The Bacterial Alarmone (p)ppGpp Activates the Type III Secretion System in Erwinia amylovora

Ancona, Veronica; Lee, Jae Hoon; Chatnaparat, Tiyakhon; Oh, Jinrok; Hong, Jong Ha; Zhao, Youfu

doi:10.1128/jb.02551-14

Cited by 50 publications

(60 citation statements)

References 75 publications

(109 reference statements)

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“…The ⌬rel Msm mutant of M. smegmatis is also elongated and multiseptate (26). Plant pathogens Erwinia amylovora and Pseudomonas syringae lacking (p)ppGpp are also elongated compared with the respective WT strains (44,45). We found that both ⌬rel Msm and ⌬dcpA mutants are elongated, multinucleate, and multiseptate.…”

Section: Discussionmentioning

confidence: 64%

Regulation of Growth, Cell Shape, Cell Division, and Gene Expression by Second Messengers (p)ppGpp and Cyclic Di-GMP in Mycobacterium smegmatis

et al. 2016

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The alarmone (p)ppGpp regulates transcription, translation, replication, virulence, lipid synthesis, antibiotic sensitivity, biofilm formation, and other functions in bacteria. Signaling nucleotide cyclic di-GMP (c-di-GMP) regulates biofilm formation, motility, virulence, the cell cycle, and other functions. In Mycobacterium smegmatis, both (p)ppGpp and c-di-GMP are synthesized and degraded by bifunctional proteins Rel Msm and DcpA, encoded by rel Msm and dcpA genes, respectively. We have previously shown that the ⌬rel Msm and ⌬dcpA knockout strains are antibiotic resistant and defective in biofilm formation, show altered cell surface properties, and have reduced levels of glycopeptidolipids and polar lipids in their cell wall (K. R. Gupta, S. Kasetty, and D. Chatterji, Appl Environ Microbiol 81:2571-2578, 2015, http://dx.doi.org/10.1128/AEM.03999-14). In this work, we have explored the phenotypes that are affected by both (p)ppGpp and c-di-GMP in mycobacteria. We have shown that both (p)ppGpp and c-di-GMP are needed to maintain the proper growth rate under stress conditions such as carbon deprivation and cold shock. Scanning electron microscopy showed that low levels of these second messengers result in elongated cells, while high levels reduce the cell length and embed the cells in a biofilm-like matrix. Fluorescence microscopy revealed that the elongated ⌬rel Msm and ⌬dcpA cells are multinucleate, while transmission electron microscopy showed that the elongated cells are multiseptate. Gene expression analysis also showed that genes belonging to functional categories such as virulence, detoxification, lipid metabolism, and cell-wall-related processes were differentially expressed. Our results suggests that both (p)ppGpp and c-di-GMP affect some common phenotypes in M. smegmatis, thus raising a possibility of cross talk between these two second messengers in mycobacteria. IMPORTANCEOur work has expanded the horizon of (p)ppGpp and c-di-GMP signaling in Gram-positive bacteria. We have come across a novel observation that M. smegmatis needs (p)ppGpp and c-di-GMP for cold tolerance. We had previously shown that the ⌬rel Msm and ⌬dcpA strains are defective in biofilm formation. In this work, the overproduction of (p)ppGpp and c-di-GMP encased M. smegmatis in a biofilm-like matrix, which shows that both (p)ppGpp and c-di-GMP are needed for biofilm formation. The regulation of cell length and cell division by (p)ppGpp was known in mycobacteria, but our work shows that c-di-GMP also affects the cell size and cell division in mycobacteria. This is perhaps the first report of c-di-GMP regulating cell division in mycobacteria. N ucleotide second messengers are utilized across all domains of life (1, 2). To counter innumerable threats and to regulate physiological functions, bacteria exploit a repertoire of signaling nucleotides, (p)ppGpp, cyclic di-GMP (c-di-GMP), c-di-AMP, cGMP, and cAMP (1-3). Each second messenger signaling module controls the response to a specific set of cues and brings about changes in gene...

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Section: Discussionmentioning

confidence: 64%

Regulation of Growth, Cell Shape, Cell Division, and Gene Expression by Second Messengers (p)ppGpp and Cyclic Di-GMP in Mycobacterium smegmatis

et al. 2016

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“…In E. amylovora , transcription of the T3SS genes is positively activated by (p)ppGpp-mediated alternative sigma factor cascade, including the master regulator HrpL, alternative sigma factor RpoN, enhancer binding protein (EBP) HrpS, ribosome-binding protein YhbH, IHF, transcriptional factor DksA and (p)ppGpp biosynthesis proteins RelA and SpoT781064; whereas GrrS/GrrA and EnvZ/OmpR systems negatively regulates T3SS gene expression13. In P. carotovorum , expression of hrpL is positively regulated by GacA/GacS- rsmB system, while RsmA negatively controls its expression43.…”

Section: Discussionmentioning

confidence: 99%

“…In order to colonize its host and cause disease, E. amylovora requires the deployment of effector proteins into the host cells by a type III secretion system (T3SS) and the production of the exopolysaccharide (EPS) amylovoran456. The T3SS in E. amylovora is encoded by the hypersensitive response and pathogenicity ( hrp ) island and is regulated by a HrpL-RpoN sigma factor cascade, which is further activated by the bacterial alarmone (p)ppGpp789101112. The T3SS is also controlled by several two-component signal transduction systems (TCST), including GacS/GacA (GrrS/GrrA) and EnvZ/OmpR systems1314.…”

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confidence: 99%

The RNA-binding protein CsrA plays a central role in positively regulating virulence factors in Erwinia amylovora

Ancona

Lee

Zhao

2016

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The GacS/GacA two-component system (also called GrrS/GrrA) is a global regulatory system which is highly conserved among gamma-proteobacteria. This system positively regulates non-coding small regulatory RNA csrB, which in turn binds to the RNA-binding protein CsrA. However, how GacS/GacA-Csr system regulates virulence traits in E. amylovora remains unknown. Results from mutant characterization showed that the csrB mutant was hypermotile, produced higher amount of exopolysaccharide amylovoran, and had increased expression of type III secretion (T3SS) genes in vitro. In contrast, the csrA mutant exhibited complete opposite phenotypes, including non-motile, reduced amylovoran production and expression of T3SS genes. Furthermore, the csrA mutant did not induce hypersensitive response on tobacco or cause disease on immature pear fruits, indicating that CsrA is a positive regulator of virulence factors. These findings demonstrated that CsrA plays a critical role in E. amylovora virulence and suggested that negative regulation of virulence by GacS/GacA acts through csrB sRNA, which binds to CsrA and neutralizes its positive effect on T3SS gene expression, flagellar formation and amylovoran production. Future research will be focused on determining the molecular mechanism underlying the positive regulation of virulence traits by CsrA.

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“…(p)ppGpp is required for SPI-1 activation under low oxygen conditions and positively regulates PI-2 genes in Salmonella enterica serovar Typhimurium (36). Recently, the regulation of the T3SS by (p)ppGpp was demonstrated in Erwinia amylovora via a sigma factor cascade (37). In B. pertussis, the production of the T3SS was significantly decreased by a deficiency of (p)ppGpp (19), and the level still was low even though the amount of glutamate was limited (Fig.…”

Section: Discussionmentioning

confidence: 99%

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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The Bacterial Alarmone (p)ppGpp Activates the Type III Secretion System in Erwinia amylovora

Cited by 50 publications

References 75 publications

Regulation of Growth, Cell Shape, Cell Division, and Gene Expression by Second Messengers (p)ppGpp and Cyclic Di-GMP in Mycobacterium smegmatis

Regulation of Growth, Cell Shape, Cell Division, and Gene Expression by Second Messengers (p)ppGpp and Cyclic Di-GMP in Mycobacterium smegmatis

The RNA-binding protein CsrA plays a central role in positively regulating virulence factors in Erwinia amylovora

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

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