*†A.R.P. current address is School of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden
Supplementary Figure S1. Duplicate SDS-PAGE gels (representing biological replicates) corresponding to those in Figure 2. The protein profiles of subcellular proteins on SDS-PAGE for five different bacteria are shown. Three types of differences in the protein profiles are indicated: (i) prominent differences are marked by red arrows; (ii) differences due to brands of media components (HI-LB and IN-LB) are marked by black dots; and (iii) minor variations in expression level are marked by green arrows. Duplicates (representing biological replicates) of these gels can be seen in Supplementary Figure S1. (A) E. coli DH5α. Subcellular proteins of E. coli DH5α showed the expression of three protein of >110 kDa, 85 kDa, and <14 kDa in the lanes HI-LB, HI-RDLB + HI-sRNA, and IN-RDLB + HI-sRNA. Also, there were minor differences in the expression levels of the >65 kDa protein and the between 35 and 25 kDa protein. (B) E. coli BL21 (DE3). A prominent protein at 65 kDa was seen only in IN-LB, but not in HI-LB. However, when IN-RDLB and HI-RDLB were supplemented with IN-sRNA, the band could be seen. (C) S. flexneri. A prominent <14 kDa protein was observed in HI-LB but not in IN-LB. It appeared when HI-RDLB and IN-RDLB were supplemented with HI-sRNA. Also, a few minor differences in several other proteins of the bacteria cultured in HI-LB and IN-LB have been indicated. (D) S. typhimurium. Two prominent proteins of >110 kDa and <85 kDa in HI-LB, and in HI-RDLB and IN-RDLB supplemented with Hi-sRNA are indicated. A few other proteins with expression level differences are also indicated. (E) EPEC A5. Similar to E. coli DH5α and S. typhimurium, a prominent protein of 85 kDa was observed in HI-LB but not in IN-LB. This protein also appeared when HI-RDLB and IN-RDLB was supplemented with HI-sRNA. The prominent <14 kDa protein seen in E. coli DH5α and S. flexneri were not seen in EPEC, even though it is a variant of the E. coli species. Protein marker, unstained protein molecular weight marker, (Fermentas Inc., Hanover, MD, USA).
Efforts to delineate the basis for variations in protein profiles of different membrane fractions from various bacterial pathogens led to the finding that even the same medium [e.g., Luria Bertani (LB) broth] purchased from different commercial sources generates remarkably dissimilar protein profiles despite similar growth characteristics. Given the pervasive roles small RNAs play in regulating gene expression, we inquired if these source-specific differences due to media arise from disparities in the presence of small RNAs. Indeed, LB media components from two different commercial suppliers contained varying, yet significant, amounts of 10–80 bp small RNAs. Removal of small RNA from LB using RNaseA during media preparation resulted in significant changes in bacterial protein expression profiles. Our studies underscore the fact that seemingly identical growth media can lead to dramatic alterations in protein expression patterns, highlighting the importance of utilizing media free of small RNA during bacteriological studies. Finally, these results raise the intriguing possibility that similar pools of small RNAs in the environment can influence bacterial adaptation.
During our analysis of subcellular protein composition in enteropathogenic Escherichia coli (EPEC), we frequently noted inconsistencies among protein profiles, even under seemingly identical growth conditions using Luria Bertani (LB) medium prepared from components purchased through either Himedia (HI-LB; Mumbai, Maharashtra, India) or Invitrogen (IN-LB; Carlsbad, CA, USA).
LB is a complex media consisting of tryptone, yeast extract, and NaCl used extensively in microbiology and cell biology studies of various processes, including host-microbe interactions (1). When we analyzed ethanol-precipitated fractions of autoclaved LB, nucleic acid species smaller than 100 bp were identified. During the production of tryptone and yeast extracts, although tissues from different biological sources are subjected to harsh treatment procedures to remove nucleic acids and other contaminants, it is difficult to achieve complete hydrolysis of all the nucleic acid fragments. Our observations correlate with those of Chen et al. (2) suggesting that the protein expression studies carried out with commercial growth media containing enzymatic digests of casein, such as LB, engendered variations in protein profiles, leading us to speculate that these nucleic acid populations identified in LB media could act in a similar fashion to many recently identified small RNAs (sRNA) regulators to alter protein expression profiles.
In recent years, various biological functions have been attributed to sRNAs (3-5), including roles in the regulation of gene expression, gene silencing, coordinating intricate stress responses (6-8), and apoptosis (9). In addition, roles in quorum sensing and bacterial cell-cell communications (10-13) are also evident based on studies from bacterial pathogens like Vibrio harveyi, Pseudomonas fluorescens (12), and Escherchia coli (14). Although the exact mechanisms remain unclear, such regulatory noncoding sRNAs (15,16) are also involved in population density-dependent activities such as virulence factor regulation (17,18) in bacterial species like Erwinia, Vibrio cholerae, V. harveyi, Salmonella entricea, and Pseudomonas aeruginosa (17). Although substantial numbers of sRNA species and their related pathways still await further study, many of these could potentially function as metabolic regulators (19).
We examined the presence of sRNAs in LB media components from two different commercial sources. Subsequently, we compared the protein profiles of E. coli DH5α, BL21 (DE3), and pathogens such as enteropathogenic E. coli (EPEC), Salmonella typhimurium, and Shigella flexneri grown in LB, RNA-depleted LB (RDLB), and RDLB supplemented with RNA isolated from LB (reconstituted LB). In all cases, we observed significant differences in protein profiles. Our results support the hypothesis that nonspecific or specific interference by sRNAs in autoclaved LB medium can contribute to changes in bacterial protein expression.
Materials and methods
Preparation of LB medium
In this study, we used yeast extract and tryptone purchased from Himedia (HI-LB) and Invitrogen (IN-LB). HI-LB was prepared by dissolving 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 1 L distilled water and adjusting the pH to 7.2 with 1 N NaOH prior to autoclave. In the case of IN-LB, 25 g LB powder (containing 10 g tryptone, 5 g yeast extract, and 10 g NaCl) were dissolved in 1 L distilled water and autoclaved after adjusting the pH to 7.2.
Preparation of RNA-depleted LB medium
RNA-depleted LB medium (RDLB) was prepared from both media (HI-LB andIN-LB) by two methods: (i) LB medium was incubated with 0.5 µg/mL RNaseA (Genie, Bangalore, Karnataka, India) at 37°C for 1.5 h to digest the RNA in the medium and then autoclaved; or (ii) the pH of the LB medium was increased from 7.2 to 11.0 using 5 M NaOH (Sisco Research Laboratories, Mumbai, Maharashtra, India), and after 15 min at room temperature, the pH was decreased to 7.2 using 11 N HCl (Sisco Research Laboratories). In both the cases, we did not notice the presence of nucleic acids when the ethanol-precipitated fractions were analyzed on agarose gels, as described below. Since the second method was not compatible with bacterial growth and protein expression studies, we adopted method (i) for all the experiments in this study.
Analysis of nucleic acid content and agarose gel electrophoresis
We removed proteins and lipids from 6 mL LB medium by two-phase extraction. The aqueous layer containing nucleic acids was obtained by centrifugation at 12,000 × g for 10 min with phenol:chloroform (1:1 v/v; Sisco Research Laboratories). RNA-free glycogen (0.2 mg/mL; SD Fine Chemicals, Mumbai, Maharashtra, India), one-tenth volume 3 M sodium acetate (Merck, Mumbai, India), and 2.5 volumes absolute ethanol (Sisco Research Laboratories) were added to the aqueous phase. After incubation of the mixture (-70°C for 30 min), it was centrifuged at 12,000 × g for 10 min. The precipitate was washed twice with 0.5 mL 70% (v/v) ethanol, air-dried, dissolved in 60 µL nuclease-free water (Fermentas, Glen Burnie, MD, USA), and divided into three parts of 20 µL each, which were individually treated with 5 µg RNaseA, 0.25 U DNase (Promega Scientific, San Luis Obispo, CA, USA) for 3 h at 37°C, and 5 µL water, respectively (20). Samples were subjected to electrophoresis in TBE buffer (0.045 M Tris-borate and 0.001 M EDTA, pH 8.3; Sisco Research Laboratories) at 100 V on 1.5% (w/v) agarose gels (Sisco Research Laboratories) containing ethidium bromide (0.5 µg/mL; Sisco Research Laboratories) (21).
Analysis of sRNA fragments by denaturing urea PAGE
Twenty-five micrograms sRNA fractions obtained from IN-LB and HI-LB were analyzed on 15% denaturing PAGE containing 6 M urea. A mixture of single-stranded (ss) DNA (oligomers of 20, 23, 40, 56 nucleotides; MicroSynth, Lindau, Germany) and ssRNA (50–1000 nucleotides; New England Biolabs, Hitchin, OTY, UK) were used as molecular size markers to deduce the size of sRNA fractions. Electrophoresis was carried out at 25 mA with 0.5 × TBE as the running gel buffer and stained with 0.5 × TBE containing ethidium bromide (0.5 µg/mL) (21).
Stability analysis of sRNAs present in the LB medium
This experiment was performed using the following preparations: (i) 1 mL RDLB was mixed with sRNAs isolated from 1 mL HI-LB medium, prior to electrophoresis; (ii) 1 mL RDLB was mixed with sRNAs isolated from 1 mL HI-LB medium and incubated at 37°C for 1.5 h; (iii) 1 mL RDLB was mixed with sRNAs isolated from 1 mL HI-LB and treated with 0.5 µg/mL RNaseA and incubated at 37°C for 1.5 h; (iv) 1 mL RDLB was mixed with sRNAs isolated from 1 mL HI-LB medium, incubated at 37°C for 1.5 h, and autoclaved; (v) 1 mL RDLB was treated with 0.5 µg/mL RNaseA, incubated for 1.5 h, and autoclaved; this preparation was again incubated with sRNAs (from 1 mL HI-LB) for 1.5 h at 37°C; (vi) 1 mL RDLB was mixed with sRNAs isolated from 1 mL HI-LB, incubated at 37°C for 1.5 h, autoclaved, and incubated with 0.5 µg/mL RNaseA at 37°C for 1.5 h. About 50 µL each preparation were analyzed on denaturating urea-PAGE.
Preparation of reconstituted LB media
Reconstitution experiments were performed to examine whether the sRNAs arising from LB medium contribute to changes in protein profiles of bacteria. In order to conduct this experiment, nucleic acids (containing sRNA) were obtained aseptically from 3 mL LB medium (from both HI-LB and IN-LB) by ethanol precipitation, as described earlier. Ethanol precipitate thus obtained from 3 mL HI-LB was added to the same volume of IN-RDLB and vice versa. Reconstituted LB media were used to culture the bacteria, and their proteins were analyzed.
Preparation of whole-cell lysates and protein profile analysis by SDS-PAGE
The five strains, E. coli DH5α and BL21 (DE3), S. typhimurium, S. flexneri, and EPEC, were cultured by incubating overnight at 37°C, 180 rpm. Approximately 3 mL RDLB and reconstituted RDLB prepared from HI-LB and IN-LB were seeded with 5% of the above cultures, respectively. The volumes corresponding to 0.7 OD at 595 nm were centrifuged in 2-mL microcentrifuge tubes at 4500 × g for 5 min. The obtained cell pellets were washed twice with 0.5 mL TE buffer (20 mM Tris-HCl, 10 mM EDTA, pH 7.4), suspended in 25 µL 4 × SSB (40% glycerol, 8% v/v β-mercaptoethanol, 32% w/v SDS, 0.02% Bromophenol blue), and heated in a dry bath for 10 min. Resulting suspensions were centrifuged at 12,000 × g for 10 min, and the supernatants were analyzed on SDS-PAGE (12% polyacrylamide; 15 × 13 cm) using Tricine as tank buffer instead of Tris-glycine (25 mM Tris-HCl, 200 mM Tricine, 0.1% w/v SDS, pH 8.3). Electrophoresis was performed at 40 mA, and the resolved proteins were stained using Coomassie Brilliant Blue (CBB) R-250 (Sisco Research Laboratories).
Results and discussion
Based on studies demonstrating protein expression regulation by sRNAs, along with their pervasive roles in cellular homeostasis (4,7,8,17,19,22), we speculated that small fragments of RNA in LB medium could cause variation in either growth or protein expression profiles. Hence, we examined the growth and protein profiles for five different bacterial species grown in LB obtained from two different manufacturers, as well as in LB media that had been RNA-depleted.
Heat-stable sRNAs in LB media
Agarose gel electrophoresis of nucleic acid extracts prepared from either HI-LB or IN-LB revealed the presence of small, heat stable nucleic acids less than 100 bp (Figure 1A). Corroborating our initial gel analysis, spectrophotometric analysis (A260/A280) revealed the presence of about 30 and 100 µg nucleic acids/mL in IN-LB and HI-LB, respectively (Figure 1A, lanes 2 and 3). These nucleic acid species were subsequently identified as RNA by virtue of their lability (Figure 1A, lane 6) to alkali and digestibility by RNaseA (Figure 1B), but not by DNase (Figure 1C). The ingredients of HI-LB (tryptone and yeast extract) also exhibited a prominent band below 100 bp (Figure 1A, lanes 4 and 5). However, denaturing PAGE analysis revealed the sizes of sRNA at ~10–80 nucleotides in the form of nondistinct bands in HI-LB and distinct bands in IN-LB (Figure 1D). Since sRNAs were observed even after autoclaving (Figure 1E, lanes 3 and 4) and following various manipulations and incubations at 37°C (Figure 1E, lane 6), we report that these sRNA fractions are quite stable.