Simultaneous catabolism of plant - derived aromatic compounds results in enhanced growth for 1 members of the Roseobacter lineage 2

18 Plant-derived aromatic compounds are important components of the dissolved organic carbon 19 pool in coastal salt marshes, and their mineralization by resident bacteria contributes to carbon 20 cycling in these systems. Members of the roseobacter lineage of marine bacteria are abundant in 21 coastal salt marshes and several characterized strains, including Sagittula stellata E-37, utilize 22 aromatic compounds as primary growth substrates. The genome sequence of S. stellata contains 23 multiple, potentially competing, aerobic ring-cleaving pathways. Preferential hierarchies in 24 substrate utilization and complex transcriptional regulation have been demonstrated to be the 25 norm in many soil bacteria that also contain multiple ring-cleaving pathways. The purpose of this 26 study was to ascertain whether substrate preference exists in S. stellata when provided a mixture 27 of aromatic compounds that proceed through different ring-cleaving pathways. We focused on 28 the protocatechuate (pca) and the aerobic benzoyl-CoA (box) pathways and the substrates known 29 to proceed through them, p-hydroxybenzoate (POB) and benzoate, respectively. When these two 30 substrates were provided at non-carbon limiting concentrations, temporal patterns of cell density, 31 gene transcript abundance, enzyme activity, and substrate concentrations indicated S. stellata 32 simultaneously catabolized both substrates. Furthermore, enhanced growth rates were observed 33 when S. stellata was provided both compounds simultaneously compared to cells grown singly 34 with an equimolar concentration of either substrate alone. This simultaneous catabolism 35 phenotype was also demonstrated in another lineage member, Ruegeria pomeroyi DSS-3. These 36 findings challenge the paradigm of sequential aromatic catabolism reported for soil bacteria and 37 contribute to the growing body of physiological evidence demonstrating the metabolic versatility 38 of roseobacters. 39 on Jne 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom Introduction 40 The structural diversity of aromatic compounds in the environment is influenced by the various 41 mechanisms that produce them, including naturally occurring abiotic (1) and biotic processes (2, 42 3) as well as those of anthropogenic origin (4). Regardless of the source, the dissolved organic 43 carbon pool containing aromatic compounds in nature is typically structurally heterogeneous (5, 44 6). This is an important consideration for microbial degradation and has received significant 45 attention in studies examining the catabolism of aromatic compound mixtures classified as 46 environmental pollutants (7). Considerably less attention has focused on microbial physiology of 47 mixtures of naturally occurring aromatic compounds, such as those derived from lignin, the 48 structural component of vascular plants (8). 49 50 Microbial mineralization of aromatic compounds plays an important role in global carbon 51 cycling and bioremediation. Bacterial aromatic catabolism is described as “catabolic funneling” 52 where upper (also called peripheral) pathways transform a diverse suite of aromatic compounds 53 into one of a limited number of intermediates that are then subject to ring cleavage (9). The β54 ketoadipate pathway is one such pathway and is a paradigm for aerobic catabolism of plant55 derived aromatics (10, 11). In organisms possessing one or both its branches, peripheral 56 pathways generate dihydroxylated intermediates, either catechol or protocatechuate. Alternative 57 aerobic ring-cleaving mechanisms are also present in bacteria, including epoxidation of CoA58 thioesterified aromatics, as occurs in the benzoyl-CoA (box) pathway for benzoate degradation 59 (12). These catabolic pathways are typically under tight transcriptional regulation (13, 14) and 60 subject to catabolite repression (15-17). 61 62 on Jne 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom Previous studies reveal substrate preferences are the norm when bacterial strains are presented 63 with a mixture of aromatic compounds (9). This phenomenon has been best characterized for 64 select soil-derived bacteria provided mixtures of benzoate and p-hydroxybenzoate (18-20). Each 65 of these compounds is ultimately processed through parallel ring-cleavage branches of the β66 ketoadipate pathway, referred to as the catechol (cat) and protocatechuate (pca) branches (21), 67 respectively. The hierarchical nature of substrate utilization profiles has been mechanistically 68 explained as cross-regulation and typically involves transcriptional regulation by pathway 69 metabolites or regulatory proteins (e.g., (22, 23)). However, the extent to which similar 70 hierarchies exist in environmentally relevant microbes is not yet clear. Furthermore, as 71 environmental bacteria are dependent upon growth substrate pools that are highly heterogeneous 72 in composition (24, 25), mixed substrate studies may provide the foundation for a better 73 understanding of bacterial catabolism in nature. 74 75 The Roseobacter lineage of marine bacteria is numerically abundant and active in the world’s 76 oceans (26, 27). Group members are most dominant in coastal environments, including salt 77 marshes heavily influenced by lignocellulosic vascular plant material (28). Roseobacter ubiquity 78 and success in the oceans has been attributed, in part, to their ability to use a large repertoire of 79 growth substrates, including aromatic compounds (29-31). Genome analyses have identified 80 several ring-cleaving pathways in roseobacters, including the box and pca pathways (30). Yet, it 81 is unknown whether roseobacters show evidence of substrate preference when presented with 82 mixtures of aromatic compounds representative of compounds derived from vascular plants 83 abundant in coastal marine habitats. 84 85 on Jne 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom Materials and Methods 86 Media and growth conditions. A marine basal medium (MBM) containing 1.5% (w/v) sea salts 87 [Sigma-Aldrich, St. Louis, MO] with 225 nM K2HPO4, 13.35 μM NH4Cl, 71 mM Tris-Cl (pH 88 7.5), 68 μM Fe-EDTA, trace metals, and vitamins was used to culture Ruegeria pomeroyi DSS-3 89 and Sagittula stellata E-37 at 30°C (32). Variovorax paradoxus EPS was also cultured at 30°C in 90 M9 basal media (33). All growth experiments used cells preconditioned on 3.5 or 7 mM acetate 91 at early stationary phase to match the carbon concentration of the new medium. Initial inocula 92 were ≤ 1% transfer volume (~10 cells ml). Benzoic acid and p-hydroxybenzoic acid were 93 obtained from Sigma-Aldrich, and sodium acetate was obtained from Fisher Scientific 94 [Waltham, MA]. All glassware was combusted minimally for 6 h at 450°C to remove trace 95 carbon, and negative (non-carbon amended) controls of cells in basal media were also 96 performed. 97 98 Nucleic acid isolation. For RNA preservation and isolation, approximately 10 cells were 99 pelleted at 5000 x g for 5 min, resuspended in 1 ml of RNAlater [Ambion, Austin, TX], and 100 preserved for 1 h at room temperature. RNAlater was removed by aspiration following 6000 x g 101 centrifugation for 5 min, and the cells were flash frozen in liquid N2 and stored at -70°C until 102 processed. Cells were lysed by agitation in the presence of low-binding 200 μm zirconium beads 103 [OPS Diagnostics, L.L.C., Lebanon, NJ], and RNA was extracted using the RNeasy Mini Kit 104 [Qiagen, Valencia, CA]. Genomic DNA was removed using the vigorous Turbo DNase (4 U) 105 [Ambion] method as described in the product manual. Nucleic acids were quantified and purity 106 was assessed with an ND-1000 spectrophotometer [NanoDrop Technologies Inc., Wilmington, 107 DE]. Reverse transcription was carried out in 60 μl volumes containing 180 ng RNA, 600 U M108 on Jne 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom MLV reverse transcriptase [Invitrogen, Carlsbad, CA], 500 μg ml random hexamers [Promega, 109 Madison, WI], 120 U RNaseOUT [Invitrogen], 500 μM dNTPs [Promega], and 10 mM DTT 110 [Invitrogen]. Initially the random hexamers in the presence of dNTPs were annealed to the RNA 111 for 5 min at 65°C, followed by an immediate transfer to ice to maintain the binding interaction. 112 Next, to protect the mRNA and increase full-length cDNA yields, DTT and RNaseOUT were 113 added and incubated for 2 min at 37°C. Finally cDNAs were generated from the RNA by M114 MLV reverse transcriptase by first activating for 10 min at 25°C followed by 50 min of synthesis 115 at 37°C. The enzymes were denatured and inactivated for 15 min with 70°C, and the remaining 116 cDNA was stored at -20°C. 117 118 Gene transcription assays. RT-qPCR was used to assess relative gene expression. Transcripts 119 diagnostic of the benzoyl-CoA pathway (boxA) and the protocatechuate branch of the β120 ketoadipate pathway (pcaH) were measured and normalized to the expression of three reference 121 genes (alaS, map, and rpoC). Primers were designed for each of these 5 genes and are shown in 122 Table S1. All primer sets were optimized for quantitative (qPCR) using the following method. In 123 25 μl qPCR volumes, a matrix of forward and reverse primer concentrations (ranging from 100124 1500 nM final concentrations) was used along with a fixed concentration of E-37 genomic DNA 125 (2.5×10 genomes reaction) and 1X SYBR Premix Ex Taq (Perfect Real Time) [Takara Bio 126 Inc., Otsu, Japan]. The qPCR amplification included an initial 95°C denaturation for 15 min, 127 followed by 40 cycles of amplification (95°C denaturation for 45 s, 58°C annealing for 45 s, and 128 72°C elongation and fluorescence detection for 15 s), and a final 72°C extension for 5 min. A 129 melting curve from 50°C to 100°C read every 1°C was performed after each reaction to ensure 130 only a single product melted around 90°C. Within each primer set matrix, the combination of 131 on Jne 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom forward and reverse primer concentrations that yielded the lowest Cq (quantification cycle) was 132 used in the subsequent RT-qPCR. 133 134 The 25 μl qPCR volumes consisted of 15% cDNA template, 577 μM oligonucleotide primers, 135 and SYBR Premix Ex Taq (Perfect Real Time) [Takara]. Each cDNA template sample was 136 amplified with primer sets to quantitate alaS, boxA, map, pcaH, and rpoC. Technical qPCR 137 triplicates were performed for each of the five primer sets used to amplify the three biological 138 triplicates at five time points. The cycling conditions were also the same as described for the 139 qPCR optimization. Non reverse-transcribed aliquots were also performed as negative controls to 140 ensure the RT-qPCR measurements represented cDNA concentration and other nucleic acids’ Cq 141 were negligible (>5 difference). Reference genes for normalization remained unchanged during 142 the sampling time points and have been successfully applied as reference genes to another 143 roseobacter in our laboratory (34). All normalized boxA and pcaH RT-qPCR data were 144 relativized to their basal expression of E-37 cells grown on 7 mM acetate according to 145 calculations described by Hellemans et al. (35). Technical replicate errors were propagated with 146 a truncated first-order Taylor series expansion. 147 148 Protocatechuate 3,4 dioxygenase (PcaHG) enzyme assays. Approximately 10 cells were 149 washed in a 4°C solution containing 1.5% sea salts [Sigma-Aldrich] and 50 mM Tris-acetate (pH 150 7.5). Rinsed and pelleted cells were suspended in 396 μl of Bugbuster Protein Extraction 151 Reagent [Novagen, Inc., Madison, WI]. Lysozyme (4 μl) was added (to a final concentration of 152 0.1 ng ml) and the cells were incubated at 30°C for 15 min. After the cell debris was removed 153 via centrifugation (21000 x g for 30 min at 4°C), crude cell lysates were assayed for 154 on Jne 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom protocatechuate 3,4-dioxygenase (PcaHG, EC 1.13.11.3) activity by measuring the kinetic loss of 155 protocatechuate at 290 nm with a DU 800 UV/Vis spectrophotometer [Beckman Coulter, Inc., 156 Brea, CA] (36). Briefly, the 1 ml assays included 400 μM protocatechuate and 50 mM Tris157 acetate (pH 7.5) along with ≥3 different volumes of lysate tested below the saturation point. 158 Protein concentrations were determined with the Coomassie Plus Protein Assay Reagent Kit 159 [Thermo Scientific Pierce, Rockford, IL] (37). The specific activity for each sample was 160 calculated using the Δε of εprotocatechuate and εβ-carboxymuconolactone (2280 cm M). E-37 cells grown 161 solely on 7 mM acetate and on 2 mM protocatechuate served as negative and positive controls. 162 163 HPLC-PDA analysis of substrate concentrations. Aromatic substrates in the spent media were 164 separated with a Waters 2695 high-performance liquid chromatography (HPLC) instrument 165 containing a reverse-phase 3.9 x 150 mm Novak-Pak C18 column [Waters Corp., Milford, MA] 166 coupled to a Waters 2996 photodiode array (PDA) detector. For the spent MBM, an isocratic 167 elution of 0.8 ml min at 25°C with the mobile phase containing 30% MeCN(aq) and 0.07% 168 phosphoric acid(aq) produced distinct peaks for benzoate (3.62 min) and POB (2.12 min). The 169 same separation conditions were used for M9 spent media with the exception of increased (2.5%) 170 phosphoric acid(aq). A ten-point serial dilution curve of authentic standards was used to determine 171 the concentration of each compound at their λmax in the MBM solution that were 230.3 and 256.2 172 nm for benzoate and POB. The peak area of each eluate was calculated with ApexTrack’s 173 integration tool using the Empower 2 Pro software package [Waters] for each of the technical 174 (HPLC-PDA machine) triplicates performed on each sample. Linear regression of the temporally 175 paired substrate concentrations was used to assess the statistical correlation between the 176 on Jne 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom catabolic/disappearance rate of each substrate using SigmaPlot 11.0 [Systat Software, Inc., 177