RocA Regulates Phosphatase Activity of Virulence Sensor CovS of Group A Streptococcus in Growth Phase- and pH-Dependent Manners

ABSTRACT The control of the virulence response regulator and sensor (CovR-CovS) two-component regulatory system in group A Streptococcus (GAS) strains regulates more than 15% of gene expression and has critical roles in invasive GAS infection. The membrane-embedded CovS has kinase and phosphatase activities, and both are required for modulating the phosphorylation level of CovR. Regulator of Cov (RocA) is a positive regulator of covR and also been shown to be a pseudokinase that interacts with CovS to enhance the phosphorylation level of CovR; however, how RocA modulates the activity of CovS has not been determined conclusively. Although the phosphorylation level of CovR was decreased in the rocA mutant in the exponential phase, the present study shows that phosphorylated CovR in the rocA mutant increased to levels similar to those in the wild-type strain in the stationary phase of growth. In addition, acidic stress, which is generally present in the stationary phase, enhanced the phosphorylation level of CovR in the rocA mutant. The phosphorylation levels of CovR in the CovS phosphatase-inactivated mutant and its rocA mutant were similar under acidic stress and Mg2+ (the signal that inhibits CovS phosphatase activity) treatments, suggesting that the phosphatase activity, but not the kinase activity, of CovS is required for RocA to modulate CovR phosphorylation. The phosphorylation level of CovR is crucial for GAS strains to regulate virulence factor expression; therefore, the growth phase- and pH-dependent RocA activity would contribute significantly to GAS pathogenesis. IMPORTANCE The emergence of invasive group A streptococcal infections has been reported worldwide. Clinical isolates that have spontaneous mutations or a truncated allele of the rocA gene (e.g., emm3-type isolates) are considered to be more virulent than isolates with the intact rocA gene (e.g., emm1-type isolates). RocA is a positive regulator of covR and has been shown to enhance the phosphorylation level of intracellular CovR regulator through the functional CovS protein. CovS is the membrane-embedded sensor and modulates the phosphorylation level of CovR by its kinase and phosphatase activities. The present study shows that the enhancement of CovR phosphorylation is mediated via the repression of CovS’s phosphatase activity by RocA. In addition, we found that RocA acts dominantly on modulating CovR phosphorylation under neutral pH conditions and in the exponential phase of growth. The phosphorylation level of CovR is crucial for group A Streptococcus species to regulate virulence factor expression and is highly related to bacterial invasiveness; therefore, growth phase- and pH-dependent RocA activity and the sequence polymorphisms of rocA gene would contribute significantly to bacterial phenotype variations and pathogenesis.

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Role of mRNA Stability in Growth Phase Regulation of Gene Expression in the Group A Streptococcus

Journal of Bacteriology ◽

10.1128/jb.01658-06 ◽

2006 ◽

Vol 189 (5) ◽

pp. 1866-1873 ◽

Cited By ~ 52

Author(s):

Timothy C. Barnett ◽

Julia V. Bugrysheva ◽

June R. Scott

Keyword(s):

Gene Expression ◽

Stationary Phase ◽

Growth Phase ◽

Group A Streptococcus ◽

Exponential Phase ◽

Northern Hybridization ◽

Late Exponential Phase ◽

Group A ◽

Phase Regulation ◽

The Stability

ABSTRACT The impressive disease spectrum of Streptococcus pyogenes (the group A streptococcus [GAS]) is believed to be determined by its ability to modify gene expression in response to environmental stimuli. Virulence gene expression is controlled tightly by several different transcriptional regulators in this organism. In addition, expression of most, if not all, GAS genes is determined by a global mechanism dependent on growth phase. To begin an analysis of growth-phase regulation, we compared the transcriptome 2 h into stationary phase to that in late exponential phase of a serotype M3 GAS strain. We identified the arc transcript as more abundant in stationary phase in addition to the sag and sda transcripts that had been previously identified. We found that in stationary phase, the stability of sagA, sda, and arcT transcripts increased dramatically. We found that polynucleotide phosphorylase (PNPase [encoded by pnpA]) is rate limiting for decay of sagA and sda transcripts in late exponential phase, since the stability of these mRNAs was greater in a pnpA mutant, while stability of control mRNAs was unaffected by this mutation. Complementation restored the wild-type decay rate. Furthermore, in a pnpA mutant, the sagA mRNA appeared to be full length, as determined by Northern hybridization. It seems likely that mRNAs abundant in stationary phase are insensitive to the normal decay enzyme(s) and instead require PNPase for this process. It is possible that PNPase activity is limited in stationary phase, allowing persistence of these important virulence factor transcripts at this phase of growth.

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The Nature of Antibacterial Adaptive Immune Responses against Staphylococcus aureus Is Dependent on the Growth Phase and Extracellular Peptidoglycan

Infection and Immunity ◽

10.1128/iai.00733-19 ◽

2019 ◽

Vol 88 (1) ◽

Author(s):

Payal P. Balraadjsing ◽

Lisbeth D. Lund ◽

Yuri Souwer ◽

Sebastian A. J. Zaat ◽

Hanne Frøkiær ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

T Cell ◽

Stationary Phase ◽

Growth Phase ◽

Cell Response ◽

Th17 Cell ◽

Exponential Phase ◽

Th1 Cell ◽

Content Type

ABSTRACT Staphylococcus aureus has evolved different strategies to evade the immune response, which play an important role in its pathogenesis. The bacteria express and shed various cell wall components and toxins during different stages of growth that may affect the protective T cell responses to extracellular and intracellular S. aureus. However, if and how the dendritic cell (DC)-mediated T cell response against S. aureus changes during growth of the bacterium remain elusive. In this study, we show that exponential-phase (EP) S. aureus bacteria were endocytosed very efficiently by human DCs, and these DCs strongly promoted production of the T cell polarizing factor interleukin-12 (IL-12). In contrast, stationary-phase (SP) S. aureus bacteria were endocytosed less efficiently by DCs, and these DCs produced small amounts of IL-12. The high level of IL-12 production induced by EP S. aureus led to the development of a T helper 1 (Th1) cell response, which was inhibited after neutralization of IL-12. Furthermore, preincubation with the staphylococcal cell wall component peptidoglycan (PGN), characteristically shed during the exponential growth phase, modulated the DC response to EP S. aureus. PGN preincubation appeared to inhibit IL-12p35 expression, leading to downregulation of IL-12 and an increase of IL-23 production by DCs, enhancing Th17 cell development. Taken together, our data indicate that exponential-phase S. aureus bacteria induce a stronger IL-12-dependent Th1 cell response than stationary-phase S. aureus and that this Th1 cell response shifted toward a Th17 cell response in the presence of PGN.

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Genetic Evidence that Legionella pneumophila RpoS Modulates Expression of the Transmission Phenotype in Both the Exponential Phase and the Stationary Phase

Infection and Immunity ◽

10.1128/iai.72.5.2468-2476.2004 ◽

2004 ◽

Vol 72 (5) ◽

pp. 2468-2476 ◽

Cited By ~ 57

Author(s):

Michael A. Bachman ◽

Michele S. Swanson

Keyword(s):

Life Cycle ◽

Rna Polymerase ◽

Stationary Phase ◽

Growth Phase ◽

Legionella Pneumophila ◽

Opportunistic Pathogen ◽

Sigma Factors ◽

Exponential Phase ◽

Posttranscriptional Control ◽

Multiple Copies

ABSTRACT The opportunistic pathogen Legionella pneumophila alternates between two states: replication within phagocytes and transmission between host amoebae or macrophages. In broth cultures that model this life cycle, during the replication period, CsrA inhibits expression of transmission traits. When nutrients become limiting, the alarmone (p)ppGpp accumulates and the sigma factors RpoS and FliA and the positive activators LetA/S and LetE promote differentiation to the transmissible form. Here we show that when cells enter the postexponential growth phase, RpoS increases expression of the transmission genes fliA, flaA, and mip, factors L. pneumophila needs to establish a new replication niche. In contrast, in exponential (E)-phase cells whose (p)ppGpp levels are low, rpoS inhibits expression of transmission traits, on the basis of three separate observations. First, rpoS RNA levels peak in the E phase, suggestive of a role for RpoS during replication. Second, in multiple copies, rpoS decreases the amounts of csrA, letE, fliA, and flaA transcripts and inhibits the transmission traits of motility, infectivity, and cytotoxicity. Third, rpoS blocks expression of cytotoxicity and motility by E-phase bacteria that have been induced to express the LetA activator ectopically. The data are discussed in the context of a model in which the alarmone (p)ppGpp enables RpoS to outcompete other sigma factors for binding to RNA polymerase to promote transcription of transmission genes, while LetA/S acts in parallel to relieve CsrA posttranscriptional repression of the transmission regulon. By coupling transcriptional and posttranscriptional control pathways, intracellular L. pneumophila could respond to stress by rapidly differentiating to a transmissible form.

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RivR Is a Negative Regulator of Virulence Factor Expression in Group A Streptococcus

Infection and Immunity ◽

10.1128/iai.00703-12 ◽

2012 ◽

Vol 81 (1) ◽

pp. 364-372 ◽

Cited By ~ 19

Author(s):

Jeanette Treviño ◽

Zhuyun Liu ◽

Tram N. Cao ◽

Esmeralda Ramirez-Peña ◽

Paul Sumby

Keyword(s):

Mutant Strain ◽

Virulence Factor ◽

Regulatory Networks ◽

Group A Streptococcus ◽

Human Keratinocytes ◽

Negative Regulator ◽

Upstream Region ◽

Content Type ◽

Factor Expression ◽

Group A

The bacterial pathogen group AStreptococcus(GAS) causes human diseases ranging from self-limiting pharyngitis (also known as strep throat) to severely invasive necrotizing fasciitis (also known as the flesh-eating syndrome). To control virulence factor expression, GAS utilizes both protein- and RNA-based mechanisms of regulation. Here we report that the transcription factor RivR (RofA-like protein IV) negatively regulates the abundance of mRNAs encoding the hyaluronic acid capsule biosynthesis proteins (hasABC; ∼7-fold) and the protein G-related α2-macroglobulin-binding protein (grab; ∼29-fold). Our data differ significantly from those of a previous study of the RivR regulon. Given thatgrabandhasABCare also negatively regulated by the two-component system CovR/S (controlofvirulence), we tested whether RivR functions through CovR/S. A comparison ofrivandcovsingle and double mutant strains showed that RivR requires CovR activity forgrabandhasABCregulation. Analysis of the upstream region ofrivRidentified a novel promoter the deletion of which reducedrivRmRNA abundance by 70%. ArivRmutant strain had a reduced ability to adhere to human keratinocytes relative to that of the parental and complemented strains, a phenotype that was abolished upon GAS pretreatment with hyaluronidase, highlighting the importance of capsule regulation by RivR during colonization. TherivRmutant strain was also attenuated for virulence in a murine model of bacteremia infection. Thus, we identify RivR as an important regulator of GAS virulence and provide new insight into the regulatory networks controlling virulence factor production in this pathogen.

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Transcriptome analysis revealed growth phase-associated changes of a centenarian-originated probiotic Bifidobacterium animalis subsp. lactis A6

10.21203/rs.3.rs-1036916/v1 ◽

2021 ◽

Author(s):

Hui Wang ◽

Jieran An ◽

Chengfei Fan ◽

Zhengyuan Zhai ◽

Hongxing Zhang ◽

...

Keyword(s):

Carbon Source ◽

Stationary Phase ◽

Gene Cluster ◽

Growth Phase ◽

Differential Expression Analysis ◽

Acid Tolerance ◽

Glucose Consumption ◽

Exponential Phase ◽

Putative Gene ◽

Bifidobacterium Animalis

Abstract Background The physiology and application characteristics of probiotics are closely associated with the growth phase. Bifidobacterium animalis subsp. lactis A6 is a promising probiotic strain isolated from the feces of a healthy centenarian in China. In this study, RNA-seq was carried out to investigate the metabolic mechanism between the exponential and the stationary phase in B. lactis A6. Results Differential expression analysis showed that a total of 810 genes were significantly changed in the stationary phase compared to the exponential phase, which consisted of 392 up-regulated and 418 down-regulated genes. The results showed that the transport and metabolism of cellobiose, xylooligosaccharides and raffinose were enhanced at the stationary phase, which expanded carbon source utilizing profile to confront with glucose consumption. Meanwhile, genes involved in NH3 production were up-regulated at the stationary phase to enhance acid tolerance during fermentation. In addition, peptidoglycan biosynthesis was significantly repressed, which is comparable with the decreased growth rate during the stationary phase. Remarkably, a putative gene cluster encoding Tad pili was up-regulated 6.5~12.1-fold, which is consistent with the significantly increased adhesion rate to mucin from 2.38–4.90% during the transition from the exponential phase to the stationary phase. Conclusions This study reported growth phase-associated changes of B. lactis A6 during fermentation, including expanded carbon source utilizing profile, enhanced acid tolerance, and up-regulated Tad pili gene cluster responsible for bacterial adhesion in the stationary phase. These findings provide a novel insight into the growth phase associated characteristics in B. lactis A6 and provide valuable information for further application in the food industry.

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Growth-phase dependence of susceptibility to antimicrobial peptides in Staphylococcus aureus

Microbiology ◽

10.1099/mic.0.044727-0 ◽

2011 ◽

Vol 157 (6) ◽

pp. 1786-1797 ◽

Cited By ~ 30

Author(s):

Miki Matsuo ◽

Yuichi Oogai ◽

Fuminori Kato ◽

Motoyuki Sugai ◽

Hitoshi Komatsuzawa

Keyword(s):

Staphylococcus Aureus ◽

Cell Surface ◽

Antimicrobial Peptides ◽

Stationary Phase ◽

Surface Charge ◽

Growth Phase ◽

Exponential Phase ◽

Dependent Manner ◽

Phase Dependence ◽

Cell Surface Charge

Bacterial cell surface charge is responsible for susceptibility to cationic antimicrobial peptides. Previously, Staphylococcus aureus dlt and mprF were identified as factors conferring a positive charge upon cell surfaces. In this study, we investigated the regulation of cell surface charge during growth. Using a group of S. aureus MW2 mutants, which are gene-inactivated in 15 types of two-component systems (TCSs), we tested dltC and mprF expression and found that two TCSs, aps and agr, were associated with dltC and mprF expression in a growth phase-dependent manner. The first of these, aps, which had already been identified as a sensor of antimicrobial peptides and a positive regulator of dlt and mprF expression, was expressed strongly in the exponential phase, while its expression was significantly suppressed by agr in the stationary phase, resulting in higher expression of dltC and mprF in the exponential phase and lower expression in the stationary phase. Since both types of expression affected the cell surface charge, the susceptibility to antimicrobial peptides and cationic antibiotics was changed during growth. Furthermore, we found that the ability to sense antimicrobial peptides only functioned in the exponential phase. These results suggest that cell surface charge is tightly regulated during growth in S. aureus.

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Group A streptococcal growth phase-associated virulence factor regulation by a novel operon (Fas) with homologies to two-component-type regulators requires a small RNA molecule

Molecular Microbiology ◽

10.1046/j.1365-2958.2001.02226.x ◽

2001 ◽

Vol 39 (2) ◽

pp. 392-406 ◽

Cited By ~ 137

Author(s):

Bernd Kreikemeyer ◽

Michael D. P. Boyle ◽

Bettina A. (Leonard) Buttaro ◽

Markus Heinemann ◽

Andreas Podbielski

Keyword(s):

Virulence Factor ◽

Growth Phase ◽

Small Rna ◽

Component Type ◽

Two Component ◽

Group A

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Taxon- and Growth Phase-Specific Antioxidant Production by Chlorophyte, Bacillariophyte, and Haptophyte Strains Isolated From Tropical Waters

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2020.581628 ◽

2020 ◽

Vol 8 ◽

Author(s):

Norazira Abdu Rahman ◽

Tomoyo Katayama ◽

Mohd Effendy Abd Wahid ◽

Nor Azman Kasan ◽

Helena Khatoon ◽

...

Keyword(s):

Fatty Acids ◽

Stationary Phase ◽

Antioxidant Capacity ◽

Growth Phase ◽

Stationary Phases ◽

Exponential Phase ◽

Antioxidant Compounds ◽

Antioxidant Compound ◽

Scavenging Capacity ◽

Β Carotene

Antioxidants found in microalgae play an essential role in both animals and humans, against various diseases and aging processes by protecting cells from oxidative damage. In this study, 26 indigenous tropical marine microalgae were screened. Out of the 26 screened strains, 10 were selected and were further investigated for their natural antioxidant compounds which include carotenoids, phenolics, and fatty acids collected in their exponential and stationary phases. The antioxidant capacity was also evaluated by a total of four assays, which include ABTS, DPPH, superoxide radical (O2•–) scavenging capacity, and nitric oxide (•NO–) scavenging capacity. This study revealed that the antioxidant capacity of the microalgae varied between divisions, strains, and growth phase and was also related to the content of antioxidant compounds present in the cells. Carotenoids and phenolics were found to be the major contributors to the antioxidant capacity, followed by polyunsaturated fatty acids linoleic acid (LA), eicosapentaenoic acid (EPA), arachidonic acid (ARA), and docosahexaenoic acid (DHA) compared to other fatty acids. The antioxidant capacity of the selected bacillariophytes and haptophytes was found to be positively correlated to phenolic (R2-value = 0.623, 0.714, and 0.786 with ABTS, DPPH, and •NO–) under exponential phase, and to carotenoid fucoxanthin and β-carotene (R2 value = 0.530, 0.581 with ABTS, and 0.710, 0.795 with O2•–) under stationary phase. Meanwhile, antioxidant capacity of chlorophyte strains was positively correlated with lutein, β-carotene and zeaxanthin under the exponential phase (R2 value = 0.615, 0.615, 0.507 with ABTS, and R2 value = 0.794, 0.659, and 0.509 with •NO–). In the stationary phase, chlorophyte strains were positively correlated with violaxanthin (0.755 with •NO–), neoxanthin (0.623 with DPPH, 0.610 with •NO–), and lutein (0.582 with •NO–). This study showed that antioxidant capacity and related antioxidant compound production of tropical microalgae strains are growth phase-dependent. The results can be used to improve the microalgal antioxidant compound production for application in pharmaceutical, nutraceutical, food, and feed industry.

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Influence of Bacterial Detachment on the Bioflotation of Malachite

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.1130.485 ◽

2015 ◽

Vol 1130 ◽

pp. 485-488

Author(s):

Ga Hee Kim ◽

So Yeon Park ◽

Kyuh Yeong Park ◽

Junh Yun Choi ◽

Si Young Q. Choi ◽

...

Keyword(s):

Fluid Flow ◽

Stationary Phase ◽

Surface Properties ◽

Growth Phase ◽

Morphological Characteristics ◽

Exponential Phase ◽

Rhodococcus Opacus ◽

Cell Detachment ◽

Flotation Process ◽

Flotation Behavior

The effects of the bacterial growth phase on the malachite flotation were investigated in a well-controlled Hallimond tube system. Rhodococcus opacus, which is one of representative hydrophobic bacteria, was employed for this study. The test results showed that the bacteria in the stationary phase exhibit two-fold greater floatability than those in mid-exponential phase. To understand the observed flotation behavior, complementary cell characterization tests (e.g., zeta potential and contact angle measurements) and cell attachment tests were conducted. Interestingly, the bacteria at both phases exhibited similar surface properties as well as almost identical amount of cells attached onto the malachite, suggesting that the growth phase dependent flotation behavior cannot be attributed to the variation of cell surface properties and the extent of cell adsorption. On the other hand, cell detachment tests revealed that the amount of cells detached from the malachite surface is greater for the mid-exponential phase than the stationary phase due to the higher fluid drag applied to the cells at the mid-exponential phase, which was explained by the differences in the size and shape of attached bacteria onto the malachite surface. Specifically, the bacteria in the mid-exponential phase had a larger size and formed loosely-packed structures like an end-to end on the malachite surface. These morphological characteristics were found to cause the bacteria of mid-exponential phase to be separated highly sensitive and easy from malachite surface due to the fluid flow. The findings from this study suggest that in the case of bioflotation using a relatively large bacteria size than the collector, it is important to consider the cell detachment by the fluid flow that occurs during a flotation process.

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Network rewiring: physiological consequences of reciprocally exchanging the physical locations and growth-phase-dependent expression patterns of the Salmonella fis and dps genes

10.1101/2020.07.08.193441 ◽

2020 ◽

Author(s):

Marina M Bogue ◽

Aalap Mogre ◽

Michael C Beckett ◽

Nicholas R Thomson ◽

Charles J Dorman

Keyword(s):

Gene Expression ◽

Protein Binding ◽

Stationary Phase ◽

Growth Phase ◽

Regulatory Network ◽

Mammalian Cells ◽

Expression Patterns ◽

Virulence Gene ◽

Exponential Phase ◽

Virulence Gene Expression

ABSTRACTThe Fis nucleoid-associated protein controls the expression of a large and diverse regulon of genes in Gram-negative bacteria. Fis production is normally maximal in bacteria during the early exponential phase of batch culture growth, becoming almost undetectable by the onset of stationary phase. We tested the effect on the Fis regulatory network in Salmonella of moving the complete fis gene from its usual location near the origin of chromosomal replication to the position normally occupied by the dps gene in the Right macrodomain of the chromosome, and vice versa, creating the strain GX. In a parallel experiment, we tested the effect of rewiring the Fis regulatory network by placing the fis open reading frame under the control of the stationary-phase-activated dps promoter at the dps genetic location within Ter, and vice versa, creating the strain OX. ChIP-seq was used to measure global Fis protein binding and gene expression patterns. Strain GX showed few changes when compared with the wild type, although we did detect increased Fis binding at Ter, accompanied by reduced binding at Ori. Strain OX displayed a more pronounced version of this distorted Fis protein-binding pattern together with numerous alterations in the expression of genes in the Fis regulon. OX, but not GX, had a reduced ability to infect cultured mammalian cells. These findings illustrate the inherent robustness of the Fis regulatory network to rewiring based on gene repositioning alone and emphasise the importance of fis expression signals in phenotypic determination.IMPORTANCEWe assessed the impacts on Salmonella physiology of reciprocally translocating the genes encoding the Fis and Dps nucleoid-associated proteins (NAPs), and of inverting their growth phase production patterns such that Fis is produced in stationary phase (like Dps) and Dps is produced in exponential phase (like Fis). Changes to peak binding of Fis were detected by ChIP-seq on the chromosome, as were widespread impacts on the transcriptome, especially when Fis production mimicked Dps. Virulence gene expression and the expression of a virulence phenotype were altered. Overall, these radical changes to NAP gene expression were well tolerated, revealing the robust and well-buffered nature of global gene regulation networks in the bacterium.

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