Novosphingobium Pentaromativorans US6-1 Research Articles

The stringent response gene rsh plays multiple roles in Novosphingobium pentaromativorans US6-1's accommodation to different environmental pollutants: Phenanthrene, copper and nZVI

The rsh based stringent response system is widely employed by bacteria to cope with environmental stresses. However, how does the stringent response involve in bacterial accommodation to environmental pollutant is largely unexplored. In this study, to comprehensively understand the roles of rsh in Novosphingobium pentaromativorans US6-1's metabolism and accommodation to different pollutants, three distinct pollutants, phenanthrene, copper and nanoparticulated zero valent iron (nZVI) were selected as exposure substances. Results indicated that rsh played important roles in US6-1's multiplication and metabolism, including survival rate at stationary phase, amino acid and nucleotide metabolism, extracellular polymeric substance (EPS) production, redox homeostasis, etc. The deletion of rsh affected phenanthrene removal rates through regulating the multiplication of US6-1 and increasing the expression of degradation related genes. The rsh mutant showed higher resistance to copper than the wild type, largely due to higher EPS production and enhanced expression of copper resistance related genes. Finally, the rsh based stringent response helped maintain the redox homeostasis when US6-1 confronted nZVI particles that exerted oxidative stress, thereby improving the survival rate. Overall, this study provides firsthand data that rsh plays multiple roles in US6-1's accommodation to environmental pollutants. The stringent response system could be a powerful tool for environmental scientists and engineers to harness bacterial activities for bioremediation purposes.

The Stringent Response Gene Rsh Plays Multiple Roles in Novosphingobiumpentaromativorans Us6-1's Accommodation to Different Environmental Pollutants: Phenanthrene, Copper and Nzvi

The Stringent Response Gene Rsh Plays Multiple Roles in Novosphingobiumpentaromativorans Us6-1's Accommodation to Different Environmental Pollutants: Phenanthrene, Copper and Nzvi

Finding novel chemoreceptors that specifically sense and trigger chemotaxis toward polycyclic aromatic hydrocarbons in Novosphingobium pentaromativorans US6-1

Bacterial chemotaxis can improve the efficiency of aromatic compound degradation, however, knowledge of how bacteria sense high-molecular-weight polycyclic aromatic hydrocarbons (HMW-PAHs), is limited. Here, the chemotactic responses of Novosphingobium pentaromativorans US6-1 to 9 aromatic compounds were investigated. The results showed that US6-1 chemotactically responded to phenanthrene (PHE), pyrene (PYR), benzo[a]pyrene (BaP) and their six metabolites. Six methyl-accepting chemotaxis proteins (MCPs) were annotated from US6-1 genome, four of which contained putative ligand-binding domains (LBDs). To confirm whether these four MCPs were involved in triggering chemotaxis toward PAHs, the MCP mutants were constructed. Observations showed a loss of the chemotactic responses to benzoate, phthalate, PHE and BaP only in the mutant ∆mcp03030. Surface plasmon resonance (SPR) assays further confirmed that MCP03030LBD specifically bound phthalate, PHE, PYR and BaP, while MCP18870LBD bound only PYR. The mutant ∆mcp03030-∆mcp18870 was then constructed and was shown to have lost the chemotactic response to 5 aromatic compounds. Combined with the effects of outer membrane transporter deletion on chemotaxis and MCP deletion on the PAH degradation, our study demonstrated that the chemoreceptors MCP03030 and MCP18870 can recognize PAHs and their metabolites in the periplasm, triggering metabolism-dependent and metabolism-independent chemotaxis, and be linked with HMW-PAH biodegradation.

Oxygen availability affects the synthesis of quorum sensing signal in the facultative anaerobe Novosphingobium pentaromativorans US6-1.

Bacterial populations rely on quorum sensing (QS) to coordinate their behaviors and are often challenged by the fluctuation in oxygen concentrations in their habitats. Oxygen is a crucial factor that affects bacterial metabolism in multiple ways. However, little is known about whether and how oxygen availability affects QS activities. To fill this gap, we used the facultative anaerobe Novosphingobium pentaromativorans US6-1 as a model system, and observed that the QS signal acyl homoserine-lactones (AHLs) were produced only in anoxic environments, such as biofilm, or liquid medium that initially contained less than 2 mg/L dissolved oxygen, but not in highly oxic environments. Comparative transcriptome analysis revealed that oxygen availability significantly affected the physiological activities in US6-1, including fatty acid metabolism, oxidative phosphorylation, citrate cycle, QS activities, and flagellar assembly. The absence of AHLs in the oxic culture was not due to degradation, but to the very low expression of the AHL synthase gene novI. High concentration of NADH during the middle log phase under static cultivation may be a trigger for AHL synthesis. This is the first report that production of AHLs is coupled with anoxic metabolism in a facultative anaerobe, which extends our knowledge on factors affecting bacterial QS occurrence. KEY POINTS: • AHL production is anoxic cultivation related. • Oxygen availability affects AHL synthesis by influencing novI expression. • Oxygen availability changes many metabolism activities including NADH production.

Acyl homoserine lactone based quorum sensing affects phenanthrene removal by Novosphingobium pentaromativorans US6-1 through altering cell surface properties

Novosphingobium pentaromativorans US6-1 can degrade polycyclic aromatic hydrocarbons (PAHs) and produce acyl-L-homoserine lactones (AHLs), which are quorum-sensing (QS) signal molecules. However, whether and how QS affects bioremediation of PAHs remain unknown. In this study, using novI and novR deletion mutants (ΔnovI and ΔnovR) and complementary strains (ΔnovI (pCM62-novI) and ΔnovR (pCM62-novR)), we proved that novI and novR constitute the QS system in US6-1. The expression of most genes encoding PAH degrading enzymes were significantly upregulated in mutants, and this finding was consistent with measurements showing that mutants had a higher phenanthrene (phe) removal efficiency (PRE) than wild-type US6-1 (WT_US6-1). Both mutants had significantly higher cell surface hydrophobicity (CSH) and lower extracellular-polysaccharide (EPS) production than WT_US6-1. The functional C–O–C groups on the cell membrane were also changed in mutants. Upregulation of spt in ΔnovI and downregulation of epsd in ΔnovR supported the measurement results of CSH and EPS production. These results indicate that the AHLs-based QS in US6-1 negatively regulates PRE and CSH and positively regulates EPS production. Correlation analysis indicates that in US6-1, QS negatively affects PRE by altering the cell surface properties, and hence, QS may serve as a target for controlling bioremediation.

Transcriptome Analysis of Novosphingobium pentaromativorans US6-1 Reveals the Rsh Regulon and Potential Molecular Mechanisms of N-acyl-l-homoserine Lactone Accumulation.

In most bacteria, a bifunctional Rsh responsible for (p)ppGpp metabolism is the key player in stringent response. To date, no transcriptome-wide study has been conducted to investigate the Rsh regulon, and the molecular mechanism of how Rsh affects the accumulation of N-acyl-l-homoserine lactone (AHL) remains unknown in sphingomonads. In this study, we identified an rshUS6–1 gene by sequence analysis in Novosphingobium pentaromativorans US6-1, a member of the sphingomonads. RNA-seq was used to determine transcription profiles of the wild type and the ppGpp-deficient rshUS6–1 deletion mutant (∆rsh). There were 1540 genes in the RshUS6–1 regulon, including those involved in common traits of sphingomonads such as exopolysaccharide biosynthesis. Furthermore, both RNA-seq and quantitative real-time polymerase chain reaction (qRT-PCR) showed essential genes for AHL production (novI and novR) were positively regulated by RshUS6–1 during the exponential growth phase. A degradation experiment indicated the reason for the AHL absence in ∆rsh was unrelated to the AHL degradation. According to RNA-seq, we proposed σE, DksA, Lon protease and RNA degradation enzymes might be involved in the RshUS6–1-dependent expression of novI and novR. Here, we report the first transcriptome-wide analysis of the Rsh regulon in sphingomonads and investigate the potential mechanisms regulating AHL accumulation, which is an important step towards understanding the regulatory system of stringent response in sphingomonads.

Novosphingobium endophyticum sp. nov. isolated from roots of Glycyrrhiza uralensis

A novel Gram-staining-negative, non-motile, rod-shaped and aerobic bacterial strain, designated EGI 60015(T), was isolated from healthy roots of Glycyrrhiza uralensis F. collected from Yili County, Xinjiang Province, Northwest China. The 16S rRNA gene sequence of strain EGI 60015(T) was found to show 97.6% sequence similarity with Novosphingobium pentaromativorans US6-1(T). The phylogenetic analysis based on 16S rRNA gene sequences showed that the strain formed a clade with N. pentaromativorans US6-1(T) in the neighbor-joining tree. Q-10 was identified as the respiratory quinone of strain EGI 60015(T). The major fatty acids were summed feature 8 (C18:1 ω6c and/or C18:1 ω7c; 55.04%), summed feature 4 (C17:1 anteiso B and/or iso I; 18.34%) and summed feature 3 (C16:1 ω6c and/or C16:1 ω7c; 8.53%). The polar lipids detected were phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and sphingoglycolipids. The DNA G+C content of strain EGI 60015(T) was determined to be 66.6 mol%. The genomic DNA relatedness value between EGI 60015(T) and N. pentaromativorans US6-1(T) (54%) was below the 70% limit for species identification. Based on the result of the molecular studies supported by its morphological, physiological, chemotaxonomic and other differentiating phenotypic characteristics, strain EGI 60015(T) was considered to represent a novel species within the genus Novosphingobium, for which the name Novosphingobium endophyticum sp. nov. is proposed. The type strain is EGI 60015(T) (=CGMCC 1.15095(T) = KCTC 42486(T) = DSM 29948(T)).

Biodegradation of polycyclic aromatic hydrocarbons by Novosphingobium pentaromativorans US6-1.

Novosphingobium pentaromativorans US6-1, a marine bacterium isolated from muddy sediments of Ulsan Bay, Republic of Korea, was previously shown to be capable of degrading multiple polycyclic aromatic hydrocarbons (PAHs). In order to gain insight into the characteristics of PAHs degradation, a proteome analysis of N. pentaromativorans US6-1 exposed to phenanthrene, pyrene, and benzo[a]pyrene was conducted. Several enzymes associated with PAHs degradation were identified, including 4-hydroxybenzoate 3-monooxygenase, salicylaldehyde dehydrogenase, and PAH ring-hydroxylating dioxygenase alpha subunit. Reverse transcription and real-time quantitative PCR was used to compare RHDα and 4-hydroxybenzoate 3-monooxygenase gene expression, and showed that the genes involved in the production of these two enzymes were upregulated to varying degrees after exposing the bacterium to PAHs. These results suggested that N. pentaromativorans US6-1 degraded PAHs via the metabolic route initiated by ring-hydroxylating dioxygenase, and further degradation occurred via the o-phthalate pathway or salicylate pathway. Both pathways subsequently entered the tricarboxylic acid (TCA) cycle, and were mineralized to CO2.

Experimental results and integrated modeling of bacterial growth on an insoluble hydrophobic substrate (phenanthrene).

Metabolism of a low-solubility substrate is limited by dissolution and availability and can hardly be determined. We developed a numerical model for simultaneously calculating dissolution kinetics of such substrates and their metabolism and microbial growth (Monod kinetics with decay) and tested it with three aerobic phenanthrene (PHE) degraders: Novosphingobium pentaromativorans US6-1, Sphingomonas sp. EPA505, and Sphingobium yanoikuyae B1. PHE was present as microcrystals, providing non-limiting conditions for growth. Total PHE and protein concentration were tracked over 6-12 days. The model was fitted to the test results for the rates of dissolution, metabolism, and growth. The strains showed similar efficiency, with vmax values of 12-18 g dw g(-1) d(-1), yields of 0.21 g g(-1), maximum growth rates of 2.5-3.8 d(-1), and decay rates of 0.04-0.05 d(-1). Sensitivity analysis with the model shows that (i) retention in crystals or NAPLs or by sequestration competes with biodegradation, (ii) bacterial growth conditions (dissolution flux and resulting chemical activity of substrate) are more relevant for the final state of the system than the initial biomass, and (iii) the desorption flux regulates the turnover in the presence of solid-state, sequestered (aged), or NAPL substrate sources.

Proteomic Characterization of Plasmid pLA1 for Biodegradation of Polycyclic Aromatic Hydrocarbons in the Marine Bacterium, Novosphingobium pentaromativorans US6-1

Novosphingobium pentaromativorans US6-1 is a halophilic marine bacterium able to degrade polycyclic aromatic hydrocarbons (PAHs). Genome sequence analysis revealed that the large plasmid pLA1 present in N. pentaromativorans US6-1 consists of 199 ORFs and possess putative biodegradation genes that may be involved in PAH degradation. 1-DE/LC-MS/MS analysis of N. pentaromativorans US6-1 cultured in the presence of different PAHs and monocyclic aromatic hydrocarbons (MAHs) identified approximately 1,000 and 1,400 proteins, respectively. Up-regulated biodegradation enzymes, including those belonging to pLA1, were quantitatively compared. Among the PAHs, phenanthrene induced the strongest up-regulation of extradiol cleavage pathway enzymes such as ring-hydroxylating dioxygenase, putative biphenyl-2,3-diol 1,2-dioxygenase, and catechol 2,3-dioxygenase in pLA1. These enzymes lead the initial step of the lower catabolic pathway of aromatic hydrocarbons through the extradiol cleavage pathway and participate in the attack of PAH ring cleavage, respectively. However, N. pentaromativorans US6-1 cultured with p-hydroxybenzoate induced activation of another extradiol cleavage pathway, the protocatechuate 4,5-dioxygenase pathway, that originated from chromosomal genes. These results suggest that N. pentaromativorans US6-1 utilizes two different extradiol pathways and plasmid pLA1 might play a key role in the biodegradation of PAH in N. pentaromativorans US6-1.