Role of (p)ppGpp in biofilm formation and expression of filamentous structures in Bordetella pertussis

Sugisaki, Kentaro; Hanawa, Tomoko; Yonezawa, Hideo; Osaki, Takako; Fukutomi, Toshiyuki; Kawakami, Hayato; Yamamoto, Tomoko; Kamiya, Shigeru

doi:10.1099/mic.0.066597-0

Cited by 42 publications

(54 citation statements)

References 49 publications

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“…6). In contrast, Bsp22 was significantly reduced by the depletion of (p)ppGpp, similar to previous results (19). Moreover, Bsp22 was slightly induced by glutamate limitation in both cell-free culture fluids and lysates (Fig.…”

Section: Consumption Of Glutamate In the Mediumsupporting

confidence: 69%

“…Since other genes encoding RSH proteins except for relA and spoT have not been found in B. pertussis (19), strain PMK21 did not synthesize detectable (p)ppGpp even in the presence of serine hydroxamate, a strong (p)ppGpp inducer, and it showed significantly decreased production of Bsp22 (19). (p)ppGpp is required for SPI-1 activation under low oxygen conditions and positively regulates PI-2 genes in Salmonella enterica serovar Typhimurium (36).…”

Section: Discussionmentioning

confidence: 99%

“…Cells accumulate (p)ppGpp, which is synthesized by RelA/SpoT homologues (RSH) (27), in response to nutrient starvation due to the activation of the stringent response. Previously, we demonstrated a significant reduction of the T3SS in strain PMK21, a (p)ppGpp-deficient mutant in which both the relA and spoT genes have been deleted, even though the expression of other virulence factors was not affected (19). Therefore, it was anticipated that glutamate limitation would elicit a stringent response in B. pertussis.…”

Section: Consumption Of Glutamate In the Mediummentioning

confidence: 99%

“…The cDNA fractions were used for quantitative PCR by the SYBR green method (SYBR premix Ex Taq GC; TaKaRa, Otsu, Japan) with a real-time PCR system 7500 (Applied Biosystems) using primer pairs described in Table 2. The data were normalized to the endogenous recA level and analyzed by the ⌬⌬C T method (where C T is threshold cycle) (19). The data were used to calculate relative fold changes in SSC medium compared to the level in medium with a modified glutamate concentration (SSC-0.2E).…”

Section: Quantitative Reverse Transcription-pcr (Qrt-pcr)mentioning

confidence: 99%

“…The extraction of total RNA and quantification of specific mRNAs were performed as described previously (19). Briefly, harvested bacterial cells were treated with RNAprotect bacterial reagent (Qiagen), and total RNA was extracted.…”

Section: Quantitative Reverse Transcription-pcr (Qrt-pcr)mentioning

confidence: 99%

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Glutamate Limitation, BvgAS Activation, and (p)ppGpp Regulate the Expression of the Bordetella pertussis Type 3 Secretion System

et al. 2016

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Bordetella pertussis is a bacterium that is considered to be highly adapted to humans, and it has not been isolated from the environment. As this bacterium does not utilize sugars, the abundant supply of glutamate in Stainer Scholte (SS) medium enables B. pertussis to grow efficiently in liquid culture in vitro, and as such, SS medium is a popular choice for laboratory experiments. However, the concentration of glutamate in the in vivo niche of B. pertussis is quite low. We investigated the bacterial response to low concentrations of glutamate to elucidate bacterial physiology via the expression of the type 3 secretion system (T3SS), and we discuss its relationship to the Bvg mode in which the two-component regulator of pathogenesis (BvgAS) is activated. Glutamate limitation induced the expression of both the T3SS apparatus and effector genes at the transcriptional level. (p)ppGpp, a modulator of the stringent response, was necessary for maximum expression of the T3SS genes. These observations indicate that the expression of the T3SS is managed by nutrient starvation. In addition, the autoaggregation ability was high in the absence of glutamate and no autoaggregation was observed in glutamate-replete medium. Taken together, glutamate-limited conditions in Bvg ؉ mode elicit the high expression of T3SS genes in B. pertussis and promotes its sessile form. IMPORTANCEBordetella pertussis is a highly contagious pathogen that causes respiratory infectious disease. In spite of the increasing use of vaccination, the number of patients with pertussis is increasing. The proteins produced in vivo often are different from the protein profile under laboratory conditions; therefore, the development of conditions reflecting the host environment is important to understand native bacterial behavior. In the present study, we examined the effect of glutamate limitation, as its concentration in vivo is much lower than that in the culture medium currently used for B. pertussis experiments. As predicted, the T3SS was induced by glutamate limitation. These results are suggestive of the importance of regulation by nutrient conditions and in the pathogenicity of B. pertussis.

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Section: Consumption Of Glutamate In the Mediumsupporting

confidence: 69%

Section: Discussionmentioning

confidence: 99%

Section: Consumption Of Glutamate In the Mediummentioning

confidence: 99%

Section: Quantitative Reverse Transcription-pcr (Qrt-pcr)mentioning

confidence: 99%

Section: Quantitative Reverse Transcription-pcr (Qrt-pcr)mentioning

confidence: 99%

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Glutamate Limitation, BvgAS Activation, and (p)ppGpp Regulate the Expression of the Bordetella pertussis Type 3 Secretion System

et al. 2016

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Biofilm Formation and Environmental Signals inBordetella

Hanawa

2016

Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria

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Amino acid supplementation for enhancing recombinant protein production in E. coli

Kumar

Chauhan

Shah

et al. 2020

Biotech & Bioengineering

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Recombinant protein production (RPP) in Escherichia coli (E. coli) often induces metabolic burden to the cells that compromise their overall growth and productivity. Amino acid starvation due to RPP is a major contributor of the metabolic burden on the cells and induces global stress response known as a stringent‐like response. In this study, the effect of amino acid supplementation in a chemically defined medium on cellular growth and recombinant pramlintide production was investigated. Based on the consumption profile, few amino acids were categorized as growth‐promoting (GP1) and protein production promoting (GP2). Feeding strategies of GP1 and GP2 were tested in shake flasks followed by scale up into the bioreactor. A 40% increase in the recombinant pramlintide (rPramlintide) production (protein concentration of 3.09 ± 0.12 g/L and yield of 227.69 ± 19.72 mg pramlintide per gram dry cell weight) was realized. Furthermore, transcriptomics data indicated the downregulation of several genes associated with global stress response and genes involved in amino acid biosynthesis in test culture, supported by proteomics analysis. These results signify that the external supply of critical amino acids decreases cellular stress during RPP and improves process productivity.

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Role of (p)ppGpp in biofilm formation and expression of filamentous structures in Bordetella pertussis

Cited by 42 publications

References 49 publications

Glutamate Limitation, BvgAS Activation, and (p)ppGpp Regulate the Expression of the Bordetella pertussis Type 3 Secretion System

Glutamate Limitation, BvgAS Activation, and (p)ppGpp Regulate the Expression of the Bordetella pertussis Type 3 Secretion System

Biofilm Formation and Environmental Signals inBordetella

Amino acid supplementation for enhancing recombinant protein production in E. coli

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