Introduction
============

Homocysteine methyltransferase (HMT) is a family of enzymes that can catalyze the methylation of homocysteine and convert homocysteine into methionine. HMT plays an important role at the final step of the methionine synthesis. This enzyme is widely distributed among microorganisms, animals and plants, and can be divided into three categories based on methyl group donors including 5-methyltetrahydrofolate: homocysteine S-methyltransferase, betaine-homocysteine methyltransferase and S-methylmethionine: homocysteine methyltransferase.

The 5-methyltetrahydrofolate: homocysteine S-methyltransferase (also known as methionine synthase) can be further divided into two sub-categories based on the catalytic conditions. One is cobalamin-dependent homocysteine S-methyltransferase. As an intermediary methyl carrier, this enzyme catalyzes the methylation of homocysteine with 5-methyltetrahydrofolate as methyl group donor and produces tetrahydrofolic acid and methionine. The activity of this enzyme can be drastically lowered without adequate cobalamin[@B1]. The other sub-category is cobalamin-independent homocysteine S-methyltransferase. This enzyme catalyzes the direct transfer of the methyl group from 5-methyltetrahydrofolate to homocysteine to produce methionine without any intermediate methyl carrier, and the catalytic process does not require the participation of cobalamin[@B2]. However, both enzymes require zinc for the activation and binding of homocysteine. The cobalamin-dependent homocysteine S-methyltransferase exists in mammalian tissues and the cobalamin-independent homocysteine S-methyltransferase is in plants, while some microorganisms may have both[@B3]. The betaine-homocysteine methyltransferase is a cytoplasmic enzyme that is present in mammals and catalyzes the conversions of betaine and homocysteine into dimethyl glycine and methionine, respectively. This reaction also requires the irreversible oxidation of choline[@B4]. S-methylmethionine: homocysteine methyltransferase encoded by the gene *mmuM* is currently only found in microorganisms including bacteria and fungus. In the biosynthesis of methionine, this enzyme can use S-methylmethionine as a donor of methyl groups to catalyze methylation of homocysteine. Two molecules of methionine are formed in this process[@B5].

However, among the three categories of HMT, only S-methylmethionine: homocysteine ​​methyltransferase has been reported to use selenocysteine ​​as a substrate to produce nontoxic selenium compound such as methylselenocysteine[@B5]. It has high a high similarity in sequence and function to the selenocysteine methyltransferase (SMT) in Se-accumulating plants[@B6]. This enzyme enables the SMT free microorganisms to enrich selenoproteins and to tolerate high concentrations of environmental selenium, even though such microorganisms do not contain SMT.

Selenium can be absorbed and metabolized through the sulfur assimilation pathway, because both selenium and sulfur elements have similar chemical properties[@B7]-[@B9]. Animals and human beings can obtain selenate or selenocysteine directly from food. In plants, marine algae, yeast and bacteria, selenate and selenite are activated by ATP sulfurylase[@B10], leading to the eventual selenium assimilation.

One of HMT genes first found in *E.coli* was once called *yagD*[@B5] and was renamed *mmuM* gene later[@B6]. *mumM*is distributed in microbial organisms and mainly encoded in bacterial chromosomes. So far, only a few bacteria have been found to carry *mmuM* genes on their plasmids[@B11]. In this work, *mmuM* gene profiles of clinically isolated pathogenic bacteria have been analyzed through high-throughput sequencing. The sequence diversity and evolution together with the molecular mechanism of horizontal transfer of this gene have also been analyzed.

Results
=======

The distribution of *mmuM*gene in bacteria
------------------------------------------

533 *mmuM* gene sequences covering 70 genera were selected from GenBank and other databases. Among them, three species with the more *mmuM* sequences were *Streptococcus* (134)*, Klebsiella* (72) and *Lactobacillus* (47). Other four genera (*Mycobacterium*, *Bacillus*, *Escherichia* and *Xanthomonas*) each had more than 20 sequences and sixty genera each had less than 10 sequences ([Supplementary Material](#SM1){ref-type="supplementary-material"}: Table S1). The frequency of *mmuM* gene varied from genus to genus. The statistics of 10 genera that had the highest *mmuM* gene frequencies in the collection showed that *Mycobacterium* ranked the first (32.22%, 29/90), while *Xanthomonas* was in the second place (31.58%, 12/38) and the third one was*Enterobacter* (17.65%, 6/34). *Streptococcus* had 17.47% (29/166), and *Klebsiella*and *Escherichia* only had 4.88% (6/123) and 1.77% (6/339), respectively (Table [1](#T1){ref-type="table"}).

Among 533 *mmuM* gene sequences collected, most of them were encoded in chromosomes. Only five sequences were located in plasmid genomes. These five *mmuM* gene-containing plasmids were: pBWB401 (NC_010180) and pMC429 (NC_018689) of *Bacillus*, pDSHI02 (NC_009956) of *Dinoroseobacter*, pIP1206 (NC_010558) of *Escherichia* and pKF3-140 (NC_013951) of *Klebsiella*. The *mmuM* gene sequences of pKF3-140 and pIP1206 were exactly the same, while the nucleotide identities of the *mmuM* genes for pBWB401 and pMC429 was 98.7 %. The *mmuM* gene sequences of pKF3-140 or pIP1206 were different from those of the other three plasmids pBWB401, pMC429 and pDSHI02 and the nucleotide sequence similarity identities with the three were 51.5%, 51.7% and 50.4%, respectively. The bacterial chromosomes that had *mmuM* genes with the highest similarities to those *mmuM* genes on plasmids were analyzed. The *mmuM* gene encoded in *Escherichia coli* genome (CP000948) showed an identity of 100% with the *mmuM* genes of pIP1206 and pKF3-140. The *mmuM* gene sequence on *Halobacillus sp*. genome (NZ_ANPF01000013) showed identities of 56.4 % and 56.3 % with those of pBWB401 and pMC429, respectively, while the *mmuM* gene on *Kitasatospora* setae genome (AP010968) only showed an identity of 52.3% with that of pDSHI02.

Taken together, *mmuM* gene is not evenly distributed over bacteria. In this study, many of this gene were collected from genera of *Streptococcus, Klebsiella*and *Lactobacillus;* however, genera with higher distribution frequencies of the gene were not them, but *Mycobacterium, Xanthomonas*and*Enterobacter*. In addition, the *mmuM* gene encoded in plasmids provides clues of its dissemination in bacteria through horizontal gene transfer.

Phylogenetic analysis of *mmuM* gene in bacteria
------------------------------------------------

All 533 *mmuM* genes were clustered based on the amino acid sequence identity with a threshold value of 80% and a total of 144 clusters were obtained ([Supplementary Material](#SM1){ref-type="supplementary-material"}: Table S1). The sequence with the highest similarity to the consensus sequence in each cluster was selected as the representative sequence used to reconstruct the phylogenetic tree. Meanwhile, 16S rRNA gene sequence in each of 70 genera was selected as a reference from which the *mmuM* gene was retrieved. The clustering results showed that the genus with the most gene clusters was *Lactobacillus* which contained 20 *mmuM* gene clusters, followed by *Streptomyces* with 13 clusters. The genera with the least gene clusters were *Escherichia* (2 clusters) and *Klebsiella*(1 cluster, [Supplementary Material](#SM1){ref-type="supplementary-material"}: Table S1). The phylogenetic analysis showed that the *mmuM* genes from the same family (eg *Enterobacteriaceae*) were located relatively closer in the phylogenetic tree, which was consistent to the locations of 16S rRNA genes in the corresponding 16S rRNA gene phylogenetic tree (Figure [1](#F1){ref-type="fig"}). It indicated that there was a correlation in evolution between *mmuM* gene and its host. Generally, the *mmuM* gene sequences from the same species were closer in the phylogenetic tree. On the contrary, the sequences of NC_010558 and NZ_BAFF01000003 which were both derived from *Escherichia* were located on two well-separated branches, while the NC_013951 sequence from *Klebsiella*was on the same branch as NC_010558 of *E. coli*was. In addition, the *mmuM* sequences of CP005991 from *Enterobacter* and NZ_JH725436 from *Kosakonia* were both located on the same branch. On the 16S rRNA phylogenetic tree, *Escherichia*and *Klebsiella* were well separated although both belonged to the same family (*Enterobacteriaceae*). It suggests that horizontal gene transfer of mmuM probably happened widely among strains, species or genera, even some phylogeneticly remote bacteria.

Gene mapping of *mmuM* in nine pathogenic bacteria genomes
----------------------------------------------------------

To investigate what frequency and coverage of *mmuM* gene are there in clinical pathogens, we used 90% indentify of amino acid sequences as a threshold to cluster 533 *mmuM*genes. As a result, 160 clusters were obtained and a reference sequence in each of these clusters was selected for gene mapping. Each reference has the highest similarity to its consensus sequence in each cluster ([Supplementary Material](#SM1){ref-type="supplementary-material"}: Table S2). The high-throughput sequencing reads of nine pathogens were mapped to 160 *mmuM* gene reference sequences, respectively. The results showed that only 5 reference sequences had positive mapping results with more than 80% coverage of the full length of reference sequences. Among those five sequences, three were mapped positively in the pooled genome sequences of *Staphylococcus aureus*and *Enterobacter cloacae*, respectively. Two were mapped positively in the pooled genome sequences of *Klebsiella pneumoniae*. For the pooled genome sequences of *Acinetobacter baumannii*, *Salmonella spp.* or *Enterococcus faecalis*, each of them had only one reference sequence mapped positively. No positive mapping result was obtained in the pooled genome sequences of *Pseudomonas aeruginosa* or *Enterococcus faecium* (Table [2](#T2){ref-type="table"}). It indicated that different pathogen maybe contained different number of the gene *mmuM*.

Among the five reference sequences, NC_013951 from *Klebsiella* had the highest positive rate. It was identified in the pooled genome sequences of *Escherichia coli*, *Enterobacter cloacae*, *Klebsiella pneumoniae* and *Salmonella spp.* Furthermore, it showed that the gene sequence in *Escherichia coli* had a higher redundancy of 195.3, while in *Klebsiella pneumoniae*, it only had a redundancy of 4.8 (Table [2](#T2){ref-type="table"}).

Verification of *mmuM* gene
---------------------------

To verify presence of the gene *mmuM* in the pathogen genomes, polymerase chain reaction (PCR) was performed using reference NC_013951 as the target gene against several genera of bacteria and the amplified products were verified by sequencing. The upstream primer sequence of *mmuM* for PCR amplification was 5\'-CGGAATTCTTGCGTTGTGCTATGGTGCT (*mmuM*-F), and the downstream primer sequence of *mmuM* was 5\'-CCAAGCTTTCAGCTTCGCGCTTTTAACG (*mmuM*-R). The sequencing results of the PCR products confirmed the mapping results that all four bacteria (*Escherichia coli*, *Enterobacter cloacae*, *Klebsiella pneumoniae* and *Salmonella spp.*) with positive mapping results harbored *mmuM* genes. 29 sequences of PCR products from more than 600 strains were completely consistent with the reference sequence of NC_013951. Among these 29 sequenced PCR products, 13 were amplified from 150 strains of *E. coli*(13/150), 13 from 185 strains of *Enterobacter cloacae* (13/185), 2 from 200 strains of *Klebsiella pneumoniae* (2/200) and 1 from 89 strains of *Salmonella spp.* (1/89). It confirmed the mapping results above that all four bacteria contained the *mmuM* Gene (NC_013951) and that the *mmuM* gene in pooled samples of *E. coli* and *Enterobacter cloacae* has a higher redundancy than in those of *Klebsiella pneumoniae* and *Salmonella*.

Comparative analysis of the structure of the plasmids with *mmuM* genes
-----------------------------------------------------------------------

As mentioned above, of 533 *mmuM* gene sequences selected from the database, only 5 genes were encoded on plasmids. The structures of the plasmids with *mmuM* genes were analyzed carefully. Comparative genomics study showed that, although pKF3-140 plasmid had the highest similarity with *E. coli* plasmids p1ESCUM and pUTI89 (with gene identities of 98% and 92%, respectively)[@B11], there was no *mmuM* gene in these two plasmids. The plasmid pIP1206[@B12], however, only showed a gene identities of 49 % with plasmid pKF3-140, but the *mmuM* gene on pIP1206 showed the highest similarity with that on pKF3-140. As mentioned above, *mmuM* gene sequences encoded by pIP1206 and pKF3-140 are identical (with an identity of 100%). Furthermore, the 21 kb flanking regions of the*mmuM* genes on both plasmids showed a nucleotide sequence identity of 99.3 % (Figure [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}).

The whole plasmid genome sequences of pKF3-140 and pIP1206 were compared with each other to further analyze the evolutionary relationship between these two bacteria. It showed that pIP1206 was about 20 kb longer than pKF3-140. There were a large number of similar regions between these two plasmids. Most of the regions were in the same orientation. Each plasmid could be divided into five blocks based on the gene functions. Block A contained more genes than the other four blocks. The genes in this region were concerned with various functions, such as the restriction modification system, replication, transfer leading regions and metabolic functions (for example, S-methylmethionine metabolism). Block B included genes related to conjugation. Block C had genes mainly for replication. Block D had genes associated with drug resistance, while block E contained the genes related to the transport system. It was interesting to mention that an about 21 kb fragment (126.2-146.8 kb) harboring *mmuM* gene in block A of pKF3-140 showed a high similarity of 99.3% to the corresponding region in the plasmid pIP1206 (159.5-139.0 kb, Figure [3](#F3){ref-type="fig"}). The other four blocks also showed sequence similarities in their homolog genes, which suggested that these two plasmids might have the same backbone origin.

Comparative genomics analysis of the *mmuM*-encoding regions between the plasmid pKF3-140 and E. coli genomes
-------------------------------------------------------------------------------------------------------------

The common 21 kb *mmuM*-encoding regions of both plasmid pKF3-140 and pIP1206 were used as a reference in searching similar fragments from all prokaryotic genome sequences available in GenBank database. The searching results revealed that the 4.9 kb *mmuM*-encoding regions of the plasmids (137560-142459 of pKF3-140 and 148065-143166 of pIP1206) were matched to six *E. coli*genomes (such as *E. coli* str. K12 substr. DH10B genome 247283-253259, Figure [2](#F2){ref-type="fig"}, [4](#F4){ref-type="fig"}). These six *E. coli* chromosomes included *E. coli*K12 strains DH10B (CP000948.1), W3110 (AP009048.1), MG1655 (U00096.2), DH1 (ME8569, AP012030.1) and DH1 (CP001637.1) and chromosome minutes 4-6 (U70214.1). The first five strains have complete genome sequences available in the database.

However, the regions in *E. coli* genomes that demonstrated similarity to 4.9 kb regions of the plasmids were about 1 kb longer. Taking *E. coli* str. K12 substr DH10B as an example, its *mmuM*-encoding region was 5977 bp (247283-253259) in length and 1077 bp (251414-252490 ) longer than the corresponding regions of the plasmids ( Figure [4](#F4){ref-type="fig"} ), although the common 4.9 kb showed a high similarity with an identity of 99.9% and contained only five different nucleotides. The common 4.9 kb sequence encoded six genes (or gene fragments) including *insA*, *insB*, *fbpC*, *mmuM*, *mmuP* and *insH-1*. The plasmids encoded a truncated *fbpC* while *E. coli* str. K12 substr. DH10B encoded not only a complete*fbpC* gene, but also a complete *fbpB* gene more (Figure [4](#F4){ref-type="fig"}). Further analysis showed that this 4.9 kb *mmuM*-encoding region comprised a transposon. The left side of the region was an insertion sequence 1 (IS1) with 23 bp flanking imperfect inverted repeats (IRs). The transposase of IS1 was encoded by*insA* and *insB* genes. The right side of the region was an insertion sequence 5 (IS5) with 16 bp flanking IRs. The transposase of IS5 was encoded by the *insH-1* gene. In the middle of the region, there were other genes including a partial *fbpC* (or*fbpB*-*fbpC* in *E. coli* genomes), *mmuM* and *mmuP* genes. The *fbpB* and *fbpC* genes were related to Fe (3+) ion absorption. By the way, on the both sides of the 4.9 kb region in these two plasmids, there were 8 bp imperfect direct repeats (DRs). Therefore, the 4.9 kb region in plasmids (or 6.0 kb in *E. coli*) was a transposon which consisted of IS1, truncated *fbpC* (or *fbpB*-*fbpC*), *mmuM*, *mmuP* and IS5. As previously reported, under certain conditions, the transposon could transpose between chromosomes and plasmids through a series of processes and could also make a horizontal gene transfer among different species or different genera of bacteria.

Discussion
==========

HMT is known as the key enzyme that catalyzes the last step in methionine biosynthesis. However, S-methylmethionine: homocysteine methyltransferase also contribute to excretion and detoxification of selenium in organisms.

Selenium is an essential trace element for growth and metabolism of animals, plants and microbial organisms. It plays a key role in certain cell processes in forms of selenocysteine and selenoenzymes[@B13]. If Se accumulation reaches a higher concentration as selenocysteine in organisms, Se will be non-specifically incorporated into other proteins, leading to toxicity effects[@B8]. Organisms usually do have selenium excretion and detoxification mechanism, which could prevent the toxicity resulted from the accumulation of selenocysteine or selenide. In eukaryotes[@B14] and prokaryotes[@B15], there are two pathways for selenium detoxification. One common way is methylation of selenocysteine or selenomethionine by methyltransferase and the other pathway is to reduce selenite into elemental selenium[@B16]. In the non-Se-accumulating plants, excessive amounts of selenocysteine can be converted into selenomethionine through the sulfur assimilation pathway. The resulted selenomethionine is methylated with methyltransferase, and eventually volatilized in the form of dimethylselenide[@B10], [@B17]. In Se-accumulating plants, selenocysteine is converted into dimethyldiselenide with the help of SMT which catalyzes methyl transfer from S-methylmethionine to selenocysteine[@B8]. In animals, the inorganic selenium ions can be methylated to dimethylselenium and trimethylselenonium which are eventually excreted via respiration or urination[@B18]. Some microorganisms also have the ability to detoxify and excrete selenium. For example, *Pseudomonas syringae* harbors trimethyl purine methylase gene (*tmp*) and can convert selenate, selenite and selenocysteine into dimethylselenide and dimethyl diselenide[@B19]. Thiopurine methyltransferase[@B20] and homologs of calichaemicin methyltransferase[@B21] found in *Pseudomonas* can also convert selenite and selenocysteine into dimethylselenide and dimethyl diselenide, respectively. S-methylmethionine: homocysteine methyltransferase found on pKF3-140 plasmid of *Klebsiella pneumoniae* KF3 was reported to enable the host*E. coli* to tolerate high concentrations of selenite[@B11]. This enzyme catalyzes the methylation of selenocysteine to form MeSeCys and the function of the enzyme is similar to that of SMT found in Se-accumulating plants.

The gene *mmuM* is widely distributed in varieties of bacteria, including both Gram-positive and Gram-negative bacilli, cocci, and mycobacteria. Of the 70 selected genera in this study, the genomes of *Mycobacterium* and *Xanthomonas* have especially high frequencies of *mmuM* gene. The gene *mmuM* in different species shows different conservative profiles. A comparative analysis of *mmuM* genes within each genus with a cut-off identity of 80% similarity of amino acid sequences revealed that all 71 *mmuM* genes in *Klebsiella* could be clustered into one cluster (71/1). 27 genes of *Escherichia* could be divided into two clusters (27/2), while *mmuM* genes from certain genera showed more varieties, for example, 134 *mmuM* genes of *Streptococcus* were clustered into 11 clusters (134/11) and 47 *mmuM* genes of *Lactobacillus* were clustered into 20 clusters (47/20). It indicated that *mmuM* genes in *Klebsiella* and *Escherichia* were more conservative than those of *Streptococcus* and *Lactobacillus*. Further analysis of the *mmuM* polymorphism by mapping all the pooled sequencing reads of nine pathogens\' genomes using the sequence of NC_013951 as the reference showed that none of the single nucleotide polymorphism (SNP) locus could be identified. In addition, sequencing of 29 PCR products of *mmuM* genes from *Escherichia coli*, *Klebsiella pneumoniae*, *Salmonella spp.*and *Enterobacter cloacae*, showed the same result as that from gene mapping. All these results further confirmed the conservation of the *mmuM* gene (NC_013951 as a reference).

The phylogenetic tree of *mmuM* genes showed that some *mmuM* genes might have closer phylogenetic relationship with those from different genera (such as *mmuM* genes between *Escherichia* and *Klebsiella*, and between *Enterobacter*and *Kosakonia*) than those in the same genus. It suggested that the *mmuM* gene in the same genus might have different origins while those in the different genera might have a common ancestor. The *mmuM* genes in *Klebsiella* are so highly conserved that they might have evolved from one common ancestor. The *mmuM* genes in *Escherichia* might have at least two origins, with one origin very close to that in *Klebsiella*.

Horizontal gene transfer (HGT) is a common biological process by which organisms acquire foreign genes or DNA fragments to across species boundaries. By rapidly introducing newly evolved donor genes into the host genomes and by avoiding the slow steps of *ab initio* gene creation, HGT therefore accelerates genome innovation and evolution[@B22], [@B23]. Some genes with special functions, such as genes for antibiotics resistance and genes for extreme environment, would be spread among organisms by HGT[@B24], [@B25]. HGT generally consists of two steps. One is a transfer process and the other is integration of foreign DNA into the host genome. The pathways for DNA transfer mainly include transformation, conjugation and transduction. HGT in bacteria is often involved with a number of mobile DNA elements such as the insertion sequence (IS), the transposon (Tn), the integron and some phages. Foreign DNA can be integrated into host cells through homologous recombination, illegitimate recombination, site-specific recombination, and the reconstruction of plasmids[@B26].

It demonstrated in this work that the*mmuM* gene also experienced the horizontal gene transfer to spread itself among bacteria. For example, the phylogenetic analysis showed that, although genetic relationship of most *mmuM* genes in the same genus or between closely related genera are closer, there exist higher homology genes among different genera than those belong to the same genus, such as the genetic relationship of the *mmuM* gene from an *Escherichia*strain (NC_010558) was closer to one from a *Klebsiella*strain (NC_013951) than that in another *Escherichia* strain (NZ_BAFF01000003). Similarly, the *mmuM* gene from *Enterobacter* (CP005991) shared the highest homology to that from *Kosakonia*(NZ_JH725436).

In addition, our study showed that the sequence similarity between plasmids pIP1206 and pKF3-140 was lower than that between pKF3-140 and p1ESCUM, or between pKF3-140 and pUTI89[@B11]. However, pKF3-140 and pIP1206 shared a 21 kb homologous region which encodes a*mmuM* gene and a number of mobile DNA elements such as insertion sequences, transposon and integron, which indicated that the 21 kb homologous region was a transferable region. It could be formed by a horizontal gene transfer. Analysis of the entire prokaryotic genomes and plasmid sequences in the NCBI database showed that a 4.9 kb fragment of the 21 kb homologous region in these two plasmids had homologous sequences in certain *E. coli* chromosomes, and the 4.9 kb fragment was identified to be a transposon. Therefore, this finding supported the hypothesis that 4.9 kb *mmuM* gene containing transposon was derived from the*E. coli* chromosome (such as *E. coli* str. K12 substr. DH10B genome, etc.) by HGT. Further HGT occurred between plasmids of *Escherichia coli* and *Klebsiella pneumonia*. As a result, the 21 kb *mmuM* gene containing regions were observed.

The comparative genomic analyses of *mmuM-*encoding plasmids and bacterial chromosomes have showed that *mmuM* gene could transfer between chromosome and plasmid genomes or among plasmid genomes. This *mmuM* gene migration among bacteria could not only enhance the methionine synthesis capability, but also enable the bacteria to tolerate a high concentration of selenium. It is also an evidence that bacteria evolved to adapt to extreme environment. Structure analysis of *mmuM*gene related sequences and the gene distribution would help to understand the molecular mechanism of dissemination and evolution of the *mmuM* gene.

Materials and methods
=====================

Collection and processing of *mmuM* gene sequences
--------------------------------------------------

*mmuM* gene sequences were obtained from NCBI Nucleotide database using *mmuM* as the key word. The resulted sequences were filtered and only sequences from bacteria were kept. The CDS of the *mmuM* gene was retrieved if the search result was a whole genome of a bacterium. Finally, 533 complete *mmuM* CDSs from 70 genera of bacteria were selected until June, 2014. The sequence retrieving, statistics analysis and other bioinformatics tools used in this study were completed with Python and Biopython scripts [@B27].

Clustering and phylogenetic analysis of *mmuM* gene sequences
-------------------------------------------------------------

The amino acid sequences of *mmuM* genes from the same genus were clustered using a cut-off threshold of 80% identity using CD-hit[@B28]. The 16S rRNA sequences were downloaded from NCBI Nucleotide database. Multiple sequence alignments were performed using MAFFT[@B29]. Phylogenetic trees were reconstructed by maximum likelihood method and the resulted trees were tested with bootstrap values of 100 replicates using PhyML3.0[@B30]. The best-fitting models of the amino acids and nucleotide substitutions were selected using Prottest3[@B31] and Modeltest3.7[@B32], respectively. Visualizing and annotating of phylogenetic trees were made using EvolView[@B33].

Collection of clinical pathogens and Hiseq 2000 DNA Sequencer sequencing
------------------------------------------------------------------------

Nine pathogens, each with about 200 strains isolated from the clinical samples in the First Affiliated Hospital of Wenzhou Medical University, China, over the years 2009-2011, were collected for sequencing. They were *Acinetobacter baumannii* (Aba), *Escherichia coli* (Eco), *Enterococcus faecalis* (Efa), *Klebsiella pneumoniae* (Kpn), *Enterococcus faecium* (Efm), *Staphylococcus aureus* (Sau), *Salmonella spp.* (Sal), *Pseudomonas aeruginosa* (Pae) and *Enterobacter cloacae* (Ecl). Bacteria were isolated and identified according to National Guide to Clinical Laboratory Procedures, and each strain was confirmed by the Vitek-60 microorganism auto-analysis system (bioMe´rieux Corporate, France). A single clone of all 9 genera of bacteria was cultured in 5 ml of liquid broth over night at 37℃, the cultures of the same species were mixed together. The genomic DNAs of the pooled bacteria were extracted and sequenced with Hiseq-2000 DNA Sequencer in Beijing Genomics Institute. The average depth of the sequencing of each bacterium was calculated (Table [3](#T3){ref-type="table"}).

Mapping of the bacterial genomes
--------------------------------

When reconstructing phylogenetic tree of the gene *mmuM* above, clustering was performed for the genes from the same genus. In order to have fewer references to reconstruct a phylogenetic tree, we used cut-off value of 80% identity. However, we selected cut-off value of 90% identity to get more references in gene mapping which can help us to map more sequence reads from pooled pathogen genomes. 533 MmuM amino acid sequences were clustered together at 90% sequence identity using CD-hit[@B28] and a total of 160 clusters were obtained. The nucleotide sequence of the *mmuM* gene with the highest similarity to the consensus protein sequence in each cluster was chosen as the reference sequence for gene mapping using SOAPaligner/soap2[@B34] ([Supplementary Material](#SM1){ref-type="supplementary-material"}: Table S2). The high-throughput sequencing reads of all nine bacteria were mapped to 160 reference sequences of the*mmuM* genes, respectively. The coverage and the redundancy were calculated according to the mapping result for each reference sequence. In this work, a coverage of 80% or higher of the full length of the reference sequence was considered as a positive result.

Detection of*mmuM* gene by PCR and sequencing
---------------------------------------------

Based on the mapping result, primers were designed using Primer Premier 5[@B35] to amplify the full length of *mmuM* gene. All strains of each species with positive *mmuM* gene mapping results were screened three times by PCR and the PCR products were confirmed by sequencing. Vector NTI[@B36] was used to assemble sequencing products and to examine the sequencing quality. Multiple sequence alignments were performed using MEGA5.2[@B37] and CLUSTAL W[@B38] to identify SNPs.

Analysis of the origin of the *mmuM*gene
----------------------------------------

The plasmid and chromosome genome sequences used in this study for extensively comparative analysis were downloaded from NCBI GenBank database (<http://www.ncbi.nlm.nih.gov>). The accession numbers of the related plasmid and the *E. coli*chromosome sequences were pKF3-140 (FJ876827/ NC_013951), pIP1206 (AM886293), p1ESCUM (CU928148), pUTI89 (CP000244) and *E. coli* str. K12 substr. DH10B (CP000948). Open reading frames (ORFs) were predicted and annotated using Glimmer3[@B39] and BASys[@B40], respectively. Insertion sequences were predicted by using IS Finder[@B41]. Nucleotide sequence was compared against the nucleotide collection database in NCBI using BlastN[@B42]. Figure [2](#F2){ref-type="fig"} was generated using GView server[@B43]. Mauve2.3.1[@B44] was used to perform comparative plasmid genome alignment. Figure [4](#F4){ref-type="fig"} was drawn using GenomeDiagram[@B27], [@B45]. Orthologous groups of genes from pKF3-140, pIP1206 and DH10B were identified using BLAST+[@B46].

Supplementary Material
======================

###### 

Tables S1 - S2.

###### 

Click here for additional data file.

This work is supported by the Natural Science Foundation of Zhejiang Province, China (LY14C060005); the Science and Technology Foundation of National Health and Family Planning Commission of China (WKJ2012-2-032); the National Natural Science Foundation of China (81401702, 81171614, 31100917); Technology Innovation Team of Zhejiang Province, China (2010R50048-13); Science and Technology Foundation of Wenzhou City, China (H20100011) and the Science and Technology Foundation of Zhejiang Province, China (2008C23074).

HGT

:   Horizontal gene transfer

HMT

:   homocysteine methyltransferase

SMT

:   selenocysteine methyltransferase.

![Phylogenetic trees. *mmuM* gene phylogenetic tree (1A) and 16S rRNA gene phylogenetic tree (1B).](ijbsv11p0011g001){#F1}

![Comparison of *mmuM* genes on plasmids and chromosomes. The comparative map was created with the genomes of four plasmids (pKF3-140, pIP1206, pESCUM and pUTI89) and two chromosomes (DH1 for*E. coli* DH1 and DH10B for *E. coli* K12 DH10B). The nucleotide sequence of pKF3-140 was used as a reference and compared with those of five query genomes. The bars within the five slots of query sequences showed that the regions hit on the reference sequence with a higher degree of similarity (70% or higher). Empty regions on the query slots indicated the parts without similar hits between the reference sequence and the query sequences. Arrow A shows the 21 kb homology region and arrow B shows the 4.9 kb homology region.](ijbsv11p0011g002){#F2}

![Genome structure comparison of pKF3-140 with pIP1206. Corresponding blocks from these two plasmids were shown according to the gene contents or sequence similarities. Blocks below the line in the plasmid pIP1206 genome indicated that the sequence in the block was in a reverse direction compared to the corresponding region in the plasmid pKF3-140. Density and height of colored lines in the box illustrated the gene density and their similarity. The denser and higher lines represented more genes and higher similarity between the sequences. Regions outside blocks were lack of detectable homology between the two plasmid genomes.](ijbsv11p0011g003){#F3}

![Comparison of the *mmuM* gene regions on pKF3-140, pIP1206 and*E. coli* DH10B. The homologue genes are marked with same color and lined together, respectively, while non-homologous genes were left blank.](ijbsv11p0011g004){#F4}

###### 

The statistics of 10 genera with higher *mmuM* gene frequencies

  Genus             Genomes with *mmuM*   Total genomes in database   Gene frequency
  ----------------- --------------------- --------------------------- ----------------
  *Mycobacterium*   29                    90                          0.3222
  *Xanthomonas*     12                    38                          0.3158
  *Enterobacter*    6                     34                          0.1765
  *Streptococcus*   29                    166                         0.1747
  *Leuconostoc*     4                     37                          0.1081
  *Lactobacillus*   21                    214                         0.0981
  *Streptomyces*    6                     66                          0.0909
  *Bacillus*        20                    283                         0.0707
  *Klebsiella*      6                     123                         0.0488
  *Escherichia*     6                     339                         0.0177

###### 

The mapping result of nine pooled genome sequences\*

          ACGE01000080   APWG01000155   CP002824   NC_007350   NC_013951   
  ------- -------------- -------------- ---------- ----------- ----------- ---------
  Aba     Coverage       \-             99.89%     \-          \-          \-
  Depth   \-             33.1190        \-         \-          \-          
  Eco     Coverage       \-             \-         \-          \-          100.00%
  Depth   \-             \-             \-         \-          195.2811    
  Efm     Coverage       \-             \-         \-          \-          \-
  Depth   \-             \-             \-         \-          \-          
  Efa     Coverage       \-             \-         \-          99.89%      \-
  Depth   \-             \-             \-         12.0309     \-          
  Ecl     Coverage       \-             100.00%    100.00%     \-          99.90%
  Depth   \-             62.3794        360.9861   \-          77.5100     
  Kpn     Coverage       \-             100.00%    \-          \-          99.00%
  Depth   \-             503.4298       \-         \-          4.8193      
  Pae     Coverage       \-             \-         \-          \-          \-
  Depth   \-             \-             \-         \-          \-          
  Sau     Coverage       97.86%         99.57%     91.96%      \-          \-
  Depth   7.3198         24.2229        2.7867     \-          \-          
  Sal     Coverage       \-             \-         \-          \-          99.90%
  Depth   \-             \-             \-         \-          21.0843     

\* only genes with coverage of more than 80% of full length were listed.

Aba: Acinetobacter baumannii, Eco: Escherichia coli, Efa: Enterococcus faecalis, Kpn: Klebsiella pneumoniae, Efm: Enterococcus faecium, Sau: Staphylococcus aureus, Sal: Salmonella spp., Pae: Pseudomonas aeruginosa, Ecl: Enterobacter cloacae.

###### 

The information of high-throughput sequencing of all nine species of pathogens

  Bacteria (accession No.)   Reference genome size(Mb)   Number of read   Strains sequenced   Depth
  -------------------------- --------------------------- ---------------- ------------------- -------
  Aba (CP000521)             3.98                        377056430        200                 47.41
  Eco (NC_000913)            4.64                        200565354        200                 21.60
  Efm (NC_017960)            2.70                        187637758        231                 30.11
  Efa (NC_004668)            3.22                        150936992        240                 19.54
  Ecl (NC_014121)            5.31                        294298898        238                 23.27
  Kpn (FO834906)             5.44                        182032468        240                 13.95
  Pae (CP000438)             6.54                        140478390        239                 8.99
  Sau (NC_002951)            2.81                        58304508         233                 8.91
  Sal (AE014613)             4.79                        132858534        201                 13.79

Aba: Acinetobacter baumannii, Eco: Escherichia coli, Efa: Enterococcus faecalis, Kpn: Klebsiella pneumoniae, Efm: Enterococcus faecium, Sau: Staphylococcus aureus, Sal: Salmonella spp., Pae: Pseudomonas aeruginosa, Ecl: Enterobacter cloacae.

[^1]: ^\#^These authors contributed equally to this work.

[^2]: Competing Interests: The authors have declared that no competing interest exists.
