Archive for July, 2018

To determine the mechanisms underlying Poly-P metabolism in strain JN459, we firstly analyzed the expression of genes involved in Poly-P metabolism in Microlunatus phosphovorus. RNA-Seq and real-time PCR analysis indicated that transcriptional levels of genes related to catabolism and anabolism of Poly-P, including ppk (MLP_47700, MLP_50300 and MLP_05750), ppgk (MLP_05430 and MLP_26610), ppx (MLP_44770), pap (MLP_23310) and ppnk (MLP_17420), exhibited significant changes during the SBR process but that only two of them (MLP_05430 and MLP_17420) were up-regulated under anaerobic conditions compared with aerobic conditions. MLP_05430 encodes PPGK, which can phosphorylate glucose using Poly-P or ATP as the phosphate donor in bacteria (Klemke et al. 2014; Tanaka, et al. 2003), and both the transcriptional and protein expression levels of MLP_05430 were higher under anaerobic conditions than under aerobic condition, suggesting that JN459 can phosphorylate glucose with Poly-P and obtain energy for growth through MLP_05430. MLP_26610 is another PPGK found in JN459, but the transcriptional level of MLP_26610 was significantly decreased under anaerobic condition, suggesting that it is not a strictly Poly-P-dependent glucokinase.

NADPH plays an essential role in lipid and nucleotide biosynthesis in bacteria, and PPNK is a Poly-P/ATP-dependent NAD kinase that can utilize ATP and Poly-P to provide NADPH, along with the pentose phosphate pathway (Lindner et al. 2010; Oyserman, et al. 2016; Sakuraba et al. 2005). M. phosphovorus harbors one ppnk gene (MLP_17420). The greater expression of MLP_17420 at the transcriptional and protein levels under anaerobic conditions versus aerobic conditions suggests that JN459 could utilize MLP_17420 to synthesize NADP using Poly-P to provide reducing power for growth under anaerobic conditions.

M. phosphovorus strain JN459 also encodes a complete PstSCAB phosphorus transport system (MLP_47720, MLP_47730, MLP_47740 and MLP_47750). Previous studies showed that PstSCAB transport systems are responsible for transporting orthophosphate into cells in low phosphorus conditions and that they are regulated by PhoU-PhoB/PhoR (Lamarche et al. 2008; Yang et al. 2012), with PhoU as a negative regulator in bacteria (Lubin et al. 2015). Transcriptome analysis of JN459 showed that the transcriptional level of PstSCAB decreased significantly under anaerobic (92 h) conditions, compared with aerobic (86 h) conditions; whereas the expression level of PhoU (MLP_47360) was high under both anaerobic (86 h) and aerobic (92 h) conditions (Table 1); the so high expression level of the negative regulator PhoU suggesting that the PstSCAB phosphorus transport system was down regulated by PhoU. At the same time, the transcriptional levels of the regulatory genes PhoB (MLP_24520) and PhoR (MLP_24530) (Table 1) showed no significant changes, which could be attributed to the high phosphorus conditions in the SBR.

Pit proteins are responsible for phosphorus transport under high phosphorus conditions (Pi > 4 μM) in bacteria (Gebhard et al. 2009; Lamarche, et al. 2008). Pit systems, composed of Pit A (constitutively expressed) and Pit B (inducibly expressed), are driven by H+ and can transport PO43- into cells under aerobic conditions and out of cells under anaerobic conditions. MLP_00530, MLP_29830 and MLP_51060 in M. phosphovorus were predicted to belong to a Pit system, the alignment analysis indicated that the amino acid sequence of MLP_00530 was similar to PitA, while MLP_29830 and MLP_51060 were similar to PitB (Kawakoshi, et al. 2012). As the transcriptional level of MLP_00530 showed no significant changes under the two conditions tested (Table 1), MLP_00530 may be of the Pit A group, which is constitutively expressed. In contrast, both MLP_29830 and MLP_51060 in JN459 decreased significantly, by almost 10-fold, under anaerobic conditions (Table 1), indicating that they may belong to the Pit B group.

In conclusion, strain JN459 accumulated phosphorus aerobically and released phosphorus anaerobically, and Poly-P granules increased in the cells during the aerobic period and decreased during the anaerobic period. Both transcriptional and protein expression analyses indicated that PPGK (MLP_05430) and PPNK (MLP_17420) may form the pathway for using Poly-P as an energy resource in M. phosphovorus under anaerobic conditions. These findings advance our understanding of phosphate metabolism in a species with strong potential for removal of phosphorus from wastewater.