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Proteomic Assessment of the Expression of Genes Related to Toluene Catabolism and Porin Synthesis in Pseudomonas stutzeri ST‑9 Esti Michael,†,‡ Margarita Gomila,§ Jorge Lalucat,§ Yeshayahu Nitzan,‡ Izabella Pechatnikov,‡ and Rivka Cahan*,† †

Department of Chemical Engineering, Ariel University, Ariel, 40700, Israel The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 52900, Israel § Microbiology, Biology Department, and IMEDEA, (CSIC-UIB) University of the Balearic Islands, Palma de Mallorca, 07122, Spain ‡

S Supporting Information *

ABSTRACT: The organization and expression of Pseudomonas stutzeri ST-9 genes related to toluene catabolism and porin synthesis was investigated. Toluene-degrading genes were found to be localized in the chromosome close to a phage-type integrase. A regulatory gene and 21 genes related to an aromatics degradation pathway are organized as a putative operon. These proteins are upregulated in the presence of toluene. Fourteen outer membrane proteins were identified as porins in the ST-9 genome. The identified porins showed that the main detected porins are related to the OmpA and OprD superfamilies. The percentage of porins in the outer membrane protein fraction, as determined by mass spectrometry, was 73% and 54% when the cells were cultured with toluene and with glucose, respectively. Upregulation of OmpA and downregulation of OprD occurred in the presence of toluene. A porin fraction (90% OprD) from both cultures was isolated and examined as a toluene uptake system using the liposome-swelling assay. Liposomes were prepared with the porin fraction from a culture that was grown on toluene (T-proteoliposome) or glucose (G-proteoliposome). There was no significant difference in the permeability rate of the different solutes through the Tproteoliposome and the G-proteoliposome. KEYWORDS: Pseudomonas stutzeri, porins, adaptive response, toluene, liposomes

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INTRODUCTION

The specific substrate-binding site comprises an advantage for solute transport in a dilute medium.4 Interestingly, contrary to most of the Gram-negative bacteria, Pseudomonas spp. mainly possess specific porins including OprB (glucose-specific), OprP (phosphate-specific), OprO (polyphosphate-specific), and OprD (basic amino acid-specific),5,6 whereas, OprF, is its general porin.5 The major general porin of P. aeruginosa, OprF, produces a large channel but allows only slow diffusion.3 Earlier studies showed that OprF is a homologue of the E. coli OmpA and that the OmpA protein population consists of two conformers, a majority (about 98%) containing “closed” channels and a minority (about 2%) containing open channels.3,2,7 OmpA occurs in the E. coli outer membrane at 100 000 copies per cell.8 It was found to be tightly regulated at the posttranscriptional level.9 When Pseudomonas sp. hDV1 was grown in media containing glucose or phenol, OmpA was found in the bacterial

The outer membrane of Gram-negative bacteria is the first cell structure that interacts with the substrate, thus it has a determinant influence on the possible toxic effects on the cell. Porins are transmembrane proteins. They comprise a majority of the outer membrane proteins and have a structure of three monomers with a channel filled with water to enable passive diffusion of water-soluble molecules.1 The molecular weight of the monomers ranges from 30 to 50 kDa. The porins exhibit high resistance to degradation by proteolytic enzymes, high temperatures, and detergents.2,3 The porins are divided into two major families, general and specific which differ in their substrate selectivity. Many Gram-negative bacteria possess mainly general porins and few specific ones. The general porins provide an aqueous environment for small hydrophilic molecules up to approximately 300 Da. The diffusion kinetics through the general porins is affected by the molecule’s shape, size, electrical charge, polarity, and its gradient across the membrane. The specific porins possess a specific substratebinding site for molecules such as carbohydrates or nucleosides. © XXXX American Chemical Society

Received: December 13, 2016

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DOI: 10.1021/acs.jproteome.6b01044 J. Proteome Res. XXXX, XXX, XXX−XXX

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Journal of Proteome Research Growth Conditions

membrane. It was suggested that OmpA’s main role is to maintain the cell wall integrity.10 OprD is a specific porin that binds several antibiotics such as imipenem and related zwitterionic carbapenems as well as basic amino acids.11 In P. aeruginosa, the OprD family includes 19members, which have a similarity of 46%−57% at the amino acid level.6,12 Several efflux pump regulators, metals, and amino acids may lead to a transcriptional and post-transcriptional regulation of OprD.13,14 It was found that benF and phaK genes encoding the P. putida putative porin are located in an operon responsible for benzoate and phenylacetic acid degradation.15 To the best of our knowledge, there is no previous report on a proteomic analysis on the porins of P. stutzeri strains, nor of P. stutzeri strains which are able to grow in the presence of toluene. Pseudomonas stutzeri is a bacterial species with a remarkable adaptation potential to many environments, including sites contaminated with organic toxic pollutants. The ability of strains of this species to use aromatic compounds as growth substrates is limited to specialized strains and is usually encoded chromosomally.16 Strain ST-9 of P. stutzeri is an isolate obtained from soil contaminated with toluene. Its whole genome has been sequenced as previously reported.17 Its length was estimated at 4.8 Mb and a total of 4.183 protein-coding genes were predicted, including a complete set of genes for the degradation of monoaromatic hydrocarbons. Transmission as well as scanning electron analyses indicated that strain ST-9 of P. stutzeri, which was grown in the presence of toluene possesses a plasmolysis space and is surrounded by an additional “material” with small vesicles in between.18 We assume that the morphological changes of P. stutzeri ST-9 are correlated to its response to the presence of toluene. The relatively new “omic” technologies which combine genomic and proteomic approaches have been applied in the present study to better understand the adaptive response of P. stutzeri ST-9 in using porins to support its resistance to the presence of toluene. The P. stutzeri ST-9 genome was investigated for the presence and organization of genes related to toluene catabolism and porin synthesis. The expression of both groups of genes was quantified in P. stutzeri ST-9 cells cultured in the presence of toluene or glucose as the sole carbon source through a proteomic approach. The outer membrane was separated and the percentage of porins as well as the porin types from both cultures were examined using mass spectrometry (MS) analysis. Additionally, porin fractions were isolated from cells of both cultures and examined as a toluene-uptake system using the liposome swelling assay.

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P. stutzeri ST-9 bacterial cells were grown in the mineral medium (MM) containing 1% glucose (MMG) or 100 mg L−1 toluene as the sole carbon source (MMT). A toluene concentration of 100 mg L−1 was chosen since a reduction in growth rate was observed when using a smaller (50 mg L−1) or a larger (250 mg L−1) toluene concentration. We assume that the smaller concentration led to a starvation effect, while the larger concentration led to bacterial damage. The cultures were grown for two generations, each lasted 25 h, at 30 °C with shaking at 170 rpm. Cytosolic Protein Separation

The bacterial cells were grown to the log phase in MMT as well as MMG, harvested at 8500g for 25 min at 4 °C in an Avanti JE, Beckman Coulter Centrifuge, Germany. The pellet was washed three times with 10 mM Tris-HCl, 10 mM MgSO4 buffer, pH 7.4 (buffer A). The pellet containing the bacterial cells was suspended with buffer A, sonicated (10 times, each 1 min, at an intensity of 100%) using a Heat Systems Ultrasonics Sonicator. The suspension was centrifuged at 8500g, and the soluble proteins of the supernatant were washed with 80% cold acetone. The precipitated cytosolic proteins were identified using MS analysis. Outer Membrane Separation

The cells were prepared as previously described for “Cytosolic Protein Separation” until the step of sonication, followed by centrifugation at 3000g for 5 min. N-Laurylsarcosine (0.5%) was added to the supernatant containing the cell envelopes in order to solubilize the inner membranes. The mixture was incubated for 30 min at room temperature and then centrifuged at 15 000g for 1 h. The resulting pellet containing the outer membranes was washed twice with buffer A, and lyophilized in a Labconco FreeZone, Kansas City, MO.19,20 The lyophilized pellet fraction containing crude outer membrane proteins (crude OMP) was further used for separation and purification of outer membrane porins. A portion of the crude OMP was washed twice in acetone (80%) to solubilize the membrane and precipitate the outer membrane proteins for mass spectrometry (MS) analysis. Separation and Purification of Outer Membrane Porins

The lyophilized pellet fraction containing the crude OMP (64 mg) was suspended in 50 mL of 20 mM Tris-HCl, pH 7.4 (buffer B), containing 0.7% w/v sodium dodecyl sulfate (SDS), incubated for 45 min at 30 °C, and then centrifuged at 15 000g19,20 (Avanti J-E, Beckman Coulter, Germany) for 1 h at 4 °C. The pellet, containing the partially purified protein, was washed once with buffer B, and centrifuged. The resulting pellet was extracted with 4 mL of buffer B containing 3.5% w/v octylglucoside (OG; Sigma, Israel) overnight at 37 °C. The mixture was centrifuged at 35 000g for 1 h at 4 °C. The supernatant containing the porins was dialyzed in buffer B for 24−48 h at 4 °C with frequent changes of buffer (porin fraction). The porin fraction concentration and profile was determined using Bradford and SDS-PAGE analyses, respectively. The proteins from the SDS-PAGE analysis were analyzed by MS. A porin fraction was lyophilized and used for the liposome swelling assay.

EXPERIMENTAL PROCEDURES

Media: Mineral Medium

Composition of mineral medium (MM) was (per liter) 2.44 g of Na2HPO4, 1.52 g of KH2PO4, 0.5 g of (NH4)2SO4, 0.2 g of MgSO4·7H2O, 0.05 g of CaCl2·2H2O, and 10 mL of trace element solution SL-4. The pH was adjusted to 7 by the addition of HCl (0.5 M) or NaOH (0.5 M). The trace element solution SL-4 contained 0.5 g of EDTA, 0.2 g of FeSO4·7H2O in 900 mL, plus 100 mL of trace element solution SL-6. Solution SL-6 contained (per liter) 0.1 g of ZnSO4·7H2O, 0.03 g of MnCl2·4H2O, 0.3 g of H3BO3, 0.2 g of CoCl2·6H2O, 0.01 g of CuCl2·2H2O, 0.02 g of NiCl2·6H2O, and 0.03 g of Na2MoO4·2H2O. All reagents and chemicals were analytical grade and were purchased from Sigma-Aldrich, Israel.

Reconstitution of Porins in Liposomes and Swelling Assay

Liposomes were prepared according to the procedure of Nikaido and Rosenberg (1983) and Wexler et al. (1992)19,21 LPhosphatidyl choline (2.4 μmol) and dicetyl phosphate (0.1 B


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Journal of Proteome Research μmol) were added to a round-bottom glass tube and dried at 50 °C (RV05ST, Werke, Germany). A porin fraction (10, 20 μg) in buffer A (final volume of 200 μL) was added to tubes containing the dried lipids and sonicated in an ultrasonic water bath (WiseClean, Wisd, USA) until the solution became translucent, after about 10 min. The preparations were incubated at 55 °C for 5 min, dried in a lyophilizer and kept in the dark for 45 min. A solution (300 μL) of 15% Dextran T40 in 5 mM Tris-HC1, pH 7.5 (buffer D), was added to the lipid layer 30 min before performing a liposome swelling assay. After incubation at room temperature, the tubes were shaken vigorously by hand to resuspend the liposomes. Liposomes with no added protein (porin fraction or BSA) were used as controls. The liposome swelling assays were performed with a Cary 200 scan spectrophotometer, Thermo, USA, and a Kinetika computer software. The liposome suspension (20− 25 μL) was added to an isotonic solution (final volume of 600 μL) of the various sugars in buffer D. Changes in optical densities at 400 nm were recorded. Since the initial rates are important, recording was begun as soon as possible after addition of the liposomes. The permeability of the porin is assumed to be proportional to the initial swelling rate, which is proportional to d[1/OD]/dt. The initial swelling rate can be calculated using the equation: d(1/OD)/dt = [1/(OD)2] × [d(OD/dt)]. The permeability rate (%) was calculated by comparing the permeability rate of the examined molecule to the permeability rate of arabinose (considered as 100%).21

LC Separation for Bands

The peptides mixture was resolved with a (7 to 45%) linear gradient of solvent B (95% acetonitrile with 0.1% formic acid) for 39 min followed by a 10 min gradient of 45 to 95% and 15 min at 95% acetonitrile with 0.1% formic acid in water at flow rates of 0.15 μL/min. Mass spectrometry was performed in a positive mode (m/z 350−2000, resolution 60 000) using repetitively full MS scans followed by collision induced dissociation (CID, at 35 normalized collision energy) of the 5 most dominant ions (>1 charges) selected from the first MS scan (isolation window, 2 m/z). The AGC settings were 5 × 105 for the full MS and 1 × 104 for the MS/MS scans. The intensity threshold for triggering MS/MS analysis was 3 × 104. A dynamic exclusion list was enabled with an exclusion duration of 30 s. LC Separation for Lysates

The peptides mixture was resolved with a (7 to 40%) linear gradient of solvent B (95% acetonitrile with 0.1% formic acid) for 120 min followed by gradient of 10 min gradient of 40 to 95% and 13 min at 95% acetonitrile with 0.1% formic acid in water at flow rates of 0.15 μL/min. Mass spectrometry was performed in a positive mode (m/z 300−1800, resolution 60 000) using repetitively full MS scan followed by collision induced dissociation (CID, at 35 normalized collision energy) of the 7 most dominant ions (>1 charges) selected from the first MS scan (isolation window, 2 m/z). The AGC settings were 5 × 105 for the full MS and 3 × 104 for the MS/MS scans. The intensity threshold for triggering MS/MS analysis was 3 × 104. A dynamic exclusion list was enabled with an exclusion duration of 30 s. The mass spectrometry data was analyzed using Proteome Discoverer 1.4 software with Sequest (Thermo) and Mascot (Matrix Science) algorithms against the P. stutzeri ST-9 genome (accession number JXJL00000000, 4183 entries) section of the NCBI-nr database with mass tolerance of 20 ppm for the precursor masses and 0.05 Da for the fragment ions. Oxidation on methionine was accepted as variable modifications and carbamidomethyl on cysteine was accepted as static modifications. Minimal peptide length was set to six amino acids and a maximum of two miscleavages was allowed. Minimum number of peptides per protein was set to 2. Peptide-level false discovery rates (FDRs) were filtered to 1% using the target-decoy strategy. Semiquantitation was performed by calculating the peak area of each peptide based on its extracted ion currents (XICs). The area of the protein is the average of the three most intense peptides from each protein. To eliminate possible contaminants from the identifications, an analysis against the entire UniprotKB database was performed. The reports from this analysis were loaded together with the reports from the analysis against P. stutzeri ST9 in the Proteome Discoverer viewer. No peptide from P. stutzeri ST9 was identified as a contaminant peptide in the general analysis. The MS analyses were performed at the Smoler Proteomics Center at the Technion, Israel.

SDS-PAGE

Protein homogeneity and molecular mass were determined by SDS-PAGE in 4% stacking and 12.5% separating polyacrylamide gels, using Dalton Mark VII-L mixtures (Sigma) as standards. Electrophoresis was carried out at 35 mV under a running buffer containing 0.025 M Tris-HCl, pH 8.3, 0.192 M glycine, and 0.1% SDS. After electrophoresis, the proteins in the gels were stained with 0.025% Coomassie brilliant blue R250 in a solution of 40% methanol and 7% acetic acid. Proteolysis and Mass Spectrometry Analysis

Proteolysis. Proteolysis of the acetone pellets: the pellets were centrifuged at 5900g for 10 min at 4 °C. The acetone was removed and the pellet was resuspended in 8 M urea, 400 mM ABC (ammonium bicarbonate), 10 mM DTT (dithiothreitol), and then reduced (60 °C for 30 min), modified with 35.2 mM iodoacetamide in 100 mM ammonium bicarbonate (in the dark at room temperature for 30 min) and digested in 2 M urea, 100 mM ABC with modified trypsin (Promega) at a 1:100 enzymeto-substrate ratio, overnight at 37 °C. An additional second trypsinization was performed for 4 h. Proteolysis of the Coomassie blue-stained band in the gel: The blue-stained band was reduced with 10 mM DTT, incubated at 60 °C for 30 min, alkylated with 10 mM iodoacetamide at room temperature for 30 min, and proteolyzed overnight at 37 °C using modified trypsin (Promega) at a 1:100 enzyme-to-substrate ratio. Mass Spectrometry Analysis. The tryptic peptides were analyzed by LC−MS/MS using an Orbitrap mass spectrometer (Thermo) fitted with a capillary HPLC (NanoLC 1D plus, Eksigent). The peptides were loaded onto a homemade capillary column (20 cm, 75 μm i.d.) packed with Reprosil C18-Aqua (Dr Maisch GmbH, Germany) in solvent A (0.1% formic acid in water).

Genomic Analysis

The amino acid sequences of the genes related to toluene catabolism and porin synthesis were analyzed and phylogenetically assigned using the BLASTP tool available at the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST/) and using the JGI webpage (http://genome.jgi.doe.gov/). A gene was considered present following the 50/50 criterion (similarity higher than 50% in at least 50% of the sequence coverage with a gene in the database). C


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Journal of Proteome Research

Figure 1. Homologous pathways for the aromatic ring cleavage in P. stutzeri ST-9, Marinobacter sp. LQ44, and P. putida plasmid pWW0. For plasmid pWW0 only the lower pathway is indicated. Genes with homologous function are indicated with the same color. The amino acid identities percentage for each gene are indicated in brackets.

Table 1. List of Genes Annotated in the P. stutzeri ST-9 Toluene Operon Pathway and in Closely-Related Strains. Percentages Identities Are Calculated against the Corresponding ST-9 Genesa

a

Gi

locus_tag

gene annotation

816240405 816240428

PK34_02515 PK34_02650

816240427 816240426 816240425

PK34_02645 PK34_02640 PK34_02635

816240424

PK34_02630

816240423 816240422 816240421 816240420 816240419

PK34_02625 PK34_02620 PK34_02615 PK34_02610 PK34_02605

816240418

PK34_02600

816240417 816240416 816240415 816240414 816240413

PK34_02595 PK34_02590 PK34_02585 PK34_02580 PK34_02575

phage integrase family protein Fis family transcriptional regulator phenol hydroxylase P0 subunit phenol hydroxylase P1 subunit monooxygenase/phenol hydroxylase P2 subunit phenol 2-monooxygenase P3 subunit phenol hydroxylase P4 subunit phenol hydroxylase P5 subunit ferredoxin catechol 2,3-dioxygenase 2 OH-muconate semialdehyde dehydrogenase 2-hydroxy-6-oxo-2,4heptadienoate hydrolase 2-keto-4-pentenoate hydratase Acetaldehyde dehydrogenase 4-hyroxy-2-oxovalerate aldolase 4-oxalocrotonate decarboxylase 2-hydroxymuconate tautomerase

Marinobacter LQ44

Pseudomonas citronelollis PI1

Marinobacter adhaerens HP15

Marinobacter algicola DG893

pWW0 TOL plasmid

82.2

100 97

77

78

64

79 96 100

97 87 91

87 84 97

85 82 92

-

98

93

90

93

-

96 98 99 95 98

87 97 95 98 73

73 96 75 86 92

79 93 88 90 90

52 80 80

96

98

89

88

71

99 99 99 93 82

97 97 97 98 100

96 95 95 95 -

90 94 94 95 -

81 81 79 83 -

Notation: -, not present.

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Statistics

Each experiment was performed at least in triplicate (unless stated otherwise). All primary data are presented as means ± standard deviations. The difference between two means was compared by ANOVA single factor analysis. The difference between the results was considered significant if the P-value was less than 0.05.

RESULTS AND DISCUSSION

The presence and organization of genes related to toluene catabolism and porins were investigated, with emphasis on those differentially expressed in cells grown on glucose or on toluene as the sole carbon and energy source. The aerobic pathways for the catabolism of aromatic hydrocarbons are organized in the upper and lower routes. D


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Table 2. MS Analysis of the Porins from Cultures Grown in the Presence of Toluene or Glucose as the Sole Carbon Source. The Genomic Context of the Detected Porins Is Indicated in the Table percentagea (%) annotated gene

length (amino acids)

MW [kDa]

Gi ST-9

locus_tag

pI

T

G

genomic context

816236112 816234930

PK34_17665 PK34_20775

OprD OprD

419 439

45.6 48.0

5.48 4.79

5.01 9.44

5.27 16.72

816237560

PK34_11635

OprD

419

45.7

4.89

12.00

28.12

816240618

PK34_00695

OprD

420

45.7

5.13

0.13

0.12

816236093

PK34_17570

OprD

419

45.7

5.99

4.71

4.32

816240236

PK34_01560

OprD

420

45.6

5.03

0.002

N.D

816237737 816238507

PK34_10855 PK34_08200

OmpA OmpA

330 260

35.4 28.7

4.79 5.45

35.70 0.62

17.32 2.29

816236199

PK34_17365

OmpA

200

21.3

4.46

29.33

22.02

816239593

PK34_04430

OmpA

217

22.2

9.54

2.27

1.62

816236488

PK34_16175

OmpA

296

32.6

10.30

0.29

0.11

816236011 816237616 816237801

PK34_17955 PK34_11920 PK34_11200

maltoporin Maltoporin Porin 4

430 407 395

47.1 45.2 42.9

4.80 5.47 4.65

0.47 N.D 0.02

0.96 0.86 0.28

located upstream of amino acid transporter system located in a short contig, closest to an hypothetical protein. In other Pseudomonas genomes it is related to heavy metal resistant proteins (Cu, Cd, Zn) not related to sugars, aromatic hydrocarbons transport, nor to heavy metal resistance not related to sugars, aromatic hydrocarbons transport, nor to heavy metal resistance. Closest to a phosphate:Na+ symporter, and downstream of two genes related to a two-component system related with the OmpR family close to mercuric ion transporter and mannitol/chloroaromatic compound transport system not related to sugars, aromatic hydrocarbons transport, nor to heavy metal resistance related to a zinc transporter, and inorganic ion transport and metabolism not related to sugars, aromatic hydrocarbons transport, nor to heavy metal resistance. Upstream is closed to a multidrug eflux pump related to the transporter of ions and amino acids (multisubunit potassium/proton antiporter, and threonine/homoserine eflux transporter) not related to sugars, aromatic hydrocarbons transport, nor to heavy metal resistance not related to sugars, aromatic hydrocarbons transport, nor to heavy metal resistance related to starch and maltose catabolism Related to glucose transport Not related to sugars, aromatic hydrocarbons transport, nor to heavy metal resistance

a

The porin percentage, a specific porin percentage of the total porins that were found in the outer membrane (an average of 3 different experiments); T, from a culture grown in the presence of toluene and G, from a culture grown in the presence of glucose. N.D., not detected, it is possible that the protein exists, but is below the threshold of the MS analysis.

The enzymes in the upper route partially oxidize the aromatic ring and the produced metabolites are further cleaved to metabolites that are introduced into the tricarboxylic acid cycle by the enzymes of the lower route. The upper route for toluene degradation can occur by oxidation of the methyl group22 or by oxygenation of the aromatic ring in different positions mediated by mono- or dioxygenases.23−25 The product of the upper pathway in toluene catabolism is catechol, which can be further metabolized by an ortho (catechol 1−2 dioxygenase) or a meta (catechol 2−3 dioxygenase) pathway. The genome sequence of strain ST-9 was previously reported.17 No plasmid was detected in the analysis. A putative operon encoding for an aromatic degradation pathway was found in contig 2 (393 712 bp). The operon comprises a fragment of 19 158 bp, which includes 21 genes, one of them with a regulatory function (positions 384977−386677; 566 amino acids) (Figure 1). The corresponding Gi accession numbers and locus tag identifiers are indicated in Table 1 and Figure 1. Interestingly, the degradative genes in strain ST-9 were localized in the chromosome, close to a phage type integrase family protein (position 359216−360700; 494 amino acids), which suggests a plausible horizontal gene event for the acquisition of this gene cluster. A closely similar integrase was found only in Pseudomonas citronelollis PI1, together with a homologous set of genes (Table 1). Gene organization of this pathway was compared with the archetypal TOL plasmid pWW0 encoding degradation of toluene and several substituted toluenes in P. putida mt-2 (Figure 1 and Table 1). The upper route is activated in the pWW0 plasmid by XylR in the

presence of toluene as effector. XylS is activated by the meta operon, but the expression of XylS can be induced directly by the presence of effectors or by high levels of expression induced by XylR.26 XylS is the operon transcriptional activator of the TOL plasmid in xyl operons. XylS activates the xyl XYZLTEGFJQKIH operon which is necessary for the degradation of m-xylene, p-xylene, and toluene. A gene encoding a Fis family transcriptional regulator was found in strain ST-9, upstream of the aromatic degradative genes (1701 bp, coordinates 384977−386677), which is 66% identical to xylR. Next to the regulator, a set of six genes was annotated as a multicomponent monooxygenase with six subunits (P0, P1, P2, P3, P4, and P5) acting as phenol hydroxylase and predicted to produce methyl-catechol. In general, the specificities of multicomponent phenolhydroxylases are broad, and the toluene monooxygenases are considered subgroups. Few significant hits were found in the sequence databases for the phenol hydroxylase genes. Only two marine strains in the JGI database showed a homologous set of genes in a best bidirectional analysis: Marinobacter adherens strain HP15, and Marinobacter algicola DG893. M. algicola is able to grow on nhexadecane and n-tetradecane, but none of these strains has been reported to grow on toluene or phenol.27,28 The closest phenol hydroxylase genes are of Marinobacter sp. LQ44 an isolated strain from marine sediment (Table 1 and Figure 1). Only four of the six subunits (P2 to P5) were found in other genomes of Pseudomonas, in strains OX1 and 2A20, with similarities between 80 and 90%. In Pseudomonas sp. OX1 (formerly P. stutzeri OX1),29 a multicomponent phenol E


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fraction was examined as an uptake system using the liposome swelling assay.20,21

hydroxylase has been described related to the wide spectrum of aromatics degraded by this strain.30 Five phenol and toluene degrading marine P. stutzeri strains were analyzed in a recent study,31 and the genome region of strain 2A20 coding for the degradation of aromatics was sequenced. The genome sequence revealed complete TMO- (toluene monooxygenase) and Xylupper pathway operons together with a phenol-degrading operon. The lower pathway in strain ST-9 is encoded by nine genes which are highly similar to the corresponding genes in the TOL plasmid (Figure 1 and Table 1). This pathway transforms 3methylcatechol to metabolites of the central metabolism (pyruvate and acetaldehyde). The highest similarity in the databases of catechol 2,3-dioxygenase of strain ST-9 has 89% similarity to the corresponding gene in Marinobacter LQ44, and 75% similarity to the corresponding gene in the naphthalenedegrading strains P. balearica DSM 6083, P. stutzeri AN10 (=CCUG 29243), and the XylE gene of the TOL plasmid pWW0. Toluene operon genes were upregulated in cells cultured on toluene. As indicated in the Supporting Information (Table S1: MS analysis of cytosolic proteins from P. stutzeri ST-9 that was grown on toluene (Tc) or glucose (Gc)) the upregulation in the MS analysis was 3−30 fold, depending on the specific protein as demonstrated in a single experiment.

Examination of the Percentage of the Porins from the Crude Outer Membrane Proteins and Identification of the Porin Types

For studying the percentage of porins from the outer membrane protein fraction, the cells were harvested, sonicated, and the inner membrane was solubilized. The mixture was centrifuged and the pellet containing the crude outer membrane protein (crude OMP) was collected. The percentage of porins in the crude OMP was 73% and 54% in the culture that was grown in the presence of toluene and in the presence of glucose, respectively. The description and percentage of each porin from the total porin of each culture (T and G, toluene and glucose, respectively) are presented in Table 2. (Table S2 Supporting Information: MS analysis of outer membrane proteins from culture that was grown on toluene (T1, T2, T3) or glucose (G1, G2, G3); three replicates). The identification of the porins from the crude OMP showed that the porins belong to two main superfamilies, OprD (with six different accession numbers) and OmpA (with five different accession numbers). The percentage of expression of maltoporin and outer membrane protein 816237801, which was ascribed to porin 4, was lower than 2% in both cultures. No data on porin 4 was found in the literature. The expressed OmpA porins were 68.21% and 43.36% in the presence of toluene and glucose, respectively. The expressed OprD porins were 31.29% and 54.55% in the presence of toluene and glucose, respectively. These results show that upregulation of OmpA and downregulation of OprD occur in the presence of toluene (Figure 2). The difference in the expression between OmpA and OprD in cells cultured in the presence of toluene or glucose was significant (P < 0.05).

Porin Genes

MS analysis revealed 65 outer membrane proteins in the outer membrane fraction. They were identified by BLAST as membrane proteins, including not only porins, but also channel proteins, ligand-gated channel proteins, ABC transporter substrate-binding proteins, multidrug transporters, or proteins associated with TonB-dependent receptors. Only 14 of these 65 proteins (Table S2 Supporting Information, MS analysis of the outer membrane proteins from culture that was grown on toluene (T1, T2, T3) or glucose (G1, G2, G3), three replicates, and Table S3 Supporting Information, 65 outer membrane proteins divided to groups according to annotation in Table S2) were identified by BLAST as porins and were further analyzed. A protein was considered a porin if it met the 50/50 criterion (similarity higher than 50% in at least 50% of the sequence coverage with a porin in the database). Main porins detected in P. stutzeri ST-9 belonged to the OmpA and OprD superfamilies. Six porins were affiliated to the OprD family, 5 to the OmpA family, 2 were annotated as maltoporins, and one was affiliated to the porin 4 family. The genomic context in the annotated genome was analyzed for the 14 porins: seven of the detected porins were not related to sugars, aromatic hydrocarbon transport, nor heavy metal resistance genes; three were related to inorganic molecule transport and metabolism or amino acids transporters; two maltoporins were detected (Gi 816236011 was related to genes involved in starch and maltose catabolism, and Gi 816237616 was related to glucose transport) (Table 2).

Figure 2. Pie chart of porins superfamily of P. stutzeri ST-9 which were found in the outer membrane proteins of the cultures that were grown in the presence of toluene (left) or glucose (right) as the sole carbon source.

Several studies have shown that exposure to aromatic hydrocarbons leads to a change in porin types as well as concentrations. A decrease in P. putida KT2440 porins: OprG, OprB, OprQ and OprF was observed when bacteria were exposed to 800 mg L−1 phenol. Exposure to phenol led to porin modifications at the post-translational level, as was observed by an increase in the porin multiple forms.32 The decrease in porin concentrations may be considered as an adaptive response to reduce the outer membrane’s permeability to phenol. It was previously hypothesized that the phenomenon of a decrease in

Porin Expression in P. stutzeri ST-9 Bacterial Cells Cultured in Toluene or Glucose as the Sole Carbon Source

In attempt to study the function of P. stutzeri ST-9 bacterial porins as an adaptive response to toluene exposure, cultures of P. stutzeri ST-9 were grown in MM containing toluene or glucose as the sole carbon source. The porins percentage in the outer membrane protein fraction, as well as the porin types from each culture, were identified.19 In addition, the porin F


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protein found in the protein band (containing the “porin fraction”) from both cultures was OprD with five different Gi accession numbers in the NCBI database (816237560, 816236112, 816236093, 816234930, and 816240618). Its percentage was 87% and 90% when the culture was grown in the presence of toluene and in the presence of glucose, respectively.

porin concentration occurs in parallel to activation of efflux pumps in attempt to reduce the concentration of the intracellular phenol. P. putida CA-3 OmpA upregulation was observed when it was grown on styrene as the sole carbon and energy source,33 as well as when P. putida KT2440 was grown on lipophilic herbicides.34 P. putida S12 OmpF was downregulated in the presence of toluene, and it was suggested that during toluene stress there is a shutting down of the OmpF channel.35 Down regulation of OprD was observed when P. aeruginosa strain PseA was grown on high amounts of hydrophobic alkanes such as n-hexane, cyclohexane, n-heptane, n-octane, n-decane, n-dodecane, and n-tetradecanea.36 Down regulation of Escherichia coli OprD was also observed in the presence of salicylate, a weak aromatic acid.37 This phenomenon indicates the crucial role of the porin OprD in the mechanism of the bacterium’s solvent tolerance.36 de Bont found that no gradient of toluene was observed from the cytoplasmic membrane to the outer membrane when the bacterium was grown on toluene.38 From our results and from the data in the literature we assume that the differences in the porin composition of cells grown with toluene and glucose is mainly an adaptive response against toluene toxicity rather than uptake for better degradation.

Examination of the “Porin Fraction” (Containing about 90% OprD) as an Uptake System Using the Liposome Swelling Assay

To gain an understanding of basic biological mechanisms, it is often necessary to reduce the number of parameters. A liposome, i.e. a spherical phospholipid bilayer, reconstituted with porins may offer an advantage for understanding the function of porins in the uptake of molecules. A liposomeswelling assay for measuring diffusion rates of solutes through porin channels was previously developed by Nikaido and Rosenberg21 and was used for studying the development of resistance to several antibiotics.19 In this study, a liposome swelling assay was performed in attempt to study whether bacterial porins serve as a channel for toluene penetration into the bacterial cells. Molecules that enter the liposome through the porins lead to liposome swelling which is monitored spectrophotometrically. The diffusion rate of several known sugars was measured to confirm that the porin fraction was indeed inserted into the liposome surface. The diffusion rate of the sugars was compared to well-known data in the literature. Liposomes reconstituted with a porin fraction (containing about 90% OprD) from the culture that was grown in the presence of toluene (designated T-proteoliposome) or glucose (designated G-proteoliposome) were used for measuring the toluene diffusion rate. Two negative controls were performed: a liposomes fraction composed solely of a phospholipid mixture (L-phosphatidylcholine and dicetyl phosphate) and a liposomes fraction that contained bovine serum albumin (BSA) instead of porins. The control liposome fractions were tested for passive diffusion of sugars and toluene through the phospholipid bilayer or through a protein (BSA) without a channel. As shown in (Figure S1 Supporting Information: liposome swelling assay for sugars and toluene diffusion rate using control liposomes constituting of a phospholipid mixture, a control experiment), no diffusion of sugars or toluene into the control liposomes was observed. The same results were obtained when BSA molecules were reconstituted into liposomes (data not shown). Insertion of 10 μg and 20 μg of the “porin fraction” into the liposomes showed that the diffusion rate of arabinose increased with the amount of incorporated protein into T-proteoliposome and G-proteoliposome (Figure 4). The diffusion rate of sugars and toluene through porins was measured in fractions of T-proteoliposome and G- proteoliposome (each proteoliposome fraction contained 10 μg of porins) (Figure 5A,B). Because of its high molecular weight, stachyose (MW 666 Da) did not permeate these liposomes. However, arabinose (MW 150 Da) had the highest penetration rate, due to its lowest molecular weight. The permeability (d(1/OD)/dt) and permeability rates (compared to arabinose which was considered as 100%) of the T-proteoliposome and G-proteoliposome for different sugars and toluene are shown in Table 3. The results shows that the relative permeation rates of the tested sugars through T-proteoliposome and G-proteoliposome is relative to their

Isolation of the “Porin Fraction” from P. stutzeri ST-9 Grown in the Presence of Toluene or Glucose as the Sole Carbon Source

The crude OMP was treated with SDS to enrich the fraction with outer membrane proteins that are stable to SDS treatment, while outer-membrane proteins that are associated with peptidoglycan remained in the SDS-insoluble fraction. This was followed by extraction with octyl-glucopyranoside, a selective solubilizer, which led to a fraction enriched with porins (“porin fraction”).19,39,40 The “porin fractions” were subjected to SDS-PAGE (12.5%) and the proteins profile is shown in Figure 3. A main protein band (designated by an

Figure 3. SDS-PAGE (12.5%). “Porin fraction” from P. stutzeri ST-9 cells that were grown in MMG (lane 1) or in MMT (lane 2) and molecular weight marker proteins (lane 3).

arrow) with an approximate molecular weight of 45 kDa was present in both cultures (MMG as well as MMT). These protein bands (same amount of proteins) were analyzed using LC−MS/MS and identified using the Proteome Discoverer software NCBI against P. stutzeri ST-9 (Table S4 Supporting Information: MS analysis of PAGE protein bands of the “porin fraction” that was isolated from culture that was grown on toluene (T, T1) or glucose (G, G1); two replicates). The main G


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MW. The diffusion rates of the sugars decrease with the increase in their size. This phenomenon has been described for several porins.40 ANOVA single factor analysis showed that there is no significant difference between the permeability and permeability rate of T-proteoliposome and G-proteoliposome for toluene. No significant difference was found in the permeability of both liposomes for the two toluene concentrations (0.001 and 0.01 M). A study of the of the toluene uptake mechanism into bacterial cells was previously performed using a toluene analogue dye (3-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)3-toluene; NBDT). The uptake of the NBDT by P. putida mt-2 and F1, decreased by 25% and 42%, respectively, as a result of adding a porin inhibitor (cadaverine). This result may indicate a regulatory involvement of porins in the uptake of toluene.41 A 40 kDa protein of P. putida CSV86 (closely resembled the OprB porin of P. putida KT2440) was extracted from a culture that was grown on glucose and reconstituted in liposomes. A rapid swelling of the liposomes was observed in the presence of an isosmotic concentration of glucose. However, when the proteoliposomes were reconstituted with the 40 kDa proteins from cells grown on benzyl alcohol, naphthalene, or succinate, no significant liposome swelling was observed in the presence of glucose.42

Figure 4. Arabinose diffusion rate through T-proteoliposome (⧫) and G-proteoliposome (■).

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CONCLUSIONS

Pseudomonas stutzeri ST-9 genes related to toluene catabolism and porin synthesis were investigated. Toluene degradative genes are localized in the chromosome close to a phage-type integrase, which suggests a horizontal gene transfer mechanism in the evolution of strain ST-9 to the toluene catabolism. A regulatory gene and 21 genes related to an aromatics degradation pathway are organized as a putative operon and were upregulated in the presence of toluene. Fourteen porins were identified in the ST-9 genome. Two main porin superfamilies were found: OmpA and OprD each contained several porins with different accession numbers. OmpA, which is known as a porin that maintains the structural integrity of the cell surface, was upregulated in the presence of toluene. A liposome-swelling assay for measuring diffusion rates of toluene through porin channels was examined with an OprD fraction. It was found that liposomes inserted with OprD led to toluene uptake. However, there was no difference in toluene uptake when the OprD fraction was isolated from a culture grown on toluene or glucose.

Figure 5. Liposome swelling assay for monitoring sugars and toluene diffusion rate using T-proteoliposome (A) and G- proteoliposome (B). stachyose (□); arabinose (◇); 0.01 M toluene (△); 0.001 M toluene (×); lactose (+); galactose (∗) and N-acetylglucose amine (○).

Table 3. Permeability (d(1/OD)/dt) and Permeability Rates of the T-Proteoliposome and G-Proteoliposome for Different Sugars and Toluene T-proteoliposome sugar

MW (Da)

arabinose galactose NAGa lactose stachyose 0.01 M toluene 0.001 M toluene

150 180 221 342 666 92 92

G-proteoliposome

permeability d(1/OD)/dt

permeability rate (%)

± ± ± ±

100 66 46 21 0 121 112

0.24 0.16 0.11 0.05 0 0.29 0.27

0.02 0.01 0.03 0.03

± 0.04 ± 0.02

permeability d(1/OD)/dt

permeability rate (%)b

± ± ± ±

100 76 52 28 0 107 114

0.29 0.22 0.15 0.08 0 0.31 0.33

0.03 0.01 0.03 0.02

± 0.04 ± 0.06

a

NAG, N-acetyl glucoseamine. bPermeability rate (%), permeability rate of different sugars compared to the permeability of arabinose which is considered as 100%. H


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(6) Stover, C. K.; Pham, X. Q.; Erwin, A. L.; Mizoguchi, S. D.; Warrener, P.; Hickey, M. J.; Brinkman, F. S.; Hufnagle, W. O.; Kowalik, D. J.; Lagrou, M.; Garber, R. L.; Goltry, L.; Tolentino, E.; Westbrock-Wadman, S.; Yuan, Y.; Brody, L. L.; Coulter, S. N.; Folger, K. R.; Kas, A.; Larbig, K.; Lim, R.; Smith, K.; Spencer, D.; Wong, G. K.; Wu, Z.; Paulsen, I. T.; Reizer, J.; Saier, M. H.; Hancock, R. E.; Lory, S.; Olson, M. V. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 2000, 406, 959−964. (7) Ried, G.; Koebnik, R.; Hindennach, I.; Mutschler, B.; Henning, U. Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12. Mol. Gen. Genet. 1994, 243, 127−135. (8) Koebnik, R.; Locher, K. P.; Van Gelder, P. Structure and function of bacterial outer membrane proteins: barrels in a nutshell. Mol. Microbiol. 2000, 37, 239−253. (9) Smith, S. G. J.; Mahon, V.; Lambert, M. A.; Fagan, R. P. A Molecular swiss army knife: OmpA structure, function and expression. FEMS Microbiol. Lett. 2007, 273, 1−11. (10) Nikolaki, A.; Papadioti, A.; Arvaniti, K.; Kassotaki, E.; Langer, J. D.; Tsiotis, G. The membrane complexome of a new Pseudomonas strain during growth on lysogeny broth medium and medium containing glucose or phenol. EuPa Open Proteomics 2014, 4, 1−9. (11) Trias, J.; Nikaido, H. Protein D2 channel of the Pseudomonas aeruginosa outer membrane has a binding site for basic amino acids and peptides. J. Biol. Chem. 1990, 265, 15680−15684. (12) Tamber, S.; Ochs, M. M.; Hancock, R. E. W. Role of the novel OprD family of porins in nutrient uptake in Pseudomonas aeruginosa. J. Bacteriol. 2006, 188, 45−54. (13) Ochs, M. M.; Lu, C. D.; Hancock, R. E.; Abdelal, A. T. Amino acid-mediated induction of the basic amino acid-specific outer membrane porin OprD from Pseudomonas aeruginosa. J. Bacteriol. 1999, 181, 5426−5432. (14) Li, H.; Luo, Y. F.; Williams, B. J.; Blackwell, T. S.; Xie, C. M. Structure and function of OprD protein in Pseudomonas aeruginosa: from antibiotic resistance to novel therapies. Int. Int. J. Med. Microbiol. 2012, 302, 63−68. (15) Nelson, K. E.; Weinel, C.; Paulsen, I. T.; Dodson, R. J.; Hilbert, H.; Martins dos Santos, V. A. P.; Fouts, D. E.; Gill, S. R.; Pop, M.; Holmes, M.; Brinkac, L.; Beanan, M.; DeBoy, R. T.; Daugherty, S.; Kolonay, J.; Madupu, R.; Nelson, W.; White, O.; Peterson, J.; Khouri, H.; Hance, I.; Chris, L. P.; Holtzapple, E.; Scanlan, D.; Tran, K.; Moazzez, A.; Utterback, T.; Rizzo, M.; Lee, K.; Kosack, D.; Moestl, D.; Wedler, H.; Lauber, J.; Stjepandic, D.; Hoheisel, J.; Straetz, M.; Heim, S.; Kiewitz, C.; Eisen, J. A.; Timmis, K. N.; Düsterhöft, A.; Tümmler, B.; Fraser, C. M. Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ. Microbiol. 2002, 4, 799−808. (16) Lalucat, J.; Bennasar, A.; Bosch, R.; García-Valdés, E.; Palleroni, N. J. Biology of Pseudomonas stutzeri. Microbiol. Mol. Biol. Rev. 2006, 70, 510−547. (17) Gomila, M.; Busquets, A.; García-Valdés, E.; Michael, E.; Cahan, R.; Nitzan, Y.; Lalucata, J. Draft genome sequence of the toluenedegrading Pseudomonas stutzeri strain ST-9. Genome Announc 2015, 3, e00567. (18) Michael, E.; Nitzan, Y.; Langzam, Y.; Luboshits, G.; Cahan, R. Effect of toluene on Pseudomonas stutzeri ST-9 morphology plasmolysis, cell size, and formation of outer membrane vesicles. Can. J. Microbiol. 2016, 62, 682−691. (19) Wexler, H. M.; Getty, C.; Fisher, G. The isolation and characterisation of a major outer-membrane protein from Bacteroides distasonis. J. Med. Microbiol. 1992, 37, 165−175. (20) Magalashvili, L.; Pechatnikov, I.; Wexler, H. M.; Nitzan, Y. Isolation and characterization of the Omp-PA porin from Porphyromonas asaccharolytica, determination of the omp-PA gene sequence and prediction of Omp-PA protein structure. Anaerobe 2007, 13, 74− 82. (21) Nikaido, H.; Rosenberg, E. Y. Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins. J. Bacteriol. 1983, 153, 241−252.

ASSOCIATED CONTENT

S Supporting Information *

. . . . . . The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jproteome.6b01044. Table (S3), 65 outer membrane proteins divided to groups according to annotation in Table S2; Table (S5), proteins page parameters, explanation of the parameters shown in Tables S1, S2, and S4; Figure (S1), liposome swelling assay for sugars and toluene diffusion rate using control liposomes constituting of a phospholipid mixture, a control experiment (PDF) Table (S1), MS analysis of cytosolic proteins from P. stutzeri ST-9 that was grown on toluene (Tc) or Glucose (Gc) (XLSX) Table (S2), MS analysis of outer membrane proteins from cultures that were grown on toluene (T1, T2, T3; three replicates) or glucose (G1, G2, G3; three replicates) (XLSX) Table (S4), MS analysis of protein bands from PAGE of the “porin fraction” that was isolated from culture that was grown on toluene (T, T1) or glucose (G, G1); two replicates (XLSX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 972-54-774-0293. ORCID

Rivka Cahan: 0000-0001-9783-4033 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support at the University of the Balearic Islands was obtained from the Spanish MINECO through project CGL2015-70925-P with FEDER cofunding. Margarita Gomila is the recipient of a postdoctoral contract from the Conselleria d’Educació, Cultura i Universitats del Govern de les Illes Balears and the European Social Fund. This research was also supported in part by the Samaria and Jordan Rift Valley Regional R&D Center and the Research Authority of the Ariel University, Israel. We also want to thank Dr. Tamar Ziv and Ms. Keren Bendalak from the “Smoler Proteomics Center at the Technion, Israel” for the excellent technical assistance and the helpful advice in mass spectrometric analysis.

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REFERENCES

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