserved among all B. cereus sensu lato strains, indicating that this regulator does not significantly contribute to the heterogeneity in virulence potentials. The CodY recognition sites are far less conserved, perhaps conferring varying strengths of CodY binding, which might modulate toxin synthesis in a strain-specific manner.

In Gram-positive bacteria, cell-cell communication mainly relies on cytoplasmic sensors of the RNPP family. Activity of these regulators depends on their binding to secreted signaling peptides that are imported into the cell. These quorum sensing regulators control important biological functions in bacteria of the Bacillus cereus group, such as virulence and necrotrophism. The RNPP quorum sensor PlcR, in complex with its cognate signaling peptide PapR, is the main regulator of virulence in B. cereus and Bacillus thuringiensis (Bt). Recent reports have shown that the global stationary phase regulator CodY, involved in adaptation to nutritional limitation, is required for the expression of virulence genes belonging to the PlcR regulon. However, the mechanism underlying this regulation was not described. Using genetics and proteomics approaches, we showed that CodY regulates the expression of the virulence genes through the import of PapR. We report that CodY positively controls the production of the proteins that compose the oligopeptide permease OppABCDF, and of several other Opp-like proteins. It was previously shown that the pore components of this oligopeptide permease, OppBCDF, were required for the import of PapR. However, the role of OppA, the substrate-binding protein (SBP), was not investigated. Here, we demonstrated that OppA is not the only SBP involved in the recognition of PapR, and that several other OppA-like proteins can allow the import of this peptide. Altogether, these data complete our model of quorum sensing during the lifecycle of Bt and indicate that RNPPs integrate environmental conditions, as well as cell density, to coordinate the behavior of the bacteria throughout growth.

Background: Host-specific environmental factors induce changes in Bacillus anthracis gene transcription during infection. A global transcription regulator, CodY, plays a pivotal role in regulating central metabolism, biosynthesis, and virulence in B. anthracis. In this study, we utilized RNA-sequencing to assess changes in the transcriptional patterns of CodY-regulated B. anthracis genes in response to three conditions of environmental starvation: iron, CO2, orglucose deprivation. In addition, we performed chromatin immunoprecipitation on newly identified CodY-mediated genes. Results: Environmental deprivation induced transcriptional changes in CodY-regulated genes in both wild-type and codY null strains, and both CodY-specific and environment-specific patterns were observed. In the iron-depleted condition, overexpression of iron homeostasis genes was observed independent of codY deletion; however, transcription of siderophore and amino acid biosynthesis genes was CodY dependent. Although CodY has a significant regulatory role in central metabolism and the carbon overflow pathway, metabolism-associated genes exhibited CodY-independent expression patterns under glucose starvation. Genes that were differentially expressed in response to CO2 availability showed CodY-dependent regulation, though their maximal expression did require a supply of CO2/bicarbonate. Conclusions: We speculate that CodY regulates the expression of environmental-responsive genes in a hierarchical manner and is likely associated with other transcription regulators that are specific for a particular environmental change. [ABSTRACT FROM AUTHOR]

The impact of gene mutations on the growth of the cells can be studied using pure cultures. However, the importance of certain proteins and pathways can be also examined via co-culturing wild type and its mutant derivative. Here, the relative fitness of a mutant strain that lacks the global nitrogen regulator, CodY, was examined in Bacillus cereus, a food poisoning Gram-positive bacterium. Fitness measurements revealed that the ΔcodY strain was outcompeted when cocultured with the wild-type ATCC 14579 under various rich laboratory medium, and also when inoculated in certain beverages. In nutrient-poor minimal medium, the ΔcodY mutant had comparable fitness to the wild-type strain. Interestingly, the relative fitness of the ΔcodY strain was antagonistic when it was cultivated in apple or orange juices due to unknown properties of these beverages, highlighting the importance of chemical composition of the test medium during the bacterial fitness measurements. [ABSTRACT FROM AUTHOR]

CodY, a global transcriptional regulator, primarily functions as a nutrient and energy sensor. It is activated by metabolic effectors like BCAA and GTP. In low G + C Gram positive bacteria, it facilitates coupling of changes in the cellular metabolite pool with those required in the transcriptome of the cell. This pleiotropic regulator controls the expression of a vast number of genes as the cell transits from exponential to the stationary phase. Earlier studies have shown that CodY is required for the virulence of Bacillus anthracis. We sought to investigate the effect of its overexpression on the physiology of B. anthracis . In our study, we found that cellular CodY levels were unchanged during this phase-transition. Expression of endogenous CodY remained the same in different nutrient limiting conditions. Immunoblotting studies revealed CodY presence in the whole spore lysate of B. anthracis indicating it to be a component of the spore proteome. We could also detect CodY in the secretome of B. anthracis . Further, CodY was overexpressed in B. anthracis Sterne strain and this led to a 100-fold decrease in the sporulation titer and a 2.5-fold decrease in the in vitro attachment ability of the bacteria. We also observed a decrease in the pellicle formation by CodY overexpressed strain when compared to wildtype bacilli. The CodY overexpressed strain showed chaining phenotype during growth in liquid media and pellicle. [ABSTRACT FROM AUTHOR]