Suppression of hesA mutation on nitrogenase activity in Paenibacillus polymyxa WLY78 with the addition of high levels of molybdate or cystine

Bacterial strains and media

Bacterial strains used in this study are listed in Table S1. P. polymyxa WLY78, mutants and the recombinant E. coli strains were grown in LB medium at 37 °C. When appropriate, antibiotics for E. coli strains and P. polymyxa WLY78 mutants were required. For P. polymyxa WLY78 mutant strains, antibiotics were added at five mg/L chloramphenicol (Cm), 12.5 mg/L tetracycline. While for E. coli, antibiotics were added at 100 mg/L ampicillin, 25 mg/L tetracycline and 25 mg/L Cm for maintenance of plasmids.

Nitrogen-limited medium (per liter contains: 10.4 g Na₂HPO₄, 3.4 g KH₂PO₄, 26 mg CaCl₂·2H₂O, 30 mg MgSO₄, 0.3 mg MnSO₄, 36 mg Ferric citrate, 7.6 mg Na₂MoO₄·2H₂O, 10 μg p-aminobenzoic acid, 10 μg biotin, four g glucose as carbon source and 0.3 g glutamic acid as nitrogen source) was used for assay of nitrogenase activity (Wang et al., 2013). When necessary, different concentrations of Na₂MoO₄ or cystine or sulfate were supplemented in the nitrogen-limited medium.

To determine nitrate reductase activity, P. polymyxa WLY78 and mutant strains were cultured in mineral salts medium (per liter contains: 9.16 g K₂HPO₄∙3H₂O, 1.0 g (NH₄)₂SO₄, 0.22 g MgSO₄∙7H₂O, 2.0 g KH₂PO₄, 8.0 g nutrient broth, 10 g KNO₃, 10 g glucose) (Sperl & Demoss, 1975).

Enzyme assays

For nitrogenase activity assays, P. polymyxa WLY78, mutants, the complemented strains and the recombinant E. coli strains were grown in 20 mL of LB media (Supplemented with antibiotics) in 50 mL flasks shaken at 200 rpm overnight at 37 °C. The cultures were collected by centrifugation, precipitations were washed three times with sterilized water and then resuspended in nitrogen-limited medium containing different concentrations of Na₂MoO₄ (supplemented with antibiotics for mutants and the engineered E. coli strains when necessary) to a final OD_{600 nm} of 0.2–0.5. The nitrogenase activity was determined by the procedure described by Xie et al. (2012).

For the assay of nitrate reductase, P. polymyxa WLY78 and mutant strains were grown in 50 mL of LB media in 100 mL flasks shaken at 200 rpm for 16 h at 37 °C. The cultures were collected by centrifugation, washed three times with sterilized water and then resuspended in mineral salts medium. The cultures were grown at 200 rpm at 37 °C anaerobically. The nitrate reductase was determined by the method described by Garzón (Garzón et al., 1992). Nitrate reductase activity was expressed as nanomoles of nitrite∙minute⁻¹∙milligram⁻¹ of cell protein.

Growth of the wild type and mutant strains

To measure growth of strains cultured in different concentrations of Na₂MoO₄, cells were grown in 20 mL of LB media in 50 mL flasks shaken at 200 rpm at 37 °C overnight. The cultures were collected by centrifugation, washed three times with sterilized water and then resuspended in sufficient nitrogen medium (nitrogen-limited medium supplemented with 100 mM NH₄Cl) containing different concentrations of Na₂MoO₄ to a start OD_{600 nm} of 0.1. After incubating the cultures for 48 h at 37 °C with shaking at 250 rpm, the maximum growth of P. polymyxa WLY78 and mutant strains were determined by absorbancy at 600 nm.

Construction of Paenibacillus hesA mutant strain

The Paenibacillus hesA mutant was constructed through homologous recombination with pRN5101 (Hou et al., 2016), which carries a temperature-sensitive region from plasmid pE194ts (Villafane et al., 1987) containing two replication origins: one is pMB1 which can be reproduced in E. coli and the other replicon is from a plasmid pE194 which can be reproduced in Gram-positive Bacillus (Gryczan, Contente & Dubnau, 1978). A 3432 bp DNA fragment, including a 1,119 bp upstream sequence and a 1,116 bp downstream fragment of the hesA coding region, was cloned to pRN5101. Then, a 1,197 bp DNA fragment, including the promoter and coding region of Cm resistant gene, was amplified from plasmid pPR9TT and inserted between upstream and downstream homologous arms in the opposite direction from the nif genes, generating plasmid pRN5101-Cm. The plasmid pRN5101-Cm was introduced into P. polymyxa WLY78 by electroporation (BTX, ECM 399) for homologous recombination. Transformants resistant to Cm were grown at 30 °C and transferred to five mL of LB media (supplemented with five mg/L Cm) in 20 mL test tubes shaken at 200 rpm for 36 h at 39 °C for about five generations. The strains were then plated onto LB agar plates supplemented with Cm and incubated for 24 h at 39 °C. Integration of the recombinant plasmid was confirmed by PCR and sequencing. The deletion mutant strain was designated as ΔhesA.

Construction of recombinant plasmids for genetic complementation assays

The genes of hesA, nifQ and moeB were PCR amplified from genomic DNA of P. polymyxa WLY78, K. oxytoca M5a1 and E. coli K12, respectively. These PCR products, being under the control of nifB promoter of P. polymyxa WLY78, were cloned to the plasmid pHY300PLK, respectively. Then the recombinant plasmids were introduced into P. polymyxa hesA mutant, respectively.

For complementation of the hesA deletion of the recombinant E. coli 78-7 which carries a nif gene cluster composed of nine genes (nifBHDKENXhesAnifV) from P. polymyxa WLY78 (Wang et al., 2013), the P. polymyxa hesA coding region, being under the control of nifB promoter of P. polymyxa WLY78 was cloned to vector pBluescript II SK (+) that contained a Cm expression cassette, producing the recombinant plasmid pBluescript II SK (+)-hesA. Primers for PCR are listed in Table S2. Recombinant plasmids and strains are listed in Table S3.

Quantitative real-time PCR analysis of nifV expression

For quantitative real-time-PCR (qRT-PCR), cultures of P. polymyxa WLY78 and ΔhesA were grown in N₂-fixing conditions (two mM glutamate and without O₂) and harvested after 20 h of incubation. Total RNA was isolated using TRIzol (cat. no. 9108; Takara Bio, Dalian, China) (Rio et al., 2010). The possibility of contamination of genomic DNA was eliminated by digestion with RNase-free DNase I (cat. no. RR047A; Takara Bio, Dalian, China). The integrity and size distribution of the RNA were verified by agarose gel electrophoresis, and the concentrations were determined spectrophotometrically. Synthesis of cDNA was carried out using RT Prime Mix according to the manufacturer’s specifications (cat. no. RR047A; Takara Bio, Dalian, China). 0.4 μg of cDNA was used for qRT-PCR. The relative transcript levels of nifV were determined with 16S rDNA as a control by SYBR Premix Ex Taq (Tli RNaseH Plus) kit (cat. no. RR820A; Takara Bio). Primers for nifV and 16S rDNA used for qRT-PCR are listed in Table S2.

Statistical analysis

Statistical analyses were performed using SPSS 20.0 (SPSS, Chicago, IL, USA). The least significant test was calculated at the 0.05 or 0.01 probability level for different treatment mean comparisons of nitrogenase activity.

A hesA gene is highly conserved in nif gene clusters among diazotrophic Paenibacillus, cyanobacteria and Frankia

Paenibacillus polymyxa WLY78 possesses a minimal nif gene cluster consisting of nine genes (nifB nifH nifD nifK nifE nifN nifX hesA and nifV). Our previous results demonstrated that the recombinant E. coli 78-7 which was generated by introducing the P. polymyxa nif gene cluster into E. coli JM109 could produce active nitrogenase (Wang et al., 2013). Notably, a hesA gene is contained within the nif gene cluster, and its predicted product HesA is a member of the ThiF-MoeB-HesA family containing an N-terminal nucleotide binding domain and a C-terminal MoeZ/MoeB-like domain. NCBI blast analyses reveal that hesA gene is significantly conserved within nif gene clusters among diazotrophic Paenibacillus, cyanobacteria and Frankia (Fig. 1). HesA from P. polymyxa WLY78 at amino acid sequence level shares more than 90% identity with those of other diazotrophic Paenibacillus spp. (Xie et al., 2014) and 46.8%, 43.1% and 49.2% identity with HesA of Frankia alni ACN14a (Chang et al., 2012), Cyanothece sp. ATCC 51142 (Welsh et al., 2008) and Anabaena sp. PCC 7120 (Mitschke et al., 2011), respectively.

Furthermore, we find that P. polymyxa HesA has 32%, 30%, 26% and 24% identity with MoeB of Pseudomonas fluorescens, K. oxytoca, E. coli and Bacillus subtilis, respectively, whereas it shows only 9% identities with NifQ of K. oxytoca and A. vinelandii and 8–18% with these NifQs from other N₂-fixing bacteria. The data indicate that HesA exhibits higher similarity to MoeB than to NifQ. Also, there are no moeB-like genes other than hesA in Paenibacillus.

The hesA mutation of P. polymyxa WLY78 leads to nearly complete loss of nitrogenase activity

As described in Materials and Methods, the ΔhesA mutant was constructed by inserting a 1,197 bp Cm resistance gene cassette into the nif gene cluster (nifBHDKENXhesAnifV) and substituting for hesA. qRT-PCR further revealed that the mRNA level of nifV gene was not affected by the hesA mutation (Fig. S1), suggesting that the insertion of Cm-cassette into nif gene cluster did not generate polarity effect.

Here, the nitrogenase activities of the ΔhesA mutant and wild-type P. polymyxa WLY78 grown in medium containing 0.25 mM sulfate as sulfur source and supplemented with 0.3 and 30 μM molybdate, respectively, were comparely analyzed (Fig. 2). The ΔhesA mutant showed a significant decrease in nitrogenase activity compared to wild type, suggesting that hesA is involved in nitrogen fixation. We noticed that the hesA mutant and P. polymyxa WLY78 also exibited some activity in medium without molybdate, which might be due to trace molybdenum in the medium.

Furthermore, we demonstrated that P. polymyxa hesA could completely restore the activity of hesA mutant to the level of wild-type Paenibacillus, confirming that hesA is required for nitrogen fixation. We also tried to complement with K. oxytoca nifQ and found that the nifQ could not suppress the effect of hesA mutation completely (Fig. 2). The data suggest that K. oxytoca nifQ could not act as a molybdenum donor for FeMo-co synthesis of nitrogenase in P. polymyxa.

The hesA mutant requires high levels of molybdate for nitrogenase activity

The molybdate requirement for nitrogenase activity in the ΔhesA mutant was studied, with P. polymyxa WLY78 as a control. As shown in Fig. 3A, P. polymyxa WLY78 had nitrogenase activity even in the absence of added sodium molybdate, because there were always traces of Mo in other chemicals, the glassware and water. The maximum activity of wild-type P. polymyxa WLY78 was obtained at 0.3 μM molybdate, and the nitrogenase activity did not increase as the concentration of molybdate increased. In contrast, the nitrogenase activity of the ΔhesA mutant improved as the molybdate concentration increased, and the maximum activity was obtained at 30 mM molybdate. The maximum activity of the ΔhesA mutant at 30 mM molybdate was 25% of that of the wild type obtained at 0.3 μM molybdate, indicating that the addition of very high levels of molybdate could partially suppress the effect of the mutation.

Diazotrophic growth of the wild-type P. polymyxa WLY78 and ΔhesA mutant showed the same dependence on the molybdate added (Fig. 3B). Compared with the wild type, the ΔhesA mutant needed higher concentration of molybdate for the maximum growth. Millimolar concentrations of molybdate obviously inhibited both activity and diazotrophic growth. Meanwhile, the ΔhesA mutant showed a higher tolerance.

Effect of two sulfur sources on nitrogenase activity of the ΔhesA mutant

Here, the effects of two sulfur sources on nitrogenase activity of the ΔhesA mutant were determined. As shown in Fig. 4A, the nitrogenase activity of the ΔhesA mutant in medium containing cystine as the only sulfur source was two to five times higher than that in medium containing sulfate at all of different concentrations of molybdenum except for a high inhibitory level of 0.3M molybdenum. However, no difference in nitrogenase activity of wild-type Paenibacillus was observed when using either sulfate or cystine as sulfur source (Fig. 4B). These results demonstrated that the use of cystine as the only sulfur source could suppress the effect of hesA mutation on nitrogenase activity, but the addition of cystine could not affect the requirement of molybdenum for maximum nitrogenase activity. The results indicated that hesA might be involved in sulfur transport.

High levels of cystine restore the nitrogenase activity of the ΔhesA mutant, but E. coli moeB cannot

As shown in Fig. 5A, the nitrogenase activity of the ΔhesA mutant was improved with the addition of cystine, and higher concentration of cystine was required for the ΔhesA mutant to reach the maximum activity of the wild type. Similarly, the activity of the ΔhesA mutant increased with the addition of sulfate, but a higher concentration (above 1.0 mM) of sulfate was required for the ΔhesA mutant to reach the maximum activity of the wild type (Fig. 5B).

In addition, we tried to complement the ΔhesA mutant with E. coli moeB and found that the expression of moeB in the mutant did not improve nitrogenase activity (Fig. 5). In fact, the nitrogenase activity in the ΔhesA mutant expressing moeB declined when the concentration of cystine exceeded 1.0 mM. The data suggested that E. coli moeB could not completely suppress the effect of the hesA mutation when sulfate or cystine was used as the only sulfur source. It has been reported that MoeB, MPT synthase sulfurase, is involved in sulfuration of MPT synthase (Leimkühler, Wuebbens & Rajagopalan, 2001; Pitterle & Rajagopalan, 1993; Rajagopalan, 1997).

A high level of molybdate is required for nitrogenase activity of the recombinant E. coli 78-7 with a hesA deletion

In order to confirm the role of the hesA in nitrogen fixation, we compared the activities of the recombinant E. coli 78-7 strain carrying a nif gene cluster (nifBHDKENXhesAnifV) of P. polymyxa WLY78 (Wang et al., 2013) and the hesA deletion strain (D-O), which was a derivative of E. coli 78-7 strain without the hesA gene. As shown in Fig. 6, the nitrogenase activity of the D-O strain was improved as the concentration of molybdate increased. The D-O strain required 10 times as much molybdate to achieve the level of activity of the E. coli 78-7 strain. The P. polymyxa hesA gene could complement the D-O strain and restore the activity of the hesA deletion strain to that of the E. coli 78-7 strain, suggesting that the addition of very high levels of molybdate can suppress the effect of the hesA mutation. The current result was consistent with the results obtained in the P. polymyxa hesA mutant.

Cystine can enhance activity of the hesA deletion in E. coli 78-7

Here, we further investigated the effect of cystine on the activities of the hesA deletion strain (D-O) which carried other nif genes (nifBHDKENXV) without hesA when it was grown in medium supplemented with 30 μM molybdate and with different concentrations of cystine. As shown in Fig. 7, cystine could enhance activity of the hesA deletion strain (D-O), but it did not have the similar effects on the complemented strain D-O (pBluescript II SK (+)-hesA).

The P. polymyxa hesA mutation does not affect nitrate reductase activity

Nitrate reductase, like nitrogenase, has been characterized as a molybdoenzyme (Taniguchi & Itagaki, 1960) and molybdenum is necessary for nitrate reductase (Lester & Demoss, 1971). Here, the nitrate reductase activities of the ΔhesA mutant and wild-type P. polymyxa WLY78 were comparatively analyzed. The ΔhesA mutant (1,048.12 ± 28.80 nmol nitrite·min⁻¹·mg⁻¹protein) and wild-type P. polymyxa WLY78 (1,057.37 ± 5.95 nmol nitrite·min⁻¹·mg⁻¹protein) showed the similar nitrate reductase activity, indicating that the hesA gene product is required for synthesis of FeMo-co, but not implicated in the biosynthesis of Mo-co.