A Serratia marcescens PigP Homolog Controls Prodigiosin Biosynthesis, Swarming Motility and Hemolysis and Is Regulated by cAMP-CRP and HexS

Shanks, Robert M. Q.; Lahr, Roni M.; Stella, Nicholas A.; Arena, Kristin E.; Brothers, Kimberly M.; Kwak, Daniel H.; Liu, Xinyu; Kalivoda, Eric J.

doi:10.1371/journal.pone.0057634

Cited by 47 publications

(50 citation statements)

References 71 publications

(105 reference statements)

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“…Serratamolide also is required for swarming motility in many strains of S. marcescens (25,42). We observed that eepR and eepS mutants were defective in swarming in the wild-type (CMS376) and K904 strain backgrounds (Fig.…”

Section: Identification Of Eepr and Eepsmentioning

confidence: 67%

“…An MBP fusion to EepR (MBP-EepR) and MBP alone were generated for affinity purification of EepR using pMal-C2 (New England BioLabs) as previously described (25). His 8 -CRP purification was described previously (18).…”

Section: Methodsmentioning

confidence: 99%

“…EMSAs were repeated at least three times. Primers and amplified regions for the oxyR, pigA, and swrW promoter regions used in EMSAs have been described previously (25). Primers for the eepR promoter are 1346 and 1884, or a biotinylated version of primer 1884, which amplify a 263-bp region of DNA just upstream of the eepR start codon, using pMQ254 as a template.…”

Section: Methodsmentioning

confidence: 99%

“…Zones of biosurfactant around colonies on swarming agar plates were measured 18 to 20 h after inoculation of the bacteria onto the surface of the agar as previously reported (23,25). Quantitative analysis of serratamolide from culture supernatants by high-performance liquid chromatography-mass spectrometry (HPLC-MS) was carried out as previously described (25). Bacterial cultures were grown in LB (10 5-ml cultures per genotype) for 20 h at 30°C.…”

Section: Methodsmentioning

confidence: 99%

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Serratia marcescens Cyclic AMP Receptor Protein Controls Transcription of EepR, a Novel Regulator of Antimicrobial Secondary Metabolites

et al. 2015

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Serratia marcescens generates secondary metabolites and secreted enzymes, and it causes hospital infections and communityacquired ocular infections. Previous studies identified cyclic AMP (cAMP) receptor protein (CRP) as an indirect inhibitor of antimicrobial secondary metabolites. Here, we identified a putative two-component regulator that suppressed crp mutant phenotypes. Evidence supports that the putative response regulator eepR was directly transcriptionally inhibited by cAMP-CRP. EepR and the putative sensor kinase EepS were necessary for the biosynthesis of secondary metabolites, including prodigiosin-and serratamolide-dependent phenotypes, swarming motility, and hemolysis. Recombinant EepR bound to the prodigiosin and serratamolide promoters in vitro. Together, these data introduce a novel regulator of secondary metabolites that directly connects the broadly conserved metabolism regulator CRP with biosynthetic genes that may contribute to competition with other microbes. IMPORTANCEThis study identifies a new transcription factor that is directly controlled by a broadly conserved transcription factor, CRP. CRP is well studied in its role to help bacteria respond to the amount of nutrients in their environment. The new transcription factor EepR is essential for the bacterium Serratia marcescens to produce two biologically active compounds, prodigiosin and serratamolide. These two compounds are antimicrobial and may allow S. marcescens to compete for limited nutrients with other microorganisms. Results from this study tie together the CRP environmental nutrient sensor with a new regulator of antimicrobial compounds. Beyond microbial ecology, prodigiosin and serratamolide have therapeutic potential; therefore, understanding their regulation is important for both applied and basic science. In order for organisms to survive and prosper, they must be able to sense their environment and effectively compete with other organisms. To respond to these environmental changes, bacteria have developed elaborate transcriptional regulatory systems that enable fine-tuning of factors that allow for their adaptation and proliferation. One of the most studied signaling systems involved in adaptation to the nutritive status of the environment is the cyclic AMP (cAMP)-associated catabolite repression system (1-4). The second messenger cAMP has been classified as an alarmone that induces positive regulation of alternative carbon transport systems in times of carbon/fuel deprivation (5). In addition to catabolite repression control, this system also can positively regulate flagellum production in unfavorable conditions (6) and activate attachment factors in nutrient-rich conditions (7).Evidence suggests that cAMP-cAMP receptor protein (CRP) can directly bind to and promote expression of secondary metabolite genes involved in antibiotic production in Streptomyces coelicolor (2). A positive or negative role for cAMP has been suggested for control of antimicrobial production in other organisms, including fungi, although dire...

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Section: Identification Of Eepr and Eepsmentioning

confidence: 67%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Serratia marcescens Cyclic AMP Receptor Protein Controls Transcription of EepR, a Novel Regulator of Antimicrobial Secondary Metabolites

et al. 2015

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“…RNA was extracted from cultures grown in LB broth to an OD 600 of 3.0 using a Qiagen RNA Easy kit, and cDNA preparations were performed as previously described (41). Two rounds of DNase treatment were performed to remove chromosomal DNA.…”

Section: Reverse Transcription-pcr (Rt-pcr)mentioning

confidence: 99%

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens

et al. 2015

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The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes ocular infections in healthy individuals. Secreted protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of protease-positive strains was observed among keratitis isolates than among conjunctivitis isolates. A positive correlation between protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined. Deletion of prtS in clinical keratitis isolate K904 reduced, but did not eliminate, cytotoxicity and secreted protease production. This indicated that PrtS is necessary for full cytotoxicity to ocular cells and implied the existence of another secreted protease(s) and cytotoxic factors. Bioinformatic analysis of the S. marcescens Db11 genome revealed three additional open reading frames predicted to code for serralysin-like proteases noted here as slpB, slpC, and slpD. Induced expression of prtS and slpB, but not slpC and slpD, in strain PIC3611 rendered the strain cytotoxic to a lung carcinoma cell line; however, only prtS induction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of both prtS and slpB genes was defective in secreted protease activity and cytotoxicity to human cell lines. PAGE analysis suggests that SlpB is produced at lower levels than PrtS. Purified SlpB demonstrated calcium-dependent and AprI-inhibited protease activity and cytotoxicity to airway and ocular cell lines in vitro. Lastly, genetic analysis indicated that the type I secretion system gene, lipD, is required for SlpB secretion. These genetic data introduce SlpB as a new cytotoxic protease from S. marcescens. Microbial keratitis (MK) is a blinding disease with poor visual outcomes even with effective antibiotics and antifungal agents (1-3). In addition to being a major cause of hospital-acquired infections, such as ventilator-associated pneumonia (4, 5), Serratia marcescens is a common cause of MK (1, 2, 6-8), yet the virulence factors involved in this process are poorly understood. In general, bacterial secreted factors, including hemolysins and proteases, contribute to the pathogenesis of bacterial corneal infections (9-12). There are several studies characterizing the importance of proteases from S. marcescens isolates in ocular infections, most recently reviewed by Matsumoto (13). Serralysin is a cytotoxic factor capable of killing mammalian cells in vitro (14,15). Purified serralysin is sufficient to cause keratitis when injected into rabbit eyes and promotes the spread of bacteria through the corneal stroma (16-18). Additionally, serralysin can degrade components of the human immune system, such as PAR-2, in vitro, and this may impact corneal pathogenesis (13,(19)(20)(21). Serralysin, and serralysin family metalloproteases, such as alkaline protease from Pseudomonas aeruginosa, can proteolyze mammalian cell surface proteins, thereby modulating cell physiology. A recent example is the protease-mediated activation of the epithelial sodium channel, lea...

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Cyclic-AMP inhibition of fimbriae and prodigiosin production by Serratia marcescens is strain-dependent

Stella

Shanks

2014

Arch Microbiol

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The cyclic-nucleotide 3’,5’-cyclic AMP (cAMP) is an ancient and wide spread regulatory molecule. Previous studies have shown that fimbria production and secondary metabolite production are inhibited by cAMP in the prokaryote Serratia marcescens. This study used genetic manipulations to test the strain specificity of cAMP-CRP regulation of fimbria production and of the red pigment, prodigiosin. A surprising amount of variation was observed, as multicopy expression of the cAMP-phosphodiesterase gene, cpdS, conferred either an increase or decrease in fimbriae-dependent yeast agglutination and prodigiosin production depending upon the strain background. Mutation of crp, the gene coding for the cAMP-receptor protein similarly conferred strain-dependent phenotypes. This study shows that three distinct biological properties, modulated by a conserved genetic regulatory molecule, can vary significantly among strains. Such variation can complicate the functional analysis of bacterial phenotypic properties which are dependent upon global genetic regulators such as cAMP.

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A Serratia marcescens PigP Homolog Controls Prodigiosin Biosynthesis, Swarming Motility and Hemolysis and Is Regulated by cAMP-CRP and HexS

Cited by 47 publications

References 71 publications

Serratia marcescens Cyclic AMP Receptor Protein Controls Transcription of EepR, a Novel Regulator of Antimicrobial Secondary Metabolites

Serratia marcescens Cyclic AMP Receptor Protein Controls Transcription of EepR, a Novel Regulator of Antimicrobial Secondary Metabolites

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens

Cyclic-AMP inhibition of fimbriae and prodigiosin production by Serratia marcescens is strain-dependent

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