Total RNA was extracted from JN459 at 86 h (aerobic) and 92 h (anaerobic). RNA sequencing libraries were constructed, and high-throughput sequencing was carried out using the HiSeq2500. The results showed significant differences in gene transcription patterns between aerobic and anaerobic conditions in JN459, with the expression of 577 genes increasing and 2037 genes decreasing significantly under anaerobic conditions. Surprisingly, almost all genes of glycolytic pathway involved in anaerobic respiration did not increase under anaerobic conditions, such as glucokinase (MLP_41670), pyruvate kinase (MLP_41150), and lactate dehydrogenase (MLP_21640), which indicated that JN459 did not only utilize glucose as the energy source. The RNA-Seq data were deposited in the NCBI Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE106860.

Multiple genes predicted to be involved in Poly-P metabolism in M. phosphovorus were differentially expressed, including genes related to Poly-P anabolism, Poly-P catabolism, and phosphorus transport (Table 1). Among these, the transcriptional level of 12 genes decreased significantly under anaerobic conditions, and only two genes, ppgk (MLP_05430) and ppnk (MLP_17420), were up-regulated, whereas four genes showed no significant changes. The distinctive high expression of ppgk (MLP_05430) and ppnk (MLP_17420) indicate that both of them may contribute to the biodegradation of Poly-P and the production of NADP+ and energy under anaerobic conditions.

Real-time PCR analysis the expression of genes involved in Poly-P metabolism

RNA-Seq data indicated that there were significant differences in transcriptional levels under aerobic and anaerobic conditions, and among those differentially expressed genes, eight genes are related to catabolism and anabolism of Poly-P, including ppk (MLP_47700, MLP_50300 andMLP_05750), ppgk (MLP_05430 and MLP_26610), ppx (MLP_44770), pap (MLP_23310) and ppnk (MLP_17420). Real-time PCR was utilized to analyze and validate the transcriptional levels of these genes at 74 h (aerobic), 80 h (anaerobic), 86 h (aerobic) and 92 h (anaerobic). Results showed that the transcriptional level of ppgk (MLP_05430) under anaerobic condition increased 3.53-fold and 2.86-fold than that under aerobic condition, and ppnk (MLP_17420) under anaerobic condition increased 2.68-fold and 2.16-fold than that under aerobic condition, the fold changes are between 80 h and 74 h, and between 92 h and 86 h, whereas the transcriptional levels of other genes decreased (Fig.4). These findings are consistent with the RNA-Seq data.

PPGK, encoded by MLP_05430, is a type of glucokinase that strictly uses Poly-P as a phosphate donor (Tanaka et al. 2003). Both RNA-Seq and real-time PCR analysis showed that the transcriptional level of MLP_05430 was much higher under anaerobic conditions than under aerobic conditions, indicating that JN459 can phosphorylate glucose with Poly-P and obtain energy for growth using MLP_05430. MLP_26610 was predicted to be another PPGK, and the amino acid similarity between MLP_26610 and MLP_05430 is 65%; however, MLP_26610 expression was significantly decreased under anaerobic conditions, indicating that MLP_26610 may be a mainly ATP-dependent and not a Poly-P-dependent glucokinase.

MLP_17420 encodes a Poly-P/ATP-dependent NAD kinase (PPNK), which can convert Nicotinamide Mononucleotide (NMN) adenine dinucleotide (NAD+) into NADP+ using Poly-P or ATP. Both RNA-Seq and real-time PCR analysis showed that MLP_17420 transcript levels increased significantly under anaerobic conditions, indicating that strain JN459 could produce reducing power using Poly-P catabolism, along with the pentose phosphate pathway, for growth.

Both PPGK and PPNK proteins increased under anaerobic conditions

Both RNA-Seq and real-time PCR analysis showed that the transcriptional levels of ppgk (MLP_05430) and ppnk (MLP_17420) increased significantly under anaerobic conditions in JN459. To determine expression at the protein level, cell pellets were collected at 74 h, 80 h, 86 h and 92 h from the SBR system and analyzed by Western blotting with polyclonal antibodies to MLP_05430 and MLP_17420. The expression of both PPGK (MLP_05430) and PPNK (MLP_17420) was higher at 80 h and 92 h than at 74 h and 86 h (Fig.5), which is consistent with the transcriptional analysis. Both Western blotting and real-time PCR results suggest that the increased expression level of PPGK (MLP_05430) and PPNK (MLP_17420) may help the bacteria catabolize Poly-P to supply energy for growth.

DISCUSSION

Phosphorus contributes to water eutrophication, and PAOs found in activated sludge are believed to aid in the removal of soluble phosphorus from wastewater. In particular, M. phosphovorus accumulates substantially more Poly-P (>10% of cell mass on a dry weight basis) than does Tetrasphaera elongata (<1%) or other proteobacterial PAOs (Jiang et al. 2012; Kawakoshi, et al. 2012; Oyserman et al. 2016; Valverde-Perez et al. 2016), while the mechanisms of Poly-P metabolism in these PAOs remain unclear. In this study, we found that strain JN459, previously identified as M. phosphovorus (Zhong, 2015), can also accumulate Poly-P aerobically and release phosphorus anaerobically, as reported in the original description of this species (Nakamura, et al. 1995).