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

Abstract

Plant-derived aromatic compounds are important components of the dissolved organic carbon pool in coastal salt marshes, and their mineralization by resident bacteria contributes to carbon cycling in these systems. Members of the roseobacter lineage of marine bacteria are abundant in coastal salt marshes, and several characterized strains, including Sagittula stellata E-37, utilize aromatic compounds as primary growth substrates. The genome sequence of S. stellata contains multiple, potentially competing, aerobic ring-cleaving pathways. Preferential hierarchies in substrate utilization and complex transcriptional regulation have been demonstrated to be the norm in many soil bacteria that also contain multiple ring-cleaving pathways. The purpose of this study was to ascertain whether substrate preference exists in S. stellata when the organism is provided a mixture of aromatic compounds that proceed through different ring-cleaving pathways. We focused on the protocatechuate (pca) and the aerobic benzoyl coenzyme A (box) pathways and the substrates known to proceed through them, p-hydroxybenzoate (POB) and benzoate, respectively. When these two substrates were provided at nonlimiting carbon concentrations, temporal patterns of cell density, gene transcript abundance, enzyme activity, and substrate concentrations indicated that S. stellata simultaneously catabolized both substrates. Furthermore, enhanced growth rates were observed when S. stellata was provided both compounds simultaneously compared to the rates of cells grown singly with an equimolar concentration of either substrate alone. This simultaneous-catabolism phenotype was also demonstrated in another lineage member, Ruegeria pomeroyi DSS-3. These findings challenge the paradigm of sequential aromatic catabolism reported for soil bacteria and contribute to the growing body of physiological evidence demonstrating the metabolic versatility of roseobacters.

Fig 1

Substrates proceeding through the protocatechuate branch of the β-ketoadipate and the aerobic benzoyl-CoA pathway in Sagittula stellata E-37. Gene designations are based on sequence homology to characterized proteins. Question marks indicate uncharacterized genes or substrates.

Fig 2

boxA and pcaH transcript abundance for E-37 grown solely on POB (2 mM) or benzoate (2 mM). cDNA copy numbers were relativized to those from cells grown on an equimolar C concentration of acetate (7 mM). The horizontal dashed line represents unchanged expression. Error bars represent the standard error of the mean for each biological triplicate.

Fig 3

Simultaneous catabolism of benzoate (1 mM) and POB (1 mM) by E-37. Panels A to C represent three biological replicates. Data for all three biological replicates are shown in separate panels due to slight variation in growth phase and sample time points among parallel cultures. The boxA and pcaH transcript abundances are expressed as percentages of those when E-37 was grown solely on benzoate and POB, respectively. Error bars represent the standard errors of the means (n = 3 per gene). PcaHG specific activity is also expressed as the percentage of activity obtained from POB-grown cells; error bars represent standard deviations of at least 3 replicates. Standard deviations of the technical variation (n = 3) for benzoate and POB concentrations are smaller than the symbol. NRQ, normalized relative quantity; O.D., optical density.

Fig 4

Growth responses to benzoate (1 mM), POB (1 mM), and a mixture of benzoate (0.5 mM) and POB (0.5 mM) by S. stellata E-37. Error bars for O.D. represent the standard deviations of biological replicates (n = 3) for each.