Characterization of LrgAB as a stationary phase-specific pyruvate uptake system in Streptococcus mutans

Abstract Background Our recent ‘-omics’ comparisons of Streptococcus mutans wild-type and lrgAB-mutant revealed that this organism undergoes dynamic cellular changes in the face of multiple exogenous stresses, consequently affecting its comprehensive virulence traits. In this current study, we further demonstrate that LrgAB functions as a S. mutans pyruvate uptake system. Results S. mutans excretes pyruvate during growth as an overflow metabolite, and appears to uptake this excreted pyruvate via LrgAB once the primary carbon source is exhausted. This utilization of excreted pyruvate was tightly regulated by glucose levels and stationary growth phase lrgAB induction. The degree of lrgAB induction was reduced by high extracellular levels of pyruvate, suggesting that lrgAB induction is subject to negative feedback regulation, likely through the LytST TCS, which is required for expression of lrgAB. Stationary phase lrgAB induction was efficiently inhibited by low concentrations of 3FP, a toxic pyruvate analogue, without affecting cell growth, suggesting that accumulated pyruvate is sensed either directly or indirectly by LytS, subsequently triggering lrgAB expression. S. mutans growth was inhibited by high concentrations of 3FP, implying that pyruvate uptake is necessary for S. mutans exponential phase growth and occurs in a Lrg-independent manner. Finally, we found that stationary phase lrgAB induction is modulated by hydrogen peroxide (H2O2) and by co-cultivation with H2O2-producing S. gordonii. Conclusions Pyruvate may provide S. mutans with an alternative carbon source under limited growth conditions, as well as serving as a buffer against exogenous oxidative stress. Given the hypothesized role of LrgAB in cell death and lysis, these data also provide an important basis for how these processes are functionally and mechanically connected to key metabolic pathways such as pyruvate metabolism.

Download Full-text

Functional characterization of a gibberellin F-box protein, PslSLY1, during plum fruit development

Journal of Experimental Botany ◽

10.1093/jxb/eraa438 ◽

2020 ◽

Author(s):

Islam El-Sharkawy ◽

Ahmed Ismail ◽

Ahmed Darwish ◽

Walid El Kayal ◽

Jayasankar Subramanian ◽

...

Keyword(s):

Fruit Development ◽

Stem Elongation ◽

Functional Characterization ◽

Feedback Regulation ◽

Transcript Abundance ◽

Independent Manner ◽

Negative Feedback Regulation ◽

Diverse Range ◽

Ga Signaling ◽

F Box Protein

Abstract Fruit development is orchestrated by a complex network of interactions between hormone signaling pathways. The phytohormone gibberellin (GA) is known to regulate a diverse range of developmental processes; however, the mechanisms of GA action in perennial fruit species are yet to be elucidated. In the current study, a GA signaling gene PslSLY1, encoding a putative F-box protein that belongs to the SLY1 (SLEEPY1)/GID2 (gibberellin-insensitive dwarf2) gene family, was isolated from Japanese plum (Prunus salicina). PslSLY1 transcript abundance declined as fruit development progressed, along with potential negative feedback regulation of PslSLY1 by GA. Subcellular localization and protein–protein interaction assays suggested that PslSLY1 functions as an active GA signaling component that interacts with the ASK1 (Arabidopsis SKP1) subunit of an SCF–ubiquitin ligase complex and with PslDELLA repressors, in a GA-independent manner. By using a domain omission strategy, we illustrated that the F-box and C-terminal domains of PslSLY1 are essential for its interactions with the downstream GA signaling components. PslSLY1 overexpression in wild-type and Arabidopsis sly1.10 mutant backgrounds resulted in a dramatic enhancement in overall plant growth, presumably due to triggered GA signaling. This includes germination characteristics, stem elongation, flower structure, and fertility. Overall, our findings shed new light on the GA strategy and signaling network in commercially important perennial crops.

Download Full-text

The Pta-AckA Pathway Regulates LrgAB-Mediated Pyruvate Uptake in Streptococcus mutans

Microorganisms ◽

10.3390/microorganisms8060846 ◽

2020 ◽

Vol 8 (6) ◽

pp. 846 ◽

Cited By ~ 1

Author(s):

Sang-Joon Ahn ◽

Shailja Desai ◽

Min Lin ◽

Kelly C. Rice

Keyword(s):

Stationary Phase ◽

Streptococcus Mutans ◽

Regulatory Mechanisms ◽

Uptake System ◽

Anaerobic Conditions ◽

Acetyl Coa ◽

Central Node ◽

Biochemical Assays ◽

Alternative Carbon Source ◽

Low Glucose

Pyruvate forms the central node of carbon metabolism and promotes growth as an alternative carbon source during starvation. We recently revealed that LrgAB functions as a stationary phase pyruvate uptake system in Streptococcus mutans, the primary causative agent of human dental caries, but its underlying regulatory mechanisms are still not clearly understood. This study was aimed at further characterizing the regulation of LrgAB from a metabolomic perspective. We utilized a series of GFP quantification, growth kinetics, and biochemical assays. We disclosed that LrgAB is critical for pyruvate uptake especially during growth under low-glucose stress. Inactivation of the Pta-Ack pathway, responsible for the conversion of acetyl-CoA to acetate, completely inhibits stationary phase lrgAB induction and pyruvate uptake, and renders cells insensitive to external pyruvate as a signal. Inactivation of Pfl, responsible for the conversion of pyruvate to acetyl-CoA under anaerobic conditions, also affected stationary phase pyruvate uptake. This study explores the metabolic components of pyruvate uptake regulation through LrgAB, and highlights its potential as a metabolic stimulator, contributing to the resuscitation and survival of S. mutans cells during nutritional stress.

Download Full-text

5-methyl Furfural Reduces the Production of Malodors by Inhibiting Sodium l-lactate Fermentation of Staphylococcus epidermidis: Implication for Deodorants Targeting the Fermenting Skin Microbiome

Microorganisms ◽

10.3390/microorganisms7080239 ◽

2019 ◽

Vol 7 (8) ◽

pp. 239 ◽

Cited By ~ 3

Author(s):

Manish Kumar ◽

Binderiya Myagmardoloonjin ◽

Sunita Keshari ◽

Indira Putri Negari ◽

Chun-Ming Huang

Keyword(s):

Carbon Source ◽

Human Skin ◽

Negative Feedback ◽

Staphylococcus Epidermidis ◽

Fermentation Process ◽

Feedback Regulation ◽

Acetolactate Synthase ◽

Skin Microbiome ◽

Negative Feedback Regulation ◽

Lactate Fermentation

Staphylococcus epidermidis (S. epidermidis) is a common bacterial colonizer on the surface of human skin. Lactate is a natural constituent of skin. Here, we reveal that S. epidermidis used sodium l-lactate as a carbon source to undergo fermentation and yield malodors detected by gas colorimetric tubes. Several furan compounds such as furfural originating from the fermentation metabolites play a role in the negative feedback regulation of the fermentation process. The 5-methyl furfural (5MF), a furfural analog, was selected as an inhibitor of sodium l-lactate fermentation of S. epidermidis via inhibition of acetolactate synthase (ALS). S. epidermidis treated with 5MF lost its ability to produce malodors, demonstrating the feasibility of using 5MF as an ingredient in deodorants targeting malodor-causing bacteria in the skin microbiome.

Download Full-text

Genetic dissection of Ragulator structure and function in amino acid-dependent regulation of mTORC1

The Journal of Biochemistry ◽

10.1093/jb/mvaa076 ◽

2020 ◽

Vol 168 (6) ◽

pp. 621-632

Author(s):

Shigeyuki Nada ◽

Masato Okada

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Feedback Regulation ◽

Genetic Dissection ◽

Independent Manner ◽

Negative Feedback Regulation ◽

Rag Gtpases ◽

And Function ◽

Mtorc1 Activation ◽

Membrane Scaffold

Abstract Ragulator is a heteropentameric protein complex consisting of two roadblock heterodimers wrapped by the membrane anchor p18/Lamtor1. The Ragulator complex functions as a lysosomal membrane scaffold for Rag GTPases to recruit and activate mechanistic target of rapamycin complex 1 (mTORC1). However, the roles of Ragulator structure in the regulation of mTORC1 function remain elusive. In this study, we disrupted Ragulator structure by directly anchoring RagC to lysosomes and monitored the effect on amino acid-dependent mTORC1 activation. Expression of lysosome-anchored RagC in p18-deficient cells resulted in constitutive lysosomal localization and amino acid-independent activation of mTORC1. Co-expression of Ragulator in this system restored the amino acid dependency of mTORC1 activation. Furthermore, ablation of Gator1, a suppressor of Rag GTPases, induced amino acid-independent activation of mTORC1 even in the presence of Ragulator. These results demonstrate that Ragulator structure is essential for amino acid-dependent regulation of Rag GTPases via Gator1. In addition, our genetic analyses revealed new roles of amino acids in the regulation of mTORC1 as follows: amino acids could activate a fraction of mTORC1 in a Rheb-independent manner, and could also drive negative-feedback regulation of mTORC1 signalling via protein phosphatases. These intriguing findings contribute to our overall understanding of the regulatory mechanisms of mTORC1 signalling.

Download Full-text

Morphological Characterization of Mycobacteriophage R1

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051281 ◽

1974 ◽

Vol 32 ◽

pp. 254-255

Author(s):

B. L. Soloff ◽

T. A. Rado

Keyword(s):

Carbon Source ◽

Stationary Phase ◽

Growth Phase ◽

Minimal Medium ◽

Morphological Characterization ◽

Logarithmic Growth Phase ◽

Complete Lysis ◽

Lysogenic Culture ◽

Logarithmic Growth

Mycobacteriophage R1 was originally isolated from a lysogenic culture of M. butyricum. The virus was propagated on a leucine-requiring derivative of M. smegmatis, 607 leu−, isolated by nitrosoguanidine mutagenesis of typestrain ATCC 607. Growth was accomplished in a minimal medium containing glycerol and glucose as carbon source and enriched by the addition of 80 μg/ ml L-leucine. Bacteria in early logarithmic growth phase were infected with virus at a multiplicity of 5, and incubated with aeration for 8 hours. The partially lysed suspension was diluted 1:10 in growth medium and incubated for a further 8 hours. This permitted stationary phase cells to re-enter logarithmic growth and resulted in complete lysis of the culture.

Download Full-text