Colony‐morphology screening uncovers a role for the <scp><i>P</i></scp><i>seudomonas aeruginosa</i> nitrogen‐related phosphotransferase system in biofilm formation

Cabeen, Matthew T.; Leiman, Sara A.; Losick, Richard

doi:10.1111/mmi.13250

Cited by 38 publications

(56 citation statements)

References 55 publications

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“…In other organisms, the PTS Ntr has long been considered a system that monitors intracellular nitrogen levels and coordinately regulates metabolism in conjunction with the carbohydrate PTS (20)(21)(22). However, recent work by others implies expanding roles for the PTS Ntr outside central metabolism; these include the regulation of biofilm formation in Pseudomonas and stress responses in Escherichia coli (23)(24)(25). We report here that loss of the PTS Ntr components in A. baumannii suppresses all phenotypes of the gigA and gigB mutant strains.…”

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confidence: 71%

GigA and GigB are Master Regulators of Antibiotic Resistance, Stress Responses, and Virulence in Acinetobacter baumannii

Gebhardt

Shuman

2017

J Bacteriol

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A critical component of bacterial pathogenesis is the ability of an invading organism to sense and adapt to the harsh environment imposed by the host's immune system. This is especially important for opportunistic pathogens, such as Acinetobacter baumannii, a nutritionally versatile environmental organism that has recently gained attention as a life-threatening human pathogen. The emergence of A. baumannii is closely linked to antibiotic resistance, and many contemporary isolates are multidrug resistant (MDR). Unlike many other MDR pathogens, the molecular mechanisms underlying A. baumannii pathogenesis remain largely unknown. We report here the characterization of two recently identified virulence determinants, GigA and GigB, which comprise a signal transduction pathway required for surviving environmental stresses, causing infection and antibiotic resistance. Through transcriptome analysis, we show that GigA and GigB coordinately regulate the expression of many genes and are required for generating an appropriate transcriptional response during antibiotic exposure. Genetic and biochemical data demonstrate a direct link between GigA and GigB and the nitrogen phosphotransferase system (PTS Ntr ), establishing a novel connection between a novel stress response module and a well-conserved metabolic-sensing pathway. Based on the results presented here, we propose that GigA and GigB are master regulators of a global stress response in A. baumannii, and coupling this pathway with the PTS Ntr allows A. baumannii to integrate cellular metabolic status with external environmental cues. IMPORTANCE Opportunistic pathogens, includingAcinetobacter baumannii, encounter many harsh environments during the infection cycle, including antibiotic exposure and the hostile environment within a host. While the development of antibiotic resistance in A. baumannii has been well studied, how this organism senses and responds to environmental cues remain largely unknown. Herein, we investigate two previously identified virulence determinants, GigA and GigB, and report that they are required for in vitro stress resistance, likely comprising upstream elements of a global stress response pathway. Additional experiments identify a connection between GigA/GigB and a widely conserved metabolic-sensing pathway, the nitrogen phosphotransferase system. We propose that coordination of these two pathways allows A. baumannii to respond appropriately to changing environmental conditions, including those encountered during infection.

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confidence: 71%

GigA and GigB are Master Regulators of Antibiotic Resistance, Stress Responses, and Virulence in Acinetobacter baumannii

Gebhardt

Shuman

2017

J Bacteriol

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“…Dilutions of the transposon insertion library from the initial transposon insertion screen were plated onto TSB agar supplemented with 0.25% (wt/vol) Congo red. Resistant mutants were isolated, and sites of insertion were determined by using random nested primer amplification with Rnd1 primers followed by Rnd2 primers, followed by sequencing (51). The specific site of insertion was determined by sequencing a PCR product amplified by using a gene-specific primer (ASD227-R-1) and a transposon-specific primer (ASD252).…”

Section: Methodsmentioning

confidence: 99%

Genome-wide screen for genes involved in eDNA release during biofilm formation by Staphylococcus aureus

DeFrancesco

Masloboeva

Syed

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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103

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Staphylococcus aureus is a leading cause of both nosocomial and community-acquired infection. Biofilm formation at the site of infection reduces antimicrobial susceptibility and can lead to chronic infection. During biofilm formation, a subset of cells liberate cytoplasmic proteins and DNA, which are repurposed to form the extracellular matrix that binds the remaining cells together in large clusters. Using a strain that forms robust biofilms in vitro during growth under glucose supplementation, we carried out a genome-wide screen for genes involved in the release of extracellular DNA (eDNA). A high-density transposon insertion library was grown under biofilm-inducing conditions, and the relative frequency of insertions was compared between genomic DNA (gDNA) collected from cells in the biofilm and eDNA from the matrix. Transposon insertions into genes encoding functions necessary for eDNA release were identified by reduced representation in the eDNA. On direct testing, mutants of some of these genes exhibited markedly reduced levels of eDNA and a concomitant reduction in cell clustering. Among the genes with robust mutant phenotypes were gdpP, which encodes a phosphodiesterase that degrades the second messenger cyclic-di-AMP, and xdrA, the gene for a transcription factor that, as revealed by RNA-sequencing analysis, influences the expression of multiple genes, including many involved in cell wall homeostasis. Finally, we report that growth in biofilm-inducing medium lowers cyclicdi-AMP levels and does so in a manner that depends on the gdpP phosphodiesterase gene.Staphylococcus aureus | biofilm | eDNA | cyclic-di-AMP

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“…In the PTS Ntr , NPr phosphorylates EIIA Ntr ; however, the EIIA Ntr is not active in transport. The PTS Ntr regulates diverse processes implicated in the metabolism of nitrogen and carbon (21), it plays a role in potassium homeostasis (22) and biofilm formation (23), and is essential for virulence in several bacteria such as S. enterica (24), B. melitensis (25), and L. pneumophila (26). In many bacteria the PTS Ntr constitutes a phosphorylation cascade that works in parallel with the sugar PTS, while in other bacteria only one of the two systems exists.…”

Section: Discussionmentioning

confidence: 99%

The Legionella pneumophila Incomplete Phosphotransferase System Is Required for Optimal Intracellular Growth and Maximal Expression of PmrA-Regulated Effectors

et al. 2017

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The nitrogen phosphotransferase system (PTS Ntr ) is a regulatory cascade present in many bacteria, where it controls different functions. This system is usually composed of three basic components: enzyme I Ntr (EI Ntr ), NPr, and EIIA Ntr (encoded by the ptsP, ptsO, and ptsN genes, respectively). In Legionella pneumophila, as well as in many other Legionella species, the EIIA Ntr component is missing. However, we found that deletion mutations in both ptsP and ptsO are partially attenuated for intracellular growth. Furthermore, these two PTS Ntr components were found to be required for maximal expression of effector-encoding genes regulated by the transcriptional activator PmrA. Genetic analyses which include the construction of single and double deletion mutants and overexpression of wild-type and mutated forms of EI Ntr , NPr, and PmrA indicated that the PTS Ntr components affect the expression of PmrA-regulated genes via PmrA and independently from PmrB and that EI Ntr and NPr are part of the same cascade and require their conserved histidine residues in order to function. Furthermore, expression of the Legionella micdadei EII Ntr component in L. pneumophila resulted in a reduction in the levels of expression of PmrAregulated genes which was completely dependent on the L. pneumophila PTS components and the L. micdadei EII Ntr conserved histidine residue. Moreover, reconstruction of the L. pneumophila PTS in vitro indicated that EI Ntr is phosphorylated by phosphoenolpyruvate (PEP) and transfers its phosphate to NPr. Our results demonstrate that the L. pneumophila incomplete PTS Ntr is functional and involved in the expression of effector-encoding genes regulated by PmrA.

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Colony‐morphology screening uncovers a role for the Pseudomonas aeruginosa nitrogen‐related phosphotransferase system in biofilm formation

Cited by 38 publications

References 55 publications

GigA and GigB are Master Regulators of Antibiotic Resistance, Stress Responses, and Virulence in Acinetobacter baumannii

GigA and GigB are Master Regulators of Antibiotic Resistance, Stress Responses, and Virulence in Acinetobacter baumannii

Genome-wide screen for genes involved in eDNA release during biofilm formation by Staphylococcus aureus

The Legionella pneumophila Incomplete Phosphotransferase System Is Required for Optimal Intracellular Growth and Maximal Expression of PmrA-Regulated Effectors

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