Horizontal gene transfer in an acid mine drainage microbial community

HGT identification and overview of HGTs among AMD microbial genomes

We first reported our identification of the genes that were horizontally transferred
among the genomes including eight organisms in AMD and nine previously isolated organisms.
Using the method of strict reciprocal best basic local alignment search tool (BLAST)
hit 20], 21] based on the accurate gene reannotation by the current computational tools 22] or annotation from the National Center for Biotechnology Information (NCBI) 23], we obtained 251 orthologous gene families that were present in more than seven genomes
in which at least one genome was from AMD, while 185 of them showed phylogenetic tree
incongruities using an approximately unbiased test (AU test) 24]. Furthermore, 66 of the 185 phylogenetic trees were excluded because the differences
in the branch length were not the same as those of the organism tree, which were probably
caused by different evolution rates and could not represent HGT events. Therefore,
119 genes [Additional file 1] with significant evolution incongruence were identified in the study. Of these 119
genes, most of them demonstrate a function related to metabolism, especially amino
acid transport and metabolism (Fig. 1). In fact, the metabolic network of E. coli has a lot of changes and growth to acquire various external nutrients in the past
100 million years caused by HGT instead of gene duplication 25]. In this case, over 66 % of transferred metabolism-related genes can build a metabolically
flexible lifestyle to facilitate the environmental adaptation in this extreme acidic
and metal-rich environment 26]. Therefore, it is reasonable to hypothesize that the 119 genes indicate possible
horizontal transfer events among genomes in AMD and the nine previously isolated organisms.

Fig. 1. Functional classification of 119 predicted transferred genes. The x-axis represents
the frequency of genes in each COG categories. The y-axis shows the four main COG
categories, and each colored bar represents a subclass in this category. (Functional
categories of subgroups of COG: [J] translation, ribosomal structure, and biogenesis;
[A] RNA processing and modification; [K] transcription; [L] replication, recombination,
and repair; [B] chromatin structure and dynamics; [D] cell cycle control, cell division,
and chromosome partitioning; [Y] nuclear structure; [V] defense mechanisms; [T] signal
transduction mechanisms; [M] cell wall/membrane/envelope biogenesis; [N] cell motility;
[Z] cytoskeleton; [W] extracellular structures; [U] intracellular trafficking, secretion,
and vesicular transport; [O] posttranslational modification, protein turnover, and
chaperones; [C] energy production and conversion; [G] carbohydrate transport and metabolism;
[E] amino acid transport and metabolism; [F] nucleotide transport and metabolism;
[H] coenzyme transport and metabolism; [I] lipid transport and metabolism; [P] inorganic
ion transport and metabolism; [Q] secondary metabolite biosynthesis, transport, and
catabolism; [R] general function prediction only; and [S] function unknown)

We further analyzed the transfer events with a clear evolutionary path in which only
AMD organisms were involved. To this end, after a rigorous manual selection from the
119 possible transfer events, 14 genes were determined to be HGT among the eight AMD
organisms (Table 1). Furthermore, possibly involved genomes among the eight AMD organisms, the possible
donor and recipient organisms, and functions of the 14 genes were identified and listed
in Table 1. It is interesting that genetic exchanges of these genes occur between bacteria and
archaea in both directions in this community. We noted that two transfer events from
bacteria to archaea, which included 11 of the 14 genes, are crucial to the development
of the AMD community. This makes a lot of sense because two iron-oxidizing chemoautotrophic
bacteria, Leptospirillum ferrodiazotrophum and Leptospirillum rubarum, dominate this relatively self-contained ecosystem 19]. With respect to the transfer events in this direction, functions of the involved
genes affect the AMD microbial community. In the next two subsections, the details
of the putative HGT events in this direction are reported. Herein it is noteworthy
that the transfer events in the other direction, i.e., from archaea to bacteria, also
suggested the significance of transferred genes to the recipients. For instance, two
genes, MesJ and Mdh, were horizontally transferred from archaea to bacteria L. ferrodiazotrophum and L. rubarum. The MesJ protein is a well-known cell-cycle protein that is directly responsible
for lysidine formation and thus is essential for decoding AUA codons in vivo27]. Mdh, which was proved to be horizontally transferred in a previous study 28], plays a key role in the cell during growth on methanol. The CcmA gene, which was transferred from archaea to the bacterium L. ferrodiazotrophum, is the first gene of an eight protein-encoded operon involved in cytochrome c maturation
and heme delivery 29]. The evolution of cytochrome c domains has been reported to involve gene transfer
events 30]. CcmA was identified as the essential gene of the cytochrome c ATPase; therefore, CcmA is likely to be transferred to assist the electron transfer process.

Table 1. Fourteen horizontally transferred genes with high reliability among eight organisms
in acid mine drainage

In this study, we focused on the 14 horizontally transferred genes that could be traced
in the AMD evolutionary process; other genes also have the possibility of horizontal
transfer among the AMD community members. HGT events are widely involved in microbial
genomes in natural communities; however, the computational identification of HGT in
genomic sequences remains very difficult in practice 11]. We could have identified more HGT events among the AMD metagenomes. However, to
more thoroughly investigate the impacts of HGTs, we selected the genes that were most
likely transferred and made a large contribution to the AMD community. A second notable
aspect of this study is that we aimed to examine HGT events between members within
the microbial community. To be sure, novel genes of a recipient genome were recognized
as being transferred by a donor species either from an external environment or internal
community. However, as a relatively self-contained ecosystem in an extreme environment,
the AMD microorganisms can demonstrate the social behaviors of both cooperation and
competition. In this light, we argued that the movement of genetic materials among
organisms within the community should be essential to their performance as a whole.
Finally, it is certain that the gene transfer events studied in this work were determined
by the strict standard of HGT identification adopted here, which also implies that
these putative HGT events were identified amongst distantly related species. HGT might
be easier between closely related species for which the barriers to the transfer more
easily can be surmounted 11]. However, because of the environmental challenges, some gene transfer events would
greatly benefit some species or the entire community, leading to a better adaptation
to the environment, even with the effort for the exchange of genetic material between
distantly related species. In other words, the HGT events reported in this manuscript
should be regarded as being nontrivial and essential to the AMD community because
of the strong selective pressure and the harsh environment in AMD.

Acquisition of antibiotic resistance in G-plasma via HGT associated with ribosomal proteins

Among the 14 horizontally transferred genes, nine were transferred from bacteria to
the archaeon G-plasma. Moreover, they encoded ribosomal proteins and the related subunits. Following, we
report our detection and analysis of the consequence of these transferred genes on
G-plasma and the AMD microbial community.

Ribosomal proteins are widely distributed in prokaryotes, and they are usually involved
in information processing (e.g., replication, transcription, and translation) or central
metabolism. Genes for ribosomal proteins are generally considered to be housekeeping
genes and relatively recalcitrant to HGT; because of this, their sequences are routinely
used as phylogenetic markers 31]. However, ribosomal genes have shown a remarkable possibility of being transferred
in many prokaryotic genomes 3]. For example, a phylogenetic study on ribosomal protein S14 revealed an unexpected
tree topology that was explained by horizontal transfer 32]. Also, ribosomal proteins L32 and L33 have phylogenetic incongruities that resulted
from gene transfer 33]. In the current study, one of the evident horizontal transfer events was that of
ribosomal protein genes. In L. ferrodiazotrophum, we found a contig (gi: 251772484) containing 10 ribosomal genes: rpsJ, rplD, rpsC, rplP, rplX, rplE, rplR, rpsE, rpsK, and rpsD. Of these genes, rpsJ, rplD, rpsC, and rplP are usually clustered as operon S10, while rplX, rplE, rplR, and rpsE are usually clustered as operon spc. All of these ribosomal genes are in the positive strand of the contig [Additional
file 2: Figure S1], and their protein name and function are listed in Table 2. Looking into the phylogenetic tree of these ribosomal genes, most of them (except
RpsK) show obvious disagreement with the species tree, strongly indicating possible horizontal
transfer events. To be specific, the ribosomal genes of G-plasma often have a close relationship with those of L. ferrodiazotrophum and L. rubarum instead of other plasma species, and they are placed in a cluster of bacteria rather
than archaea in the gene tree. The same pattern holds for the ribosomal genes of both
the spc operon (rpsE in Fig. 2, others in Additional file 2: Figures S2–S4) and S10 operon [Additional file 2: Figures S5–S8]. Therefore, ribosomal genes of G-plasma show a high possibility of being transferred from bacteria. Considering the close
relationship and the same habitat as Leptospirillum, it makes sense that L. ferrodiazotrophum and L. rubarum are the donor of these ribosomal genes for the archaeon G-plasma.

Table 2. Ribosomal gene information

Fig. 2. Phylogenetic tree of the rpsE gene family. As a conserved gene, radA is used as an outgroup. The rpsE gene of G-plasma is located in a cluster of bacteria, indicating possible horizontal transfer

Notably, these genes were identified as transferred clusters, including two known
operons, from bacteria to archaea as shown in Additional file 2: Figure S1. This feature is clearly consistent with the theory of a selfish operon,
in which operons are viewed as mobile genetic entities that are constantly disseminated
via HGT, although their retention could be favored by the advantage of the co-regulation
of functionally linked genes 34]. As a result, functional information for these transferred genes may be inferred
by comparing them with the related or analogous genes. An earlier study focusing on
the horizontal transfer of rps14, which is also a member of the spc operon, led to the conclusion that antibiotic resistance can be conferred because
rps14 is known to be involved in antibiotic resistance through the binding of puromycin
besides its major role in the assembly of ribosomal 30S subunits 32], 35]. In the current study, although rps14 does not appear in both G-plasma and the two Leptospirillum bacteria, we noted that rpsE, which is in both G-plasma and Leptospirillum, has a function that is similar to that of rps14, i.e., antibiotic resistance. Therefore, we may safely conclude that G-plasma can acquire this function through horizontally transferred spc operon genes. In addition, the S10 operon has two genes, rplP and rplD, that are involved in antibiotic resistance. These observations led to the explanation
that G-plasma acquired these genes for antibiotic resistance. As predicted by the selfish operon
model 34], these transferred gene clusters should allow cells in the AMD environment to demonstrate
the metabolic benefits of antibiotic resistance; moreover, they can enhance the fitness
of G-plasma as the recipient species.

Another question of interest is how the transferred genes associated with antibiotic
resistance influence the microbial community in AMD. We may just as well learn this
point from their role in L. ferrodiazotrophum and L. rubarum, the two most dominant organisms in AMD. The amount of antibiotic resistance proteins
in L. rubarum was correlated with the growth stage of biofilms 36]. In early growth stage samples, L. rubarum dominates the community, and its repressors of antibiotic resistance genes are abundant.
With the growth of biofilms, increasing types of microorganisms compete for limited
resources (e.g., nitrogen, oxygen, and phosphate), forcing L. rubarum to reinforce its competitiveness and self-protection. A previous study demonstrated
that antibiotic resistance proteins from L. rubarum were more abundant during late-stage growth 36]. Clearly, antibiotic resistance is closely related to the population of individual
species in the natural microbial community. It is interesting to note that G-plasma is the dominant archaeon in AMD. Based on proteomic data across 28 microbial community
samples, G-plasma constituted about 9.0?±?4.9 % of the community 37]. Deduced by analogy, G-plasma and other archaea should also encounter a similar situation, and the archaeon with
a competing advantage will survive easier than archaea without the advantage. As shown
by the population distribution, G-plasma is no doubt a strong competitor. The horizontal acquisition of antibiotic resistance
genes is a possible advantage that protects and enhances its competitiveness, making
G-plasma the largest group among AMD archaea. As a result, both these genes and their horizontally
transferred relatives that provide antibiotic resistance influence the growth of species
and shape the population structure of the AMD microbial community.

Taken together, the transferred gene clusters of ribosomal genes are associated with
antibiotic resistance, leading to the fitness improvement of the archaeon G-plasma and the community structure in AMD. It should be pointed out that the requirement
of antibiotic resistance, as the selective pressure, may force microorganisms to overcome
the possible difficulties of horizontal transfer because these horizontally acquired
antibiotic resistance genes are of great importance in the life of G-plasma in the toxic environment of the AMD, and they may be vital in making G-plasma the largest group of AMD archaea.

Possible impact of gadBC operon transfer on acid resistance in Ferroplasma

In this subsection, we discuss the putative horizontal transfer of the gadB gene of the gadBC operon from bacteria to two archaea of the Ferroplasma genus. The gadBC operon corresponds to the function of acid resistance. Acid resistance is perceived
as an essential property of microorganisms living in AMD. The glutamate decarboxylase
(GAD) system, which is common in bacteria and some eukaryotic genomes, has been extensively
studied for its major role in acid resistance in organisms such as in E. coli, Shigella flexneri, and Listeria monocytogenes38]–41]. An essential role of biochemical pathways is to yield cell–cell messengers 41]. The GAD system is usually composed of three genes, gadA, gadB, and gadC. The gadA and gadB genes usually have high sequence similarity and encode two biochemically indistinguishable
glutamate decarboxylases, and the gadC gene encodes a glutamate/GABA antiporter. The gadB and gadC genes are organized into a functionally important operon, gadBC42]. By producing alkaline ?-aminobutyrate and utilizing an intracellular proton, functions
that involve the gadBC operon, cells can adapt to low pH 43].

Through investigation of the phylogenetic tree of gadB, we found that there was an obvious incompatibility with the organism tree, and gadB from Ferroplasma fer1 and Ferroplasma fer2 were misplaced within the cluster of bacteria; the closest relatives were genes from
L. ferrodiazotrophum and L. rubarum (Fig. 3). This incompatibility indicates a possible transfer event. Moreover, transposase
genes were found in the neighborhood of the gadBC operon in both F. fer1 and F. fer2, providing additional evidence for the HGT events 44]. Because of the low conservation and poor annotation quality of the gadC gene in public databases, it is difficult to estimate the true representation of
gadB in most microbial genomes even with examples of its potential role in a few well-known
organisms such as E. coli and S. flexneri. Although the gadC gene in both F. fer1 and F. fer2 is located close to that of L. ferrodiazotrophum and L. rubarum in the phylogenetic tree, we did not analyze its confused and incongruous evolution
in the current study. However, because there was only one transcription start site
for the gadBC operon regardless of the inducing condition 45] and no obvious promoter could be found upstream of gadC, we cannot exclude the possibility of the same transfer event including the gadC gene, leading to the transfer of both the gadB and gadC gene. Furthermore, the prediction of 13 transmembrane passes with TMHMM 2.0 46] inferred the integral function of gadC in Ferroplasma. An ancestor of Ferroplasma originally acquired the gadB gene by horizontal transfer (transferred as the gadBC operon), and L. ferrodiazotrophum and L. rubarum might be the donors because of their close relationship and presence in the same
habitat in which they overcame the acid environment to use more resources.

Fig. 3. Phylogenetic tree of the gadB gene family. As a conserved gene, radA is used as an outgroup. The gadB genes of Ferroplasma fer1 and Ferroplasma fer2 are located in a cluster of bacteria, indicating possible horizontal transfer

On the basis of the function of the GAD system, it is reasonable to suggest that the
acquisition of the gadBC operon allowed F. fer1 and F. fer2 to better resist the extreme acid environment. Although there are other mechanisms
to resist a low-pH environment 47], the horizontally transferred gadBC operon plays an important role in the efficient utilization of a proton by F. fer1 and F. fer2 to resist the extreme acidic environment. Combined with the putative transfer events
of the spc operon and S10 operon from bacteria to G-plasma, there seems to be a tendency toward the movement of genes with operon structures
from bacteria to archaea. In general, this agrees with the selfish operon hypothesis
34]. However, the two cases described in this report indicate that most transferred genes
involved in the same operon have a closely functional relationship. Moreover, they
play an essential role in the requirements of antibiotic or acid resistance in recipient
archaea. This differs from the viewpoint that the majority of genes in transferred
operons are nonessential genes with related functions 34], 48]. In an environment that seems so harsh, organisms have to retain their beneficial
genetic resources to survive the external stresses of the environment.