The sigma factor σ⁵⁴ is required for the long-term survival of Leptospira biflexa in water

Comparison of EBP and σ⁵⁴ regulatory pathways among Leptospira species

Both saprophytic and pathogenic Leptospira encode a single copy of rpoN gene (encodes for σ⁵⁴), which is located on the large chromosome of the genome. L. biflexa σ⁵⁴ sequence shares ∼60% identity at the amino acid level with that of pathogenic and intermediate leptospiral species. Phylogenetic analysis showed that the Leptospira σ⁵⁴ was evolutionary separated from other bacteria, even from other Spirochaetaceae genera including Borrelia and Treponema (Fig. 1). Leptospira σ⁵⁴ has all three conserved functional domains, Region I, II and III (Fig. 2A), despite that the overall identities of Leptospira σ⁵⁴ at the amino acid level are less than 30% with a typical σ⁵⁴ such as in Enterobacteriaceae.

Activation of σ⁵⁴–controlled genes requires a prokaryotic enhancer binding protein. Both pathogenic and saprophytic Leptospira have the activator EbpA for activation of σ⁵⁴-controlled transcription initiation (Fouts et al., 2016; Hu et al., 2016) (Fig. 2B). EbpA is an FhlA (formate hydrogen lyase activator)-like EBP (group III), which includes two GAF (cGMP-specific phosphodiesterases, Adenylyl cyclases and FhlA) domains (Hopper and Bock, 1995). In addition, pathogenic Leptospira species have an activator EbpB that is not present in saprophytic Leptospira (Fouts et al., 2016; Hu et al., 2016). EbpB is an NtrC-like EBP (group I), which is activated by phosphorylation at the RR domain by a cognate histidine kinase (Doucleff et al., 2005). Thus, pathogenic and saprophytic Leptospira share an EbpA-σ⁵⁴ regulatory network, whereas pathogenic Leptospira have an additional EbpB-σ⁵⁴ network.

Targeted inactivation and complementation of rpoN in L. Biflexia

We first attempted to inactivate rpoN in pathogenic Leptospira. Despite multiple attempts, no mutant could be obtained in. L. interrogans strain Lai 56601, Fiocruz L1–130 or L495. We thus focused on saprophytic Leptospira, and successfully inactivated the rpoN gene (LEPBI_I1650) by allelic exchange in L. biflexa serovar Patoc strain Patoc1 (Fig. 3A). The lack of rpoN expression and the product σ⁵⁴ in the rpoN mutant were demonstrated by quantitative reverse transcription-PCR (qRT-PCR) (Fig. 3B) and immunoblotting analyses (Fig. 3C). A complemented strain for the rpoN mutant was also achieved by transforming a shuttle vector containing a wild-type rpoN gene driven under the hsp10 promoter of L. biflexa (Fig. 3A), and restoration of σ⁵⁴ was confirmed in the complementary strain (rpoN/pJJ003) (Fig. 3B and C).

Transcriptome analysis for the identification of σ⁵⁴-regulated genes

To identify genes influenced by σ⁵⁴, we performed whole-transcriptome analysis for wild-type L. biflexa and the rpoN mutant. The results revealed that expression of 20 genes was lower in the rpoN mutant than that in the wild-type strain (with a cut-off of over twofold of changes in expression), suggesting that these genes were positively regulated by σ⁵⁴ (Table 1). The analysis also identified 36 genes whose expression was negatively regulated by σ⁵⁴ (Table 2). Among those genes that were σ⁵⁴-dependent, several genes are predicted involving in nitrogen transport and metabolism, including genes encoding ammonia transporter AmtB1 (LEPBI_I1767), AmtB2 (LEPBI_I2408), nitrogen regulatory protein P-II (LEPBI_I2407), Nark-like nitrate/nitrite antiporter (LEPBI_I2771) and NtrX family nitrogen assimilation regulator (LEPBI_I1654). Further qRT-PCR analysis confirmed that expressions of these genes were significantly downregulated in the rpoN mutant, while the complemented strain could fully restore their expression (Fig. 4). Note that we also examined the patter of gene regulation in the rpoN mutant at both 30°C and room temperature, and a similar pattern was observed.

The σ⁵⁴ regulon is important for leptospiral survival under the nitrogen starvation or environmental water conditions

To investigate whether σ⁵⁴ is important for nitrogen utilization of L. biflexa, wild-type, the rpoN mutant and the complemented strains were cultured either under the nitrogen-rich conditions (regular Ellinghausen-McCullough-Johnson-Harris (EMJH) medium, containing 5 mM NH₄Cl) (Fig. 5A), or nitrogen-poor conditions in which NH₄Cl in the EMJH medium was replaced with 5 mM of glutamine (Fig. 5B), arginine (Fig. 5C) or alanine (Fig. 5D). The result showed that all L. biflexa strains grew well when glutamine, a known major amide-donor amino acid, was used as a nitrogen source. However, both wild-type and the mutant grew poorly when arginine or alanine was used as a nitrogen source, suggesting that L. biflexa cannot utilize these poor nitrogen sources (Fig. 5C and D).

We then examined whether there is a difference in survival between wild-type and the rpoN mutant strains under the long-term nitrogen starvation conditions. After spirochetes were incubated under the nitrogen-starvation conditions for one week, cells were plated on regular EMJH solid media for colony count. As shown in Fig. 5E, wild-type cells recovered well, whereas the rpoN mutant could no longer recover and only few tiny colonies were found. This data suggests that the rpoN mutant has a defect in survive under the long-term nitrogen starvation conditions.

Leptospira is known to be capable of surviving in environmental water for a long time. Since the rpoN mutant could not survive during nitrogen starvation, we argued that it should be incapable to survive in water. To this end, wild-type, the rpoN mutant and the complemented strain of L. biflexa were incubated in spring water, and samples were collected weekly and plated on the solid EMJH media for quantitation of cell viability. The rpoN mutant formed tiny colonies, and became no longer detectable at week 4 after incubation in water (Fig. 6), whereas wild-type and the complementary strains grew normally and remained detectable at week 6 (Fig. 6). This result indicates that σ⁵⁴ is also important to the viability of Leptospira in water.

amtB1, amtB2 and narK are induced under the nitrogen starvation condition, and are controlled by σ⁵⁴ indirectly

To begin elucidating the mechanisms underlying how the RpoN regulon governs the survival of L. biflexa in the nitrogen-limiting mediums and in water, we focused on σ⁵⁴-regulated genes, amtB1, amtB2 and narK. We examined if the expression of these genes is induced under the nitrogen-limiting conditions (Fig. 7A–C). In wild-type L. biflexa, their expressions were significantly upregulated under the nitrogen limiting conditions. The expression of these genes in the rpoN mutant could not be detected under any of the conditions tested, further supporting the notion that the expression of these genes is absolutely dependent on σ⁵⁴.

σ⁵⁴ recognizes a highly conserved −24/-12 promoter sequence (TGGCA < 6bp > TTGCT/A). Further analysis of the upstream sequences of amtB1, amtB2 and narK (including the upstream sequences of the putative operons in which these genes may reside) did not identify a putative σ⁵⁴-type promoter, a result that is consistent with recent reports of genome-wide analysis of σ⁵⁴-type promoters in published bacteria genomes (Francke et al., 2011; Bonocora et al., 2015). These data suggest that the expression of amtB1, amtB2 and narK are not directly under the control of σ⁵⁴.

rcfA encoding a tetratricopeptide repeat protein is induced under the nitrogen starvation condition, and is under the direct control of σ⁵⁴

Transcriptome analysis revealed that the expression of LEPBI_I1011, which encodes a hypothetical protein containing a tetratricopeptide repeat (TPR, pfam13424) domain, was dramatically downregulated in the rpoN mutant (Table 1). Further qRT-PCR analysis confirmed this finding (Fig. 4). Similar to other predicted nitrogen utilization-related genes, expression of LEPBI_1011 was significantly upregulated in ammonia starvation but downregulated when ammonia is in excess, and such regulation is dependent on σ⁵⁴ (Fig. 7D). These data suggest that LEPBI_I1011 is a novel protein involved in nitrogen regulation of L. biflexa. We hereby rename it as RpoN-controlled factor A (RcfA).

Promoter analysis of the upstream sequence of rcfA revealed that there is a putative σ⁵⁴-type promoter sequence (TGGCA < 6bp > TTGCA) that is virtually identical to the consensus sequence of the σ⁵⁴-type promoter, located 33bp upstream of the ATG translational start codon of RcfA (Fig. 8A). Based on the predicted transcriptional start site, transcription from this promoter will yield a rcfA mRNA containing a 21 bases of untranslated sequence, which includes a putative CGGAGG ribosomal binding site (Shine-Dalgarno sequence).

To examine if σ⁵⁴ of L. biflexa binds to the putative rcfA promoter, we purified recombinant RpoN protein (σ⁵⁴) of L. biflexa and performed the in vitro electrophoretic mobility shift assays (EMSAs). To this end, a 40 bp oligonucleotide encoding the predicted −24/-12 region of the promoter of rcfA (Fig. 8A) was end-labeled with ³²P and incubated with varying amounts of purified RpoN. The results showed that σ⁵⁴ bound to the rcfA promoter region in a dose-dependent fashion (Fig. 8B). Moreover, the binding of RpoN to labeled probe was inhibited by the addition of 200-fold excess of specific cold competitor DNA (Fig. 8C), and was not affected by the addition of 100-fold excess of non-specific salmon sperm DNA. Noted that no DNA shift was observed in the EMSAs using promoter fragments of LEPBI_I2408, LEPBI_I1767 and LEPBI_I2771 (data not shown). These results suggest that rcfA is directly under the control of σ⁵⁴.

Since EbpA is the essential activator for genes with σ⁵⁴-type promoter, we examined EbpA binding to the upstream region of rcfA (-450-50 relative to the ATG start code). The EMSA results demonstrated that EbpA was capable of binding to the upstream region of rcfA in a dose-dependent manner (Fig. 9A). The binding of EbpA to rcfA was specific, as (1) the binding was not affected with addition of nonspecific competitor poly(dI-dC) (Fig. 9B) and (2) EbpA did not bind to the upstream region of glpF that has a σ⁷⁰-type promoter (Fig. 9C).

Bacterial strains and culture conditions

Leptospira biflexa serovar Patoc strain Patoc 1 (Paris) was maintained and grown in EMJH medium at 30°C. For the rpoN mutant and its complemented strains, spectinomycin and gentamycin were added to a final concentration of 50 μg ml⁻¹ respectively. For the modified EMJH, NH₄Cl (5 mM) in the standard EMJH medium was replaced with the same concentration of glutamine, arginine, or alanine, with the pH adjusted to 7.4. The constructed suicide and shuttle vectors were maintained in E. coli strain DH5α, and E. coli strain β2163 was used as the donor for E. coli- Leptospira conjugation.

Construction of the rpoN mutant and the complementation strains

The RpoN (LEPBI_I1650)-coding gene disruption mutant was created by allelic exchange in L. biflexa by transforming with the suicide vector pLb-RpoN-KO (Fig. 3A). To construct the knockout plasmid, both the upstream and downstream 1700-bp fragments of rpoN were PCR amplified from L .biflexa genomic DNA. The resulting DNA fragments were then cloned upstream and downstream of a spectinomycin-resistance marker (aadA) driven by a constitutively expressed Borrelia promoter flaB via HindIII/MluI and NheI/XmaI restriction sites respectively. The resulting suicide plasmid was confirmed by both restriction enzyme digestion and sequencing followed by electroporation. Positive Leptospira transformants were selected via proper antibiotic resistance, and rpoN inactivation was verified by qRT-PCR and immunoblot analysis.

A shuttle vector, pJJ003, was constructed for the complementation of rpoN (Fig. 3A) based on the E. coli-Leptospira shuttle vector pCJSpLe94 (Picardeau, 2008). To this end, the spectinomycin-resistance marker in pCJSpLe94 was first replaced by a gentamycin-resistance marker originated from plasmid pSL94PfGenta (Poggi et al., 2010) to construct pJJ002. Then, an hsp10-promoter driving rpoN cassette was inserted into plasmid pJJ002. The resulting plasmid, pJJ003, was sequenced before it was transformed into the L. biflexa rpoN mutant by conjugation as described (Picardeau, 2008). The transformants with both gentamycin and spectinomycin resistance were selected and subjected to qRT-PCR and immunoblot analysis to confirm the restoration of RpoN expression.

RNA extraction, microarray construction, scanning and data analysis

The annotation of the L. biflexa strain Patoc 1 (Paris) genome (http://www.ncbi.nlm.nih.gov/genome/?term=NC_010602) was used to design 60-mer oligonucleotides using the web-based application eArray (Agilent). Three different probes were designed for each open read frame, based on the optimization of melting temperature (T_m), secondary structure and homology to other sites in the whole genome. In total, 11,166 oligonucleotides were designed, representing 3722 (out of 3730) putative open reading frames of the L. biflexa genome. They were printed in triplicate on each subarray, which were in turn printed in quadruplicate on the slide. The oligonucleotide synthesis and microarray print, as well as the scanning and data analysis, were completed by MOgene.

For the RNA extraction, both the wild-type and the rpoN mutant strains were cultivated in EMJH medium at room temperature and harvested at the mid-logarithmic growth. Total RNA was extracted from two biological replicates using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Digestion of contaminating genomic DNA in the RNA samples was performed using RNase-Free DNase I (New England Biolabs), and removal of DNA was confirmed by PCR amplification using primers specific for flaB of L. biflexa. cDNA was synthesized and labeled with Cy3 or Cy5 by use of the Amersham Post-Labeling Kit according to the manufacturer's instructions, with minor modifications. Briefly, 5 μg of total RNA was converted to cDNA by use of CyScript RT in the presence of 1 μl of random nonanucleotides (Amersham Biosciences). Each cDNA sample was labeled with Cy3 and Cy5 separately. Cy3- or Cy5-labeled cDNA from the parental strain was then combined with the Cy5- or Cy3-labeled cDNA from the mutants. Labeled probes were purified and then used in the microarray experiment. With two pairs of samples plus dye switching, we made a total of four hybridized slides. Hybridized slides were then scanned on an Axon 4000B microarray scanner using GenePix Pro 6.1 (Molecular Devices). The image was analyzed using the GenePix program, and data were then analyzed with Acuity 4.0 (Molecular Devices) by using the ratio-based normalization method. A 2-fold change cutoff value was used to select candidate genes.

qRT-PCR

RNA samples were extracted from leptospiral cultures grown to middle log phase (around 1 × 10⁸ cells per ml) at 30°C using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocols. The cDNA was synthesized using the SuperScript III Reverse Transcriptase with random primers (Invitrogen) from 1 μg RNA. qRT-PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) on an ABI 7000 sequence detection system. The flaB gene of L. biflexa was used as a reference. The relative transcription level was determined by the threshold cycle (2^–ΔΔCt) method (Livak and Schmittgen, 2001). All samples were run in triplicate, and the student t test was performed to determine the statistical significance between the expression levels of different groups.

Measurement of cell growth and viability on solid medium

To investigate the growth abilities of Leptospira strains in the mediums with different nitrogen sources, the tested strains were first grown in regular EMJH medium to late log phase. The cells were harvested by centrifugation and washed once with ammonia-free EMJH to avoid carryover of residual nitrogen sources. Then cells were subcultured in the modified fresh EMJH with different nitrogen sources at an initial concentration of 10⁶ spirochetes per ml. Every medium was independently analyzed two times. Those nitrogen sources that supported growth were further analyzed with three independent replicates each time. All cultures were incubated at 30°C and the cell numbers were numerated every 24 hr under a dark-field microscope. The viabilities of leptospires were enumerated by plating diluted culture samples on solid EMJH medium.

Water survival test

The long-term water survival abilities of Leptospira strains were investigated by a modified method (Smith and Turner, 1961; Trueba et al., 2004). Since Leptospira cannot survive in distilled water, the spring waters used in the study were commercially available drinking water in North American (pH 7.2, Kirkland Signature, USA) with trace mineral contents. The waters were filter sterilized to avoid modifying the chemical equilibrium. In brief, leptospiral cells grown to logarithmic phase (3 × 10⁸ cells per ml) were harvested by centrifugation. The supernatants were thoroughly drained, and the cells were resuspended in the original volume (15 ml) of water. All cultures were incubated at 30°C, and the viabilities of leptospires were enumerated every week by plating 100 μl diluted culture samples on solid EMJH medium. The plates were incubated at 30°C for 2 weeks, and the efficiency of plating was determined by dividing the total number of colonies, irrespective of size, by the number of colonies expected based on the cell counting via dark-field microscopy. Culturable cells were also detected by inoculating 0.5 ml culture into 5 ml fresh EMJH and incubating for 4 weeks. The typical gyrations in liquid media were used to identify live leptospires.

EMSA

EMSAs were performed to determine protein: DNA interactions as described previously (Hellman and Fried, 2007). For RpoN binding, a 50-bp putative promoter fragment containing an engineered EcoRI site was PCR amplified. The resulting fragment was purified and digested with EcoRI, and then labeled with ³²P using Taq DNA polymerase by filling in the EcoRI sites with [α-³²P]dATP. For EbpA binding, a 500-bp DNA fragment upstream of the gene LEBI_I1011 (−450 and +50 bp relative to the ATG start codon) was PCR amplified from the genome of L. biflexa with primers with containing an engineered EcoRI site, and then labeled as above. Labelled fragments (0.2 pmol) and various amounts of purified RpoN or EbpA were mixed in 10 μl binding reactions in the presence of 100 μg ml⁻¹ salmon sperm DNA for 30 min at room temperature. Some reactions included unlabeled promoter competitors or non-specific competitors for competition studies. The reactions were analyzed on non-denatured polyacrylamide gels, and then the gels were dried and exposed in a cassette using an X-ray film for autoradiography.

Protein expression and antisera preparation

For expression and purification of RpoN of L. biflexa, the rpoN gene was PCR amplified from genomic DNA of L. biflexa and cloned into the expression vector pET100 (Invitrogen). The resulting plasmid, pJJ032, was transformed into E. coli BL21DE3 (Novagen), and the C terminal His₆ tagged RpoN was expressed with the induction of 0.4 mM IPTG. The recombinant RpoN protein was further purified using Ni-NTA affinity chromatographic column (NEB) according to the manual. To obtain the recombinant protein EbpA, the full-length ebpA gene was PCR amplified by PCR application. The PCR product was cloned into pGEX-4T-2 vectors (Amersham Pharmacia Biotech) via BamHI and NotI (NEB) digestion. The resulting plasmid, pGEX-4T-2ebpA, was transformed into E. coli BL21(DE3) for the expression of glutathione S-transferase (GST) fused recombinant EbpA protein. The protein was expressed under the induction of 0.5 mM IPTG, and purified by glutathione superflow agarose (Pierce) according to the manual. To increase the solubility of the recombinant proteins, all induction was conducted at 16°C. The expressed and purification of recombinant proteins were monitored by SDS polyacrylamide gel electrophoresis.

To obtain the recombinant protein GroEL, the full-length groEL gene was PCR amplified from L. interrogans strain Lai genomic DNA. The product was digested with NdeI and XhoI endonucleases before it was inserted into pET42a and transformed into E. coli BL21DE3 (Novagen). The His₆ tagged recombinant GroEL protein was expressed under the induction of 1 mM isopropy-β-Dthiogalactoside (IPTG), and the soluble GroEL was purified using Ni-NTA affinity chromatographic column (NEB) according to the manual.

For antisera production, New Zealand rabbits were immunized intradermally on days 1, 14, 21 and 28 with 2 mg leptospiral RpoN and GroEL respectively, which were pre-mixed with Freund's adjuvant. Fifteen days after the last immunization, the sera were collected to separate the IgGs by ammonium sulfate precipitation plus a DEAE-52 column (Sigma) purification. Phosphate buffer (10 mM, pH 7.4) was used for elution and the titer of the IgGs binding was detected by immunodiffusion test.

Immunoblot analysis

Leptospira strains were inoculated into EMJH medium and grown to late logarithmic phase before they were harvested by centrifugation at 7000 g and washed twice with phosphate buffered saline (PBS, 50 mM, pH7.4). The pellets were collected for immunoblotting using the SuperSignal West Pico chemiluminescent substrate with home-made antibodies according to the manufacturer's instructions (Pierce).