N-Carbamoylputrescine Amidohydrolase of Bacteroides thetaiotaomicron, a Dominant Species of the Human Gut Microbiota

Polyamines are bioactive amines that play a variety of roles, such as promoting cell proliferation and protein synthesis, and the intestinal lumen contains up to several mM polyamines derived from the gut microbiota. In the present study, we conducted genetic and biochemical analyses of the polyamine biosynthetic enzyme N-carbamoylputrescine amidohydrolase (NCPAH) that converts N-carbamoylputrescine to putrescine, a precursor of spermidine in Bacteroides thetaiotaomicron, which is one of the most dominant species in the human gut microbiota. First, ncpah gene deletion and complemented strains were generated, and the intracellular polyamines of these strains cultured in a polyamine-free minimal medium were analyzed using high-performance liquid chromatography. The results showed that spermidine detected in the parental and complemented strains was depleted in the gene deletion strain. Next, purified NCPAH-(His)6 was analyzed for enzymatic activity and found to be capable of converting N-carbamoylputrescine to putrescine, with a Michaelis constant (Km) and turnover number (kcat) of 730 µM and 0.8 s−1, respectively. Furthermore, the NCPAH activity was strongly (>80%) inhibited by agmatine and spermidine, and moderately (≈50%) inhibited by putrescine. This feedback inhibition regulates the reaction catalyzed by NCPAH and may play a role in intracellular polyamine homeostasis in B. thetaiotaomicron.


Introduction
Polyamines are aliphatic amines with two or more amino groups and are found in almost all living organisms, from prokaryotes to higher plants and animals, and their intracellular concentrations are in the mM range [1]. The most common polyamines are putrescine, spermidine, and spermine.
In recent years, it has become clear that polyamines contribute significantly to extending the healthy life span of various organisms. The first report on the extension of animal lifespan through polyamine ingestion was a 2009 study using mice [2]. The study reported that feeding diets containing high concentrations of putrescine, spermine, and spermidine increased blood polyamine levels and reduced aging in the kidneys and liver, resulting in an extension of the lifespan [2]. In the same year, experiments with Caenorhabditis elegans, Drosophila melanogaster, and mice showed that the administration of spermidine was effective in extending the lifespan, and an enhancement in histone acetylation and export occurs through a putrescine-ornithine antiporter activity [24], while the uptake is dependent on the membrane potential [25]. The spermidine transporter MdtJI has also been reported in E. coli to prevent toxicity from the accumulation of excess spermidine in the bacteria [26].
E. coli has two pathways to metabolize putrescine to succinate via GABA. The first is a pathway with γ-aminobutyraldehyde as a reaction intermediate. In this pathway, putrescine is metabolized to GABA without γ-glutamylation [27]. One other pathway is called the Puu pathway [28], in which putrescine is γ-glutamylated and metabolized to γ-Glu-GABA via γ-glutamyl-γ-aminobutyraldehyde, followed by hydrolysis of the γglutamyl bond to GABA and Glu by PuuD [29]. In this pathway, putrescine in the medium is imported to the cell through the transporter PuuP [21]. First, PuuA uses ATP to bind glutamate to one amino group of putrescine, producing γ-glutamylputrescine [30]. Next, γ-glutamylputrescine is oxidized by PuuB to γ-glutamylγ-aminobutyraldehyde. It is then believed to be further oxidized by PuuC to γ-glutamyl-GABA. The γ-glutamyl group is then cleaved by PuuD, releasing glutamate and GABA [31]. GABA is then deaminated by PuuE to form succinate semialdehyde [32]. Finally, succinate semialdehyde is oxidized by YneI to form succinate [32].
In contrast to polyamine metabolic pathways and transporters being well studied in E. coli, which is not the dominant species in the human gut, few studies have examined polyamine biosynthetic pathways in the predominant gut bacterial species. Furthermore, the polyamine biosynthetic pathway in E. coli differs from the pathway predicted to be present in the most predominant gut microbiota species [33]. In Enterococcus faecalis, ranked 54th among the 56 most abundant species of commensal gut microbiota in Europeans [34], an agmatine-putrescine antiporter (AguD [35]) takes up agmatine into bacterial cells. Then, agmatine is hydrolyzed into N-carbamoylputrescine and ammonia-catalyzed by agmatine deiminase (AguA [36]), and N-carbamoylputrescine is converted to putrescine via a reaction catalyzed by putrescine transcarbamylase (AguB [36]). Putrescine produced from a series of biosynthetic pathways within the bacterial cell is exported by AguD to the outside of the bacterial cell [35].
Bacteroides thetaiotaomicron is ranked 8th among the 56 most abundant species of commensal gut microbiota in Europeans [34]. Phylogenetically, the phylum Bacteroidota accounts for approximately 43% of the 56 most abundant species of human commensal gut microbiota, and the genus Bacteroides accounts for approximately 36% [34]. Therefore, analysis of B. thetaiotaomicron can significantly help to understand polyamine metabolism throughout the human commensal gut microbiota. In addition, B. thetaiotaomicron has been reported to possess anti-inflammatory properties, enhance mucosal barrier function, and restrict pathogen invasion [37]. The administration of B. thetaiotaomicron in autoimmune inflammatory bowel disease mouse models protects against weight loss, histological changes in the colon, and inflammatory markers [38]. B. thetaiotaomicron has been reported to induce NF-κB-relaxed aspartate-auxotrophic-PPARγ complexes in colon cancer cell line (Caco-2) cells in vitro and to downregulate NF-κB-induced inflammatory genes such as TNFα [39]. Furthermore, the NF-κB pathway regulates T cell differentiation in asthma by controlling the expression of inflammatory genes [40], especially those encoding IL-6 and TNF-α, suggesting the potential role of B. thetaiotaomicron in this regulation [41]. However, the genus Bacteroides has been reported to have potentially detrimental effects on health. The proportion of genus Bacteroides increased in the gut microbiota of immigrants to the U.S., and a decrease in bacterial enzymes is related to the breakdown of plant fibre and obesity [42]. In addition, the Bacteroides enterotype is more common in patients with depression [43].
A polyamine biosynthetic pathway via N-carbamoylputrescine, previously reported in C. jejuni [33], is predicted to be present in B. thetaiotaomicron by the BLAST analyses. In the predicted pathway ( Figure 1) arginine is decarboxylated by SpeA to form agmatine. Next, agmatine is converted to N-carbamoylputrescine with the liberation of ammonia by a reaction catalyzed by agmatine iminohydrolase (AIH [33]). Then, N-carbamoylputrescine is converted to putrescine with the liberation of ammonia and carbon dioxide by Ncarbamoylputrescine amidohydrolase (NCPAH [33]). The synthesized putrescine is converted to carboxyspermidine by the reductive condensation of putrescine and aspartateβ-semialdehyde catalyzed by carboxyspermidine dehydrogenase (CASDH [33]). Finally, carboxyspermidine is decarboxylated by carboxyspermidine decarboxylase (CASDC) to form spermidine. Additionally, B. thetaiotaomicron is also predicted to take up extracellular spermidine by PotABCD, a homolog of the E. coli spermidine transporter [44].
A polyamine biosynthetic pathway via N-carbamoylputrescine, previously reported in C. jejuni [33], is predicted to be present in B. thetaiotaomicron by the BLAST analyses. In the predicted pathway ( Figure 1) arginine is decarboxylated by SpeA to form agmatine. Next, agmatine is converted to N-carbamoylputrescine with the liberation of ammonia by a reaction catalyzed by agmatine iminohydrolase (AIH [33]). Then, N-carbamoylputrescine is converted to putrescine with the liberation of ammonia and carbon dioxide by Ncarbamoylputrescine amidohydrolase (NCPAH [33]). The synthesized putrescine is converted to carboxyspermidine by the reductive condensation of putrescine and aspartateβ-semialdehyde catalyzed by carboxyspermidine dehydrogenase (CASDH [33]). Finally, carboxyspermidine is decarboxylated by carboxyspermidine decarboxylase (CASDC) to form spermidine. Additionally, B. thetaiotaomicron is also predicted to take up extracellular spermidine by PotABCD, a homolog of the E. coli spermidine transporter [44]. The pathway is shown with the corresponding enzymes, which were predicted by BLASTP analyses in our previous study [45]. NCPAH, the enzyme analyzed in this study, is indicated by red characters, while the enzyme analyzed in our previous study is shown by blue characters. The other predicted enzymes are shown by orange characters. The abbreviations are as follows. SpeA: arginine decarboxylase; AIH: agmatine deiminase/iminohydrolase; NCPAH: N-carbamoylputrescine amidohydrolase; CASDH, carboxyspermidine dehydrogenase; CASDC: carboxyspermidine decarboxylase; PotABCD: ATP-binding casseOe transporter for spermidine.
In a previous study, we revealed that B. thetaiotaomicron accumulates spermidine as its sole polyamine and that CASDC is essential for converting carboxyspermidine to spermidine [45], but NCPAH, which is predicted to biosynthesize putrescine, the precursor of spermidine, remains unstudied. Here, we performed biochemical and genetic analyses of Figure 1. Polyamine biosynthetic pathway in B. thetaiotaomicron. The pathway is shown with the corresponding enzymes, which were predicted by BLASTP analyses in our previous study [45]. NCPAH, the enzyme analyzed in this study, is indicated by red characters, while the enzyme analyzed in our previous study is shown by blue characters. The other predicted enzymes are shown by orange characters. The abbreviations are as follows. SpeA: arginine decarboxylase; AIH: agmatine deiminase/iminohydrolase; NCPAH: N-carbamoylputrescine amidohydrolase; CASDH, carboxyspermidine dehydrogenase; CASDC: carboxyspermidine decarboxylase; PotABCD: ATPbinding cassette transporter for spermidine.
In a previous study, we revealed that B. thetaiotaomicron accumulates spermidine as its sole polyamine and that CASDC is essential for converting carboxyspermidine to spermidine [45], but NCPAH, which is predicted to biosynthesize putrescine, the precursor of spermidine, remains unstudied. Here, we performed biochemical and genetic analyses of predicted NCPAH of B. thetaiotaomicron. As the genus Bacteroides is predominant and represents 30% of all bacteria in the human intestinal lumen, the results of this study provide a better understanding of total gut bacterial polyamine production.

Disruption and Complementation of Ncpah in B. thetaiotaomicron
Gene disruption of ncpah in B. thetaiotaomicron was performed using the previously established method [46]. DNA cloning was conducted with the In-Fusion cloning HD kit (Takara Bio USA, Mountain View, San Jose, CA, USA). The upstream and downstream regions (750 bp each) of ncpah were PCR-amplified from the JCM 5827 T genome as template using the primer pairs Pr-MS46/47 and Pr-MS48/49, respectively. The resulting two DNA fragments were ligated by overlap PCR using a primer pair Pr-MS46/49 and inserted into the SalI site of pExchange-tdk [46]. The resulting plasmid pMSK5 of MS108 was transferred by bacterial conjugation to B. thetaiotaomicron ∆tdk, and then ncpah knockout (∆ncpah) was obtained by the double-crossover event as described previously [45,46]. Introduction of the gene disruption into the target locus was verified by Sanger sequencing of DNA fragment PCR-amplified from the genome of the disruptant as template.
The ncpah-complemented strain was generated as follows. The rpoD (sigma 70 factor gene) promoter and ncpah gene were PCR-amplified from JCM 5827 T genome as template using the primer pairs Pr-MS52/53 and Pr-MS50/51, respectively. These two DNA fragments were inserted into the PstI and NotI site of pNBU2-bla-ermGb [46], and then the resulting plasmid pMSK6 of MS110 was inserted into the NBU2 att1 site on the chromosome of B. thetaiotaomicron ∆tdk ∆ncpah as described previously [45,46] to obtain the ncpah-complemented strain (∆ncpah att1::ncpah + ). The insertion of the plasmid into the targeted locus was verified by genomic PCR.

High-Performance Liquid Chromatography (HPLC) Analysis of Polyamines in Cells and Culture Supernatant
Polyamines in the cells and culture supernatant of B. thetaiotaomicron were analyzed as reported previously [45]. Specifically, cells of B. thetaiotaomicron strains (parental strain, ∆ncpah, and ∆ncpah att1::ncpah + ) were grown overnight in liquid GAM and harvested with centrifugation at 3400× g for 3 min. After washing once with the polyamine-free minimal medium, the cells were inoculated into 20 mL of the same fresh minimal medium to give an initial optical density at 600 nm (OD 600 ) of 0.03. The bacterial strains were then grown at 37 • C for 30 h, during which the growth was monitored by measuring OD 600 with a spectrophotometer. Cultures were collected at the appropriate times, after which cells and supernatants were obtained by centrifugation at 18,700× g for 5 min at 4 • C.
The cells and supernatants were used for polyamine analysis. Supernatants were mixed with trichloroacetic acid at a final concentration of 10% (w/v) and centrifuged twice at 18,700× g for 5 min at 4 • C, after which the resulting supernatants were filtered through a Cosmonice filter W (Nacalai Tesque Inc., Kyoto, Japan) and used for subsequent HPLC analysis. Similarly, the cells were washed once with phosphate-buffered saline (18,700× g, 4 • C, 5 min), resuspended in 300 µL of 5% (w/v) trichloroacetic acid, and incubated in boiling water for 15 min. The samples were then centrifuged at 18,700× g, 4 • C for 5 min to separate cell debris and supernatants, the latter of which were filtered through a Cosmonice filter W (Nacalai Tesque Inc., Kyoto, Japan) and used for subsequent HPLC analysis. Cell debris, which was dissolved in 300 µL of 0.1 N NaOH, was used to measure protein concentration by the Bradford method using bovine serum albumin as a standard (Bio-Rad protein assay kit; Bio-Rad Laboratories, Inc., Hercules, CA, USA).
For HPLC analysis, a cation exchange column (#2619PH, 4.6 × 50 mm; Hitachi, Tokyo, Japan) was used as in our previous report [45]. The polyamines were derivatized with o-phthalaldehyde with the postcolumn method and were detected with a fluorescence detector at λ ex 340 nm and λ em 435 nm. The concentration of each polyamine was calculated based on a standard curve created using standards of known concentrations. The standards used and their retention times were as follows: agmatine, 33.7 min; cadaverine, 20.5 min; N-carbamoylputrescine, 6.2 min; putrescine, 15.2 min; spermidine, 26.0 min; and spermine, 38.1 min. As a result, the concentration of polyamines in the culture supernatant was shown as µM, while that of intracellular polyamines was expressed as nmol/mg cellular protein.

Expression, Purification, and Characterization of Recombinant NCPAH
Recombinant NCPAH of B. thetaiotaomicron was expressed as a C-terminal His 6 -tagged form. The ncpah gene was PCR-amplified from JCM 5827 T genome as template using the primer pair Pr-MS435/436 and inserted into the NdeI and XhoI site of pET23b (Novagen). The resulting plasmid pMSK106 was introduced into E. coli BL21(DE3). This strain was designated MS821.
MS821, an E. coli strain for His 6 -tagged NCPAH overexpression (Table 1), was grown in LB medium supplemented with ampicillin at 25 • C with shaking at 140 rpm. When OD 600 reached to~0.5, a final concentration of 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the culture. After further 24 h incubation, the cells were harvested by centrifugation, resuspended in 50 mM potassium phosphate buffer (pH 8.0) containing 8 mM imidazole, and disrupted by sonication and centrifuged to obtain cell-free extract. The cell-free extract was applied to a Ni-NTA spin column (Qiagen, Hilden, Germany) equilibrated with lysis buffer (NPI-10 buffer containing 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0). The spin column was washed twice with wash buffer (NPI-20 buffer containing 50 mM NaH 2 PO 4 , 300 mM NaCl, and 20 mM imidazole, with pH 8.0) and eluted with elution buffer (NPI-500 buffer containing 50 mM NaH 2 PO 4 , 300 mM NaCl, 500 mM imidazole, pH 8.0) to yield recombinant NCPAH. The buffer of the resulting eluate was replaced with 50 mM potassium phosphate buffer (pH 8) using an Amicon Ultra-0.5 centrifugal filter unit (30 kDa cut off; Millipore, Billerica, MA, USA), in which addition of 400 µL of the same buffer followed by centrifugation (14,000× g for 10 min at 4 • C) was repeated five times. The purity of the protein was verified using SDS-polyacrylamide gel electrophoresis (Supplementary Figure S2). The protein concentration was determined with a Bradford assay using bovine serum albumin as a standard.

Enzymatic Assay Using Recombinant NCPAH
An enzymatic assay was performed at 50 • C for 40 min in a 400 µL reaction mixture containing 50 mM MES-NaOH buffer (pH 7.0), 20 ng/µL NCPAH, and 0-1.5 mM N-carbamoylputrescine. The reaction was started by adding different concentrations of N-carbamoylputrescine. Then, 100 µL aliquots were taken at 0, 20, and 40 min and the reactions were stopped by heating for 3 min at 95 • C. Activity was measured by quantifying ammonia released from N-carbamoylputrescine using indophenol blue method (calorimetric method) as described previously [48]. The standard curve was created by measuring the absorbance at 640 nm using known concentrations of NH 4 Cl. The kinetic parameters were determined by curve fitting the experimental data under different concentrations of N-carbamoylputrescine to Michaelis-Menten equation (GraphPad Prism v8.4.3).
The optimal temperature was determined by changing the reaction temperature within 20-60 • C. The enzymatic assay was performed for 60 min in a 400 µL reaction mixture containing 50 mM MES-NaOH buffer (pH 6.5), 20 ng/µL NCPAH, and 1 mM N-carbamoylputrescine, and the activity was measured with the indophenol blue method as mentioned above.
The optimal pH was determined by using different buffers (MES-NaOH buffer for pH 5.5-7.0; HEPES-NaOH buffer for pH 7.0-8.0; and TAPS-NaOH buffer for pH 8.0-9.0). The assay was conducted at 50 • C for 60 min in a 100 µL reaction mixture containing 50 mM buffer, 20 ng/µL NCPAH, and 1 mM N-carbamoylputrescine. The reaction was stopped by adding 100% (w/v) trichloroacetic acid to give a final concentration of 10% (w/v). After centrifugation at 21,487× g for 10 min, the supernatant was filtered using Cosmonice filter W (Nacalai Tesque Inc.) and subjected to HPLC, in which the activity was measured by quantifying the concentration of putrescine. The standard curve was created based on known concentrations of putrescine.
The effect of polyamines and their derivative compounds on the enzymatic activity was examined by adding 1 mM arginine, agmatine, putrescine, or spermidine into the reaction mixture. The assay was conducted at 50 • C for 40 min in a 100 µL reaction mixture containing 50 mM MES-NaOH buffer (pH 7.0), 5 ng/µL NCPAH, and 1 mM N-carbamoylputrescine. The activity was measured by quantifying putrescine using HPLC.

Disruption of Ncpah Abolishes Accumulation of Intracellular Spermidine in B. thetaiotaomicron
To examine the physiological role of ncpah in the bacterial growth and polyamine production of B. thetaiotaomicron, we generated a ncpah deletion strain (∆ncpah) and a ncpah-complemented strain of B. thetaiotaomicron. Growth of the ∆ncpah strain was slower in polyamine-free medium compared with those of parental and ncpah-complemented strains (Figure 2A). The generation time was longer in the ∆ncpah mutant strain (144.4 ± 0.3 min) compared to parental strain and ncpah-complemented strains (112.7 ± 0.4 and 116.8 ± 0.7 min), but was indistinguishable between the parental and ncpah-complemented strains. We also confirmed that the parental strain produced intracellular spermidine as the sole polyamine, and the concentration of spermidine was decreased from exponential to stationary phases (from 56.7 to 36.2 nmol/mg cellular protein) ( Figure 2B; Supplementary Figure S1). While the ability of ∆ncpah to produce spermidine was severely decreased (<6 nmol/mg cellular protein), the complementation of ncpah restored the production of spermidine (36.1-55.7 nmol/mg cellular protein). These results indicate that ncpah is involved in spermidine biosynthesis and contributes to growth in B. thetaiotaomicron. Another finding was that the ∆ncpah strain intracellularly produced the two unidentified putative amine compounds ( Figure 2C,D), which were neither N-carbamoylputrescine, putrescine, cadaverine, spermidine, agmatine, nor spermine (Supplementary Figure S1).

NCPAH Converts N-carbamoylputrescine to Putrescine and the Activity Is Regulated by Polyamines and the Polyamine Precursor Agmatine
NCPAH was characterized using the purified recombinant NCPAH-(His)6 (Supplementary Figure S2). An enzymatic assay using indophenol blue method showed that NCPAH converts N-carbamoylputrescine to putrescine (Supplementary Figure S3). Next, the effect of reaction temperature (20-90 °C) on the enzymatic activity was examined using indophenol blue method, and the result showed the optimal temperature was 50 °C (Figure 3A), in which the activity was 10 µmol NH3/min/mg, and the enzymatic activity was decreased at over 70 °C, probably due to the heat denaturation. Additionally, the ef-

NCPAH Converts N-carbamoylputrescine to Putrescine and the Activity Is Regulated by Polyamines and the Polyamine Precursor Agmatine
NCPAH was characterized using the purified recombinant NCPAH-(His) 6 (Supplementary Figure S2). An enzymatic assay using indophenol blue method showed that NCPAH converts N-carbamoylputrescine to putrescine (Supplementary Figure S3). Next, the effect of reaction temperature (20-90 • C) on the enzymatic activity was examined using indophenol blue method, and the result showed the optimal temperature was 50 • C ( Figure 3A), in which the activity was 10 µmol NH 3 /min/mg, and the enzymatic activity was decreased at over 70 • C, probably due to the heat denaturation. Additionally, the effect of pH (5.5-9.0) on the activity was examined using HPLC to measure putrescine levels, and the results showed the optimal pH was 7.0, at which the activity was 11.6 µmol putrescine/min/mg ( Figure 3B), and the activity was decreased to less than 40% either under pH 6.0 or over pH 8.5. The kinetic analysis, in which the NCPAH reactions were performed with varying concentrations of N-carbamoylputrescine and the initial velocity of NH 3 formation was analyzed with the indophenol blue method (Supplementary Figure S3), showed that NC-PAH has a substrate-saturation curve with N-carbamoylputrescine as a substrate (fitting to the Michaelis-Menten equation) ( Figure 3C). The Michaelis constant (K m ) and turnover number (k cat ) were 730 µM and 0.8 s −1 , respectively, resulting in 1.0 s −1 mM −1 of the catalytic activity (k cat /K m ). Furthermore, the effect of polyamines and their derivatives on the enzymatic activity was examined, and the addition of agmatine and spermidine at a final concentration of 1 mM inhibited the NCPAH reaction by over 80%, with the greatest extent of inhibition. In addition, the inhibitory effect of putrescine on the NCPAH reaction is approximately 50%, and there was no obvious inhibitory effect of arginine on the NCPAH reaction ( Figure 3D).  Figure 3C). The Michaelis constant (Km) and turnover number (kcat) were 730 µM and 0.8 s −1 , respectively, resulting in 1.0 s −1 mM −1 of the catalytic activity (kcat/Km). Furthermore, the effect of polyamines and their derivatives on the enzymatic activity was examined, and the addition of agmatine and spermidine at a final concentration of 1 mM inhibited the NCPAH reaction by over 80%, with the greatest extent of inhibition. In addition, the inhibitory effect of putrescine on the NCPAH reaction is approximately 50%, and there was no obvious inhibitory effect of arginine on the NCPAH reaction ( Figure 3D).

Discussion
In this study, we aimed to demonstrate the importance of NCPAH, an enzyme involved in the polyamine biosynthetic pathway in B. thetaiotaomicron. We cultured the parental strain, the ∆ncpah and the ncpah-complemented strain of B. thetaiotaomicron in polyaminefree minimal medium and compared the intracellular polyamine profiles and growth. Only spermidine was present in the parental strain (Supplementary Figure S1), which is consistent with previous studies on polyamines produced by B. thetaiotaomicron [45,49,50]. In contrast, intracellular spermidine was significantly reduced in the ∆ncpah strain, whereas reduced spermidine levels in the ncpah-complemented strain were restored to levels similar to those in the parental strain ( Figure 2B). These results suggested that ncpah plays an important role in spermidine biosynthesis in B. thetaiotaomicron.
The activity of NCPAH of B. thetaiotaomicron was found to be inhibited by the addition of polyamines or the polyamine precursor agmatine belonging to the polyamine biosynthetic pathway of B. thetaiotaomicron ( Figure 3D). Putrescine, a product of NCPAH, inhibited moderately (by approximately 50%) the activity of NCPAH. It is noteworthy that the reaction catalyzed by NCPAH was inhibited significantly more strongly (over 80%) by spermidine, the product of a two-step later reaction than by putrescine, the direct product. Threonine dehydrogenase is known to be 50% feedback inhibited by its reaction products [51]. However, NCPAH was more inhibited by the reaction product compared to threonine dehydrogenase. Note that such feedback inhibition has not been reported in previous reports on NCPAH from other organisms. For example, NCPAH from P. aeruginosa was not inhibited by 1 mM of arginine, ornithine, putrescine, or spermidine [52]. Of note, biochemical analyses of NCPAH have been reported in Arabidopsis thaliana [53], Selenomonas ruminatium [54], C. jejuni [33], and Medicago truncatula [55], but feedback inhibition studies of NCPAH derived from these species have not been reported. Spermidine has stronger physiological activity than putrescine and is known to have adverse effects on the organism when accumulated in excessive amounts. This suggests that feedback inhibition of B. thetaiotaomicron in the cells prevents the excessive accumulation of spermidine. On the other hand, the kinetic parameters of NCAPH in B. thetaiotaomicron are comparable to those of NCPAH in P. aeruginosa [52] and Selenomonas ruminatium [54], which have been reported in previous studies (Table 3). Taken together, the results suggest that the activity of the polyamine biosynthetic enzyme NCPAH, whose enzyme activity is comparable to NCPAH in other organisms, is tightly controlled by intracellular polyamines and the polyamine precursor agmatine, supporting the existence of an in vivo polyamine homeostasis employing a feedback mechanism so far identified only in B. thetaiotaomicron cells. ncpah deletion strains accumulated two unidentified compounds whose retention times differed from those of N-carbamoylputrescine, putrescine, agmatine, carboxyspermidine, spermidine, and spermine (Supplementary Figure S1). These are not found in the parental and ncpah complementary strains. The purification and structural determination of these molecules could lead to the identification of a novel spermidine biosynthetic pathway in B. thetaiotaomicron. In the ncpah deletion strain, a trace amount of spermidine was present in the cells despite the deletion of ncpah (Supplementary Figure S1). In this experiment, the strains were precultured in medium containing polyamines, washed once with main culture medium containing no polyamines, and then their suspensions were added to the main culture medium, so it is thought that almost no spermidine was brought in from the preculture. It is also unlikely that spermidine was brought in from cells of the ncpah deletion strain that were precultured in the GAM medium (containing polyamines). Since the intracellular spermidine concentration of ncpah deletion strains precultured in the GAM medium was unknown, we used the intracellular spermidine concentration of wild-type B. thetaiotaomicron when cultured in the GAM medium for 24 h [56] for discussion. For the washed bacterial suspension obtained from the preculture solution, the spermidine in the bacteria corresponding to OD 600 = 0.03, the turbidity of the first outbreak of the main culture, was roughly estimated to be 0.47 nmol/mg. In contrast, the intracellular spermidine concentrations found in the ncpah deletion strains ranged from 2 nmol/mg to 6 nmol/mg, suggesting that more spermidine was detected in the ncpah deletion strains than was brought in from the preculture. It has been reported that P. aeruginosa ncpah mutants grew slightly on media supplemented with N-carbamoylputrescine as the sole carbon source [57]. Furthermore, another paper reported that crude extracts of ncpah mutants exhibit a slight N-carbamoylputrescine amidohydrolase activity [58]. Taken together, it was suggested that an alternative pathway in P. aeruginosa converts N-carbamoylputrescine to putrescine. In fact, it has been reported that in ncpah-deficient strains of P. aeruginosa, the accumulated N-carbamoylputrescine induces acetylpolyamine amidohydrolase, which in turn activates a pathway that converts N-carbamoylputrescine, not the original substrate, into putrescine [59]. However, a homology search for acetylpolyamine amidohydrolase revealed that there is no corresponding enzyme in B. thetaiotaomicron (data not shown). Therefore, in B. thetaiotaomicron, it is suggested that there is a different pathway to that reported in P. aeruginosa [59], which uses N-carbamoylputrescine as a substrate to produce putrescine through a side reaction of acetylpolyamine amidohydrolase. It has also been reported that in C. jejuni, a deficiency of the enzyme CASDH, which converts putrescine to carboxyspermidine, results in a significant accumulation of the downstream product spermidine [33]. In C. jejuni, the deletion of casdh is thought to activate the alternative pathway, and similarly, in B. thetaiotaomicron, the subject of this paper, the deletion of ncpah may activate the alternative pathway. This activation could be due to unidentified compound(s) that accumulate in ncpah-deficient strains.
B. thetaiotaomicron does not export polyamines in vitro [45]; there are no data on the production of polyamines in the gut. To clarify this, it is necessary to generate gnotobiotic mice monocolonized with B. thetaiotaomicron and analyse the production of polyamines by B. thetaiotaomicron in the intestinal lumen.

Conclusions
∆ncpah and complemented strains were generated, and the intracellular polyamines of these strains were cultured in a polyamine-free minimal medium. Spermidine, which was detected in the parental and complemented strains, was depleted in the gene deletion strain. The purified NCPAH-(His) 6 was characterised and observed to be capable of converting N-carbamoylputrescine to putrescine. In addition, we observed that agmatine and spermidine, which are produced in the polyamine biosynthetic pathway, strongly inhibited the activity of NCPAH. This feedback inhibition is suggested to regulate the reaction catalysed by NCPAH and may play a role in the intracellular polyamine homeostasis of B. thetaiotaomicron. Bacteria of the genus Bacteroides occupy >30% of the lumen of the human colon. Therefore, the findings on B. thetaiotaomicron may explain a significant part of the polyamine dynamics in the human intestinal lumen.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/biomedicines11041123/s1, Figure S1: Polyamine profiles in the cells of parental and ∆ncpah mutant strains of B. thetaiotaomicron; Figure S2. SDS-PAGE analysis of purified recombinant NCPAH-(His) 6 ; Figure S3. Determination of reaction rates of NCPAH at different substrate concentrations.