Production of catechol siderophores by bacteria selected from feces of a healthy female adult through screening NRPS A domain


 Background: Catechol siderophores, which are widespread in the human gastrointestinal tract (GIT) and play important roles in maintaining iron-nutrition balance between microorganisms and host, are small molecules with a high affinity for ferric iron and are assembled by nonribosomal peptide synthetases (NRPS). In this study, we select strains encoding NRPS A domain gene from feces of a health female adult, predict its products type, and check it out in vitro . Results: Firstly, eight bacteria were determined encoding NRPS A domain gene, and then two kind of catechol siderophores, Bacillibactin and Enterobactin, were predicted according to NRPS A domain substrate specificity. Secondly, Bacillibactin and Enterobactin were checked out in cultured medium aerobic incubated with selected strains of E. Coli, Bacillus sp. and B. Cereus. For the yield of catechol siderophores, B. cereus Gut 16 secreted highest yield of Bacillibactin and E. coli Gut 07 produce highest yield of Enterobactin. Conclusion: By presence determination and activity prediction of NRPS A domain, two siderophores, Bacillibactin and Enterobactin, were checked out finally being secreted by selected strains cultured in a aerobic medium. Further research on the potential probiotic property is necessary to affirm the application in biological industry, as well as to elucidate their mechanism in human gut. Keywords : Catechol siderophores, Enterobactin, Bacillibactin, NRPS A domain, Feces, Probiotics


Background
Siderophores are small organic molecules produced by microorganisms under iron-limiting conditions which enhance the uptake of iron to the microorganisms [1,2]. More than 500 kinds of siderophore proteins have been discovered, and most of them are divided into three categories: catecholates, hydroxamaces, and carboxylates [3]. The iron-chelating ability of catechol siderophore is much higher than that of other iron-binding proteins secreted by vertebrate host cells [4].
Catechol siderophores belong to non-ribosomal peptides and are synthesized by NRPSs which is a class of giant enzymes with modular domains [5][6][7][8]. Further and more importantly substrate identification specificity reportedly exists in the NRPS adenine structure domain (NRPS A domain) can be used to predict the function and type of NRPS products [9,10].
In recent years, more and more studies have found that siderophores have shown unique efficacy in the prevention and treatment of gastrointestinal diseases. Many microorganisms that produce siderophores are critical to the health of their hosts. These microorganisms show great adaptability in the low-iron environment of the GIT and can compete with pathogenic bacteria for iron to inhibit the growth of pathogenic microorganisms [11]. Probiotic E. coli Nissle 1917 can compete with pathogenic bacteria in the gastrointestinal environment by secreting siderophores, thus inhibiting intestinal pathogenic bacteria [12]. Some microorganisms, such as Lactobacillus and Bifidobacterium, which can not synthesize siderophores by themselves, are co-cultured with iron-producing microorganisms in simulated gastrointestinal environment, in which siderophores-producing bacteria play a probiotic role as iron supplements to increase the number and growth rate of these strains significantly, thus maintaining the balance of the gastrointestinal flora in the GIT [13][14][15]. Studies have further shown that siderophores function in plant nutrition and protection, fish disease prevention and medical treatment [12,[16][17][18][19][20].
Most screened and isolated siderophores high-yielding strains originate from the ocean and plant rhizosphere soil, while the research on isolation and screening of siderophores high-yielding strains in human GIT from feces is less. In the present study, identified positive strains of NRPS A domain gene were screened from healthy adult feces. The categories and capacity producing siderophores were then analyzed to lay the foundation for application in future biological industry, also for further study on their mechanism of action in gastrointestinal nutrition and health.

Positive strains containing the NRPS A domain gene
Twenty-one strains of facultative anaerobes from feces were isolated. Positive amplification products of NRPS A domain gene were found in all 21 bacteria. The amplification product size was 800 bp, consistent with the expected size of the target fragment (Fig. 1).

Species identification of strains containing the NRPS A domain
Comparative analysis of 16S rRNA gene sequence was performed, and the duplicated ones were excluded. Finally eight strains of bacteria remained, including Gut 01, Gut 07, Gut 12, Gut 13, Gut 20, Gut 03, Gut 14, and Gut 16. The sequencing results of the 16S rRNA gene sequence of eight strains were submitted to NCBI. The serial login number of GenBank was KU156682-KU156689 (Table 1) .

Product type of NRPS A domain
The amino acid sequences of the NRPS A domain of eight strains were submitted to NCBI. The serial login number of GenBank was KU156690-KU156697( Fig. 3 and Table 2).  3 and Table 2).  (Fig. 4b).

Discussion
In the present study, detecting gene and predicting product of NRPS A domain positively from bacteria, which was selected in feces of a female health adult and cultured in a aerobic medium, was proved to be helpful to found out catechol siderophore from microbiome of human gastrointestinal habitat. Bacillibactin and Enterobactin, two kind of catechol siderophore, were checked out and measured in the supernatant of a succinic acid medium with eight catechol siderophore-producing In the present work, B. cereus Gut 16 with a high production of Bacillibactin and E. coli Gut 07 with a high production of Enterobactin were screened in vitro from feces of a health adult, indicating that these two strains play a critical role, good or bad, in human gut by siderophore pathway, also showing their application prospects in biological technology. So future research on probiotic property of these selected strains is such a necessary and urgent issue because it looks like that whether a siderophore-producing strain is good or bad depends on its probiotic properties may have. Zawadzka et al. reported that the pathogenic role of Bacillus largely depended on the siderophores it secreted and the iron-transport protein on the membrane. So the siderophore was identified as one of the virulence factors [24]. On the contrary, Cursino et al. reported that E. coli strain H22 has several sets of iron-collection systems that have antagonistic effects on intestinal microorganism in vivo and in vitro. They also suggested that the E. coli strain H22 can be a potential probiotics for livestock and humans [25]. Some other researchers also reported that siderophores have beneficial effect in preventing and treating gastrointestinal diseases [26][27][28][29].

Conclusions
Two siderophores, Bacillibactin and Enterobactin, were family checked out by presence determination and activity prediction of NRPS A domain, were determinated to be secreted by selected strains cultured in a aerobic medium. Further research on the potential probiotic property is necessary to affirm the application in biological industry, as well as to elucidate the mechanism in human gut.

Screening and identification of strains containing NRPS A domain gene
To screen positive strains containing NRPS A domain, the whole genome of the purified strains were extracted using a genome-wide extraction kit (Tiangen Biotech Beijing Co., Ltd.) and stored at -20 °C before use. By using CODEHOP software [30], primers were designed according to the NRPS gene conservative region (adenine structure domain, A domain). The primers were F2 and AR1 ( Table 3). To identify the NRPS A domain-positive strains, the 16S rRNA gene of NRPS A domain-positive strains were amplified using the universal primer 27F/1492R (Table 3). After purification, the products were connected with pmd19-T carrier (TaKaRa, Japan) and then imported into Escherichia coli DH5α (Sangon Biotech Shanghai Co., Ltd.) sensing cells. Positive clones were sequenced. The carrier sequence was removed from the sequencing results, and then the 16S rRNAs of the strains were analyzed with NCBI Blast. Their classification status was preliminarily determined. The 16S rRNA gene sequence with high similarity was downloaded as the reference sequence for phylogenetic analysis, and the comparison was conducted with Clustal W software. After the phylogenetic tree was constructed using MEGA 5.2 software (through the neighbor-joining method, setting up bootstrap to 1000 times) [31], the 16S rRNA gene sequence was submitted to the GenBank database. Table 3 List of primers used for amplifification of NRPS gene fragments and 16S rRNA gene sequences 5′-TACGGYTACCTTGTTACGACTT-3′

Prediction of product type of NRPS A domain
The NRPS A domain gene of the positive strains were amplified. The product was purified and imported in E. coli DH5α (Sangon Biotech Shanghai Co., Ltd.) after being connected with pmd19-t vector (TaKaRa, Japan). The recovered plasmid from positive clones was sequenced. To verify if the NRPS A domains were sequenced correctly, the carrier sequence was removed from the sequencing results in advance. The Conserved Domain Database (CDD) software in NCBI was used for analysis.
For the prediction of product type of NRPS A domain, the nucleic acid sequence was translated into amino acid sequence by using the open-reading-frame finder software in NCBI, and amino acid sequence analysis was performed using the UniProtKB/Swiss-Prot online database. The amino acid sequence of the NRPS A domain with high similarity was downloaded as the reference sequence, and comparison was conducted with Clustal W software. The phylogenetic tree was constructed using MEGA 5.2 software through the neighbor-joining method, setting up bootstrap to 1000 times, and then the type of secondary metabolites of NRPS was predicted finally [32,33]. The amino acid sequence of the NRPS A domain was submitted to the GenBank database.

Confirmation of producing catechol siderophores of selected Strain
Qualitative analysis of siderophores production The detection of siderophores was determined using CAS method [34]. The solid medium for iron