In this work, we targeted an iron-starved subpopulation in biofilms and compared its proteomic profile with that of the whole system. With the low K-state entry rate and high randomness, we challenged BONCAT to specifically capture gene and pathway regulations in K-state cells and compared the proteomic profile with that of the whole population.
Introduction
The methionine surrogates azidohomoalanine (Aha, Figure 1.S1A) and homopropargyl glycine (Hpg, Figure 1.S1B) have been successfully used to explore the proteomic profiles in various biological systems (9-12). In addition to Anl, several other mutant aaRSs have been developed for the methionine surrogates 2-aminooctinoic acid (Aoa, Figure 1.S1E) (15) and propargylglycine (Pra, Figure 1.S1F) (16), and for the phenylalanine ( Phe , Figure 1.S1G) surrogate p-azidophenylalanine (Azf, Figure 1.S1H) (17).
Conclusions
Dieterich DC, Lee JJ, Link AJ, Graumann J, Tirrell DA, Schuman EM (2007) Labeling, detection, and identification of newly synthesized proteomes by bioorthogonal noncanonical amino acid labeling. Grammel M, Zhang MZM, Hang HC (2010) An orthogonal alkynyl amino acid reporter for selective labeling of bacterial proteomes during infection.
Abstract
Introduction
Many studies have been performed to analyze the global expression profile of biofilms by identifying and quantifying transcriptomes using microarrays and RNAseq (6-10). To control expression, we chose the promoter of the pyoverdine synthetase F (pvdF) gene, which restricts protein labeling to iron-deprived biofilm cells.
Results
Upregulation of FliC in conjunction with downregulation of the flagellar assembly pathway deserves further investigation. any of the Ptrc:nll-metRS samples). Several proteins that provide protection against peroxides are found to be significantly upregulated in the subpopulation (Figure 2.3E).
Materials and Methods
The generality of the method points to straightforward extension to other microbial communities and higher-order systems. Overnight cultures of the wild-type, PpvdF:nll-metRS and Ptrc:nll-metRS strains in LB medium were diluted 1:100 in 0.9% NaCl and inoculated into flow cells (1 mm × 4 mm × 40 mm; Stovall) . For proteomic profiling of the biofilms, diluted overnight cultures of the PpvdF:nll-metRS and Ptrc:nll-metRS strains were inoculated into silicone rubber tubing (6 mm inner diameter, 20 cm length; McMaster-Carr).
Before injection, the peristaltic pump was turned off, the tubes upstream of the flow cells were clamped, and the downstream tubes were loosened; reagents were injected into the flow cell channels through the tubes with 30G needles. To block free thiol groups, biofilms were incubated with 100 mM chloroacetamide for 30 min in the dark at 37°C. DBCO agarose resin (25 μl of a 50% slurry; Click Chemistry Tools) was washed three times with 0.8% SDS in 1× PBS (w/v), resuspended in 25 μl of the same SDS wash buffer and added to each time added. lysate.
During the second wash with each solution, the resin samples were incubated with the wash solution for either 10 min (solutions I and iii), or 30 min (solution ii). For biofilm experiments with 3 h Anl labeling time, digested samples were subjected to LC-MS/MS analysis on a nanoflow LC system, EASY-nLC 1000, (Thermo Fisher Scientific) coupled to an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Bremen, Germany).
Acknowledgements
2006 ) Clustering of Pseudomonas aeruginosa transcriptomes from planktonic cultures, developing and mature biofilms reveals distinct expression profiles. Vasil ML, Ochsner UA (1999) Response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence. Gasser V, Guillon L, Cunrath O, Schalk I (2015) Cellular organization of siderophore biosynthesis in Pseudomonas aeruginosa: evidence for siderosomes.
Heinrichs DE, Poole K (1993) Cloning and sequence analysis of a gene (pchR) encoding an AraC family activator of pyochelin and ferripiochelin receptor synthesis in Pseudomonas aeruginosa. Dihydroaeruginoic acid synthetase and pyochelin synthetase, products of the pchEF genes, are induced by extracellular pyochelin in Pseudomonas aeruginosa. Xu KD, Stewart PS, Xia F, Huang CT, McFeters GA (1998) Spatial physiological heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen availability.
Filloux A, Michel G, Bally M (1998) GSP-dependent protein secretion in Gram-negative bacteria: the Xcp system of Pseudomonas aeruginosa. -Osorio AC, Williamson KS, Franklin MJ (2010) Heterogeneous rpoS and rhlR mRNA levels and 16S rRNA/rDNA (rRNA gene) ratios within Pseudomonas aeruginosa biofilms sampled by laser capture microdissection. subpopulations in Pseudomonas aeruginosa biofilms.
Summary of Contributions
Abstract
Fluctuations in the expression of ComK, the key transcriptional regulator of competence, result in bistable activation of multiple pathways that drive a subpopulation of cells to become competent ( 4 , 5 ). Based on studies of ComK-dependent regulation, researchers have further discovered that the same cells that acquire competence can also exhibit different expression patterns related to cell division, cell shape maintenance, detoxification, central carbon metabolism, pH homeostasis, etc. The changes in various aspects of cellular physiology in addition to competence development, differentiate competent cells from their vegetative counterparts and spores, defining them as under a separate cell state called K-state (6, 8).
However, comprehensive characterization of K-state and its gene and pathway regulations has yet to be achieved. To interpret K-state-specific changes in gene expression, the abundance of respective mRNAs was directly compared between the wild type and the comK knockout strains, both under K-state-inducing conditions. The low abundance of mRNAs in cells further limited the ability to detect transcripts of downregulated genes ( 6 , 8 ).
In addition, knockdown of comK inevitably disrupted the native cell state, biasing the detection against genes that are affected but not completely dependent on ComK ( 6 , 8 ). We compared the proteomic profiles of K-state cells and the whole population and analyzed the dynamic proteomic response of the K-state subpopulation.
Results
As a control, wild-type W168 cells were grown and labeled in the same way both before and after t0. The Pveg:nll-metRS strain, which constitutively expresses NLL-MetRS in the entire population regardless of cell state, was used to serve as a positive control. As a result, it is reasonable that the extent of protein labeling in PcomF:nll-metRS.
We found that RecA was more than 5-fold more abundant in the K-state cells (Figure 3.2E), and proteins involved in SOS response (GO: 0009432) were either upregulated or exclusively identified in the subpopulation, consistent with the proposed regulation mechanism of SOS response in K-state cells. Within the pathway, most of the upregulated proteins are involved in the transport of amino acids, peptides and vitamins while two (TagG and TagH) were for transport of teichoic acids (TAs). Consistent with their study, we found that TagG was highly upregulated and TagH was also slightly upregulated in the K-state subpopulation.
However, TAs have recently been found to be also crucial in the development of competence in B. Many proteins that are highly regulated in the subpopulation, with known functions in other cellular states, have not previously been associated with the K-state.
Materials and Methods
The culture was immediately diluted with an equal volume of starvation medium 2 (SM2) and further grown for 2 h to reach maximal competence. Cells were harvested, resuspended in 4% paraformaldehyde in 1× PBS to achieve an OD600 value of about 2, and incubated for 15 min at room temperature. Cell cultures were incubated with 1 mM Anl (Iris Biotech) by diluting 100 mM Anl stock (dissolved in double distilled water [pH 7]) in cell cultures for 1 h at 37°C with agitation (250 rpm).
Cells were pelleted at 4℃, resuspended in lysis buffer (4 w/v% SDS/PBS), and treated with protease inhibitor (cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail; . Roche). To visualize Anl incorporation, cell lysates were incubated in the dark with 15 μM alkyne-TAMRA, 250 μM CuSO4, 1.25 mM THPTA, 5 mM aminoguanidine hydrochloride, and 5 mM sodium ascorbate for 1 h at room temperature. For BONCAT enrichment, each lysate containing 3 mg of protein was incubated in the dark with 100 mM chloroacetamide for 30 min at 65 ℃ with shaking (1200 rpm) to block the free thiol group in cysteines.
25 μL of DBCO agarose resin 50% slurry (Click Chemistry Tools) was washed three times with 0.8% SDS/PBS (w/v), resuspended in original volume with 0.8% SDS/PBS, and added to each lysate. The digested samples were subjected to LC-MS/MS analysis on a nanoflow LC system, EASY-nLC 1200, (Thermo Fisher Scientific) coupled to a Q Exactive HF Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a Nanospray Flexion source.
Acknowledgements
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Raymond-Denise A, Guillen N (1992) Expression of the Bacillus subtilis dinR and recA genes after DNA damage and during competence. LV, Fisher SH (1996) Bacillus subtilis gabP gene expression is regulated independently in response to nitrogen and amino acid availability. Slack FJ, Serror P, Joyce E, Sonenshein AL (1995) A gene required for nutritional repression of the Bacillus subtilis dipeptide permease operon.
LV, Ferson AE, Fisher SH (1997) Expression of the Bacillus subtilis ureABC operon is controlled by multiple regulatory factors including CodY, GlnR, TnrA and SpoOH. Baranova N, Danchin A, Neyfakh A (1999) Mta, a global MerR-type regulator of Bacillus subtilis multidrug efflux transporters.
Supplementary Tables
Columns F-K give the LC-MS/MS iBAQ values for each protein in the PpvdF:nll-metRS (columns F-H) and Ptrc:nll-metRS (columns I-K) strains. Columns L-Q give the normalized LC-MS/MS LFQ values for each protein in the PpvdF:nll-metRS (columns L-N) and Ptrc:nll-metRS (columns O-Q) strains. Columns F-H give the LC-MS/MS iBAQ values for each protein identified in the PpvdF:nll-metRS samples.
Columns F-K give the LC-MS/MS iBAQ values for each protein in the PpvdF:nll-metRS (columns F-H) and Ptrc:nll-metRS (columns I-K) strains. Columns F-K give LC-MS/MS ion intensities for each protein in the PcomF:nll-metRS strain without (columns F-H) and with (columns I-K) K-state induction. Columns M-R give LC-MS/MS normalized LFQ values for each protein in the PcomF:nll-metRS strain without (columns M-O) and with (columns P-R) K-state induction.
Columns E-J provide LC-MS/MS iBAQ values for each protein in strains PcomF:nll-metRS (columns E-G) and Pveg:nll-metRS (columns H-J). Columns K-P provide LC-MS/MS normalized LFQ values for each protein in the PcomF:nll-metRS. columns K-M) and Pveg:nll-metRS (columns N-P) strains.
