Benzo[a]pyrene might be transported by a TonB-dependent transporter in Novosphingobium pentaromativorans US6-1

Highlights

• TonB-dependent transport systems respond positively to the pressure of BaP in US6-1.

• The TBDT gene tbdt-11 is responsible for the transportation of BaP in US6-1.

• TBDT genes might be involved in regulating PAHs absorption and degradation in US6-1.

Abstract

Sphingomonads are well known for their ability to efficiently degrade polycyclic aromatic hydrocarbons (PAHs), but little is known about the mechanism of PAH uptake and transport across the cell membrane. RNA sequencing analysis of a sphingomonad, Novosphingobium pentaromativorans US6–1 showed that 38 TonB-dependent transporter (TBDT) genes were significantly upregulated under 5-ring PAH-benzo[a]pyrene (BaP) stress. In order to reveal whether TBDTs are involved in uptake and transport BaP in US6–1, the key TBDT genes were deleted to generate mutants. The results showed that the growth status of these mutants was not different from that of the wild-type strains, but the PAH degradation ability decreased, especially for the mutant strain Δtbdt-11, which did not encode the tbdt-11 gene. Meanwhile, the cell surface hydrophobicity (CSH) of Δtbdt-11 was found to be significantly lower than that of the wild-type strain under BaP stress. Furthermore, the transcriptional activity of genes encoding PAH degradative enzymes was found to be greatly reduced in Δtbdt-11. Confocal microscopy observations showed that US6–1 could transport BaP across the outer membrane, but this transport capacity was significantly reduced in Δtbdt-11 and wild-type US6–1 treated with PMF uncoupler, further confirming that the tbdt-11 gene was associated with PAH active transport.

Introduction

Polycyclic aromatic hydrocarbons (PAHs), cyclic organic compounds with two or more fused benzene rings containing only carbon and hydrogen, are of significant concern due to their toxic, mutagenic and carcinogenic properties (Ghosal et al., 2016). PAHs with four or more rings are called high-molecular-weight PAHs (HMW PAHs) which are largely considered to be genotoxic (Nzila, 2018). Among them, Benzo[a]pyrene (BaP) with five rings is the most carcinogenic generally used as an environmental indicator for PAHs. In the natural environment, the main attenuation process of PAHs is microbial degradation (Chen and Ding, 2012). However, as typical hydrophobic organic pollutants, PAHs are relatively insoluble in water but soluble in many organic solvents. Thus, PAHs are considered to be unavailable to most microorganisms (Peng et al., 2008). Many studies reported that PAH-degrading bacteria can enhance the bioavailability of PAHs by producing biosurfactants, extracellular polymeric substances, or forming biofilms (Park et al., 2003, Johnsen and Karlson, 2004). There are also studies found that some Gram-negative bacteria can improve uptake rates of PAHs through a PAH-inducible proton-driven active transport mechanism to transport PAHs across the membrane (Kallimanis et al., 2007). Therefore, the uptake of PAHs could be considered the first step in the biodegradation of PAHs.

TonB-dependent transporters (TBDTs) are bacterial outer membrane (OM) proteins that bind and transport ferric chelates, vitamin B12, nickel complexes, carbohydrates (Schauer et al., 2008), as well as lethal agents including phages, microcins, and colicins (Cadieux et al., 2003). TBDTs bind their substrates with high affinities and rely on the inner membrane (IM) TonB system (TonB-ExbB-ExbD) to supply energy in the form of proton motive force (PMF) (Noinaj et al., 2010). The structure of TBDTs is composed of a 22-strand β-barrel, which contains a hatch domain with changeable conformations (Chimento et al., 2005). Hydrophobic compounds could bind to multiple sites in β-barrel proteins (van den Berg et al., 2004). IM TonB interacts with the hatch domain of TBDTs, which may cause local unfolding of the hatch, temporarily generating a wide channel through the hatch for the transport of large substrates (Shultis et al., 2006). TBDTs are among the most widespread OM proteins in Gram-negative bacteria, especially PAH-degrading sphingomonads (Tang et al., 2012). For instance, Sphingomonas wittichii RW1, a sphingomonads, currently contains the most TBDTs among all microorganisms (Miller et al., 2010, Colquhoun et al., 2012). These TBDTs may be used by sphingomonads to uptake diverse substrates, including aromatic compounds.

The OM of sphingomonad cells contains hydrophobic glycosphingolipids (GSLs) instead of lipopolysaccharides (LPS) commonly found in Gram-negative bacteria. The hydrophobicity feature of GSLs has been proposed contributing to the cell–substrate interaction and aid in the uptake of substrates into cells, thereby enhancing PAH biodegradability (Waigi et al., 2015). On the other hand, PAHs are degraded by intracellular enzymes that are either bound to membranes or located in the periplasmic space, therefore, the transmembrane transport of these compounds is necessary for their metabolism. A macromolecule ABC transport system for HMW hydrophobic polymers was identified in Sphingomonas strain A1 growing on alginate (Mishima et al., 2001). Sphingobium herbicidivorans MH is also capable of utilizing such uptake systems in the active transport of the herbicide dichlorprop (Zipper et al., 1998). Thus, active transport systems may be used by sphingomonads for the uptake of PAHs.

Novosphingobium pentaromativorans US6–1, a sphingomonad, is capable of efficiently degrading multiple PAHs, such as naphthalene, phenanthrene (PHE), pyrene and even BaP (Sohn et al., 2004). 4-Hydroxybenzoate 3-monooxygenase, salicylaldehyde dehydrogenase, and PAH ring-hydroxylating dioxygenase α-subunit were identified in US6–1 based on proteome analysis, suggesting that PAHs are initially degraded by ring-hydroxylating dioxygenase, and further metabolized through the o-phthalate pathway or salicylate pathway (Lyu et al., 2014). 1-DE/LCMS/MS analysis of US6–1 further revealed that PHE induced the strongest upregulation of ring-hydroxylating dioxygenase, biphenyl-2,3-diol 1,2-dioxygenase, and catechol 2,3-dioxygenase in plasmid pLA1; however, p-hydroxybenzoate induced the activation of the protocatechuate 4,5-dioxygenase on the chromosome of US6–1, suggesting that US6–1 could utilize two different extradiol pathways (Yun et al., 2014). On the other hand, p-hydroxybenzoate also induced the production of OM vesicles in US6–1, and these vesicles had a high portion of OM proteins and periplasmic proteins such as TBDTs (Yun et al., 2017). In addition, the main quorum sensing (QS) signal in US6–1 is N-acyl-L-homoserine lactone (AHL), which is positively regulated by Rsh Regulon (Lu and Huang, 2018). Chen and Huang reported that cell surface hydrophobicity (CSH) and PHE removal efficiency were negatively regulated while extracellular-polysaccharide production was positively regulated by the AHLs-based QS in US6–1 (Chen and Huang, 2020). However, although the uptake of bacteria is the first limiting step of PAH degradation, research on the efficient uptake of PAHs by US6–1 has never been reported.

In a previous study, the genome sequence of US6–1 was determined and the genome database is accessible from the public NCBI database (Choi et al., 2015). Interestingly, most TBDT genes in US6–1 are near the PAH degradative dioxygenase genes, indicating that TBDTs may be involved in PAH degradation. Our preliminary strand-specific RNA sequencing experiments revealed that 13 TBDT and TonB system genes were significantly upregulated under BaP stress after 6 days. Thus, we hypothesized that TBDTs may be involved in the active transport of PAHs across the membrane. In order to test this hypothesis, we first considered that the uptake and transport of PAHs occur mainly in the early stage of PAH degradation, so the transcriptional activity of TBDT genes in the early stage of BaP addition was analyzed. Mutants of three representative sensitive genes were generated, and the PAH degradation rates of these strains were tested. Thereafter, the growth status of strains, the membrane adsorption and uptake efficiency of PAHs were further investigated for US6–1 wild type strains and mutants. These results are useful for understanding the roles of TBDTs in PAH degradation by US6–1.