SutA is a bacterial transcription factor expressed during slow growth inPseudomonas aeruginosa

Abstract

Microbial quiescence and slow growth are ubiquitous physiological states, but their study is complicated by low levels of metabolic activity. To address this issue, we used a time-selective proteomelabeling method [bioorthogonal noncanonical amino acid tagging (BONCAT)] to identify proteins synthesized preferentially, but at extremely low rates, under anaerobic survival conditions by the opportunistic pathogen Pseudomonas aeruginosa. One of these proteins is a transcriptional regulator that has no homology to any characterized protein domains and is posttranscriptionally upregulated during survival and slow growth. This small, acidic protein associates with RNA polymerase, and chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing suggests that the protein associates with genomic DNA through this interaction. ChIP signal is found both in promoter regions and throughout the coding sequences of many genes and is particularly enriched at ribosomal protein genes and in the promoter regions of rRNA genes. Deletion of the gene encoding this protein affects expression of these and many other genes and impacts biofilm formation, secondary metabolite production, and fitness in fluctuating conditions. On the basis of these observations, we have designated the protein SutA (survival under transitions A).Pseudomonas aeruginosa | slow growth | transcription | proteomics | BONCAT T he cosmopolitan bacterium Pseudomonas aeruginosa is notorious as an opportunistic pathogen of burn wounds, medical devices, and the lungs of cystic fibrosis (CF) patients. The bacterium's genome is large and encodes an unusually high proportion of regulators (1). Compared with Escherichia coli, P. aeruginosa possesses more σ factors that direct RNA polymerase (RNAP) to promoter regions (24 vs. 7), more DNAbinding activators and repressors that enhance or prevent RNAP binding and transcription (∼550 vs. 150) (2, 3) and more small, noncoding RNAs (ncRNAs) that modulate the stability or translation of target transcripts (200 vs. 100) (4, 5). Much effort has been directed toward understanding the mechanisms by which this regulatory capacity governs the behaviors-such as quorum sensing, protein secretion, secondary metabolite production, and biofilm formation-that contribute to P. aeruginosa virulence.The physiological states of bacteria involved in chronic infections are substantially different from those most often studied in standard laboratory experiments; chronic infections are characterized by slow growth rates imposed by limited nutrients or oxidants or by host immune responses. Direct measurements of in situ microbial growth rates in the context of lung infections in CF patients have revealed doubling times of several days (6). Measurements of expectorated sputum show that hypoxic and anoxic zones exist within infected CF airways and can experience dramatic fluctuations in redox potential (7); P. aeruginosa strains isolated from the CF lung show gene expression patterns consistent with adaptations to hypoxia (8), suggesting ...

The dormancy‐specific regulator, SutA, is intrinsically disordered and modulates transcription initiation in Pseudomonas aeruginosa

“…Because we found that SutA cross‐linked most efficiently to E (Fig. and Supporting Information, Figs and ) and because our previous ChIP‐Seq results suggested that SutA could interact with either initiating or elongating RNAP in vivo (Babin et al , ), we focused on the interaction between SutA and E. First we used the homobifunctional reagent bis(sulfosuccinimidyl)suberate (BS 3 ), which cross‐links primary amines that are within about 25 Å of each other (Rappsilber, ). We added BS 3 to complexes formed with purified E and SutA (Supporting Information, Fig.…”

The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase

“…The precipitated protein was removed by centrifugation, and the concentration of liberated flavin was determined using the FAD extinction coefficient of ⑀ 450 ϭ 11,300 M Ϫ1 cm Ϫ1 . Proteomics-In-gel digests were performed as described previously (63), and LC-MS/MS analysis was performed at the Caltech Proteome Exploration Laboratory (64).…”

Biofilms 2015: Multidisciplinary Approaches Shed Light into Microbial Life on Surfaces

“…By performing a proteomic study of Pseudomonas aeruginosa to determine which proteins are actively being made under anaerobic survival conditions, she described the discovery of a small, acidic protein, SutA (survival under transitions), that is posttranscriptionally upregulated during slow growth. SutA associates with RNA polymerase and regulates the expression of genes required for ribosome biogenesis and others involved in biofilm development, secondary metabolite production, and fitness under fluctuating conditions (2). With this insight underscoring the importance of studying biofilm properties beyond the lab, the first session provided a basis for understanding processes involving the formation of biofilms and the dynamics of different naturally occurring biofilm communities.…”