Volume 11 Supplement 11
Large number of phosphotransferase genes in the Clostridium beijerinckii NCIMB 8052 genome and the study on their evolution
© Li et al; licensee BioMed Central Ltd. 2010
Published: 14 December 2010
Clostridium beijerinckii is a valuable bacteria species which has the ability of ABE (acetone, butanol and ethanol) production. It has been shown that Phosphotransferase (PTS) is an important and common system for both carbohydrate uptake and phosphorylation in bacteria, but detailed study of the system, especially its fructose/mannose/sorbose family is scant.
In the genome of Clostridium beijerinckii NCIMB 8052, a model strain recently sequenced, there are large number of PTS genes, among them 9 complete sets belong to the fructose/mannose/sorbose family of its enzyme II complex. Our study, based on evidences provided by phylogenetic relationship, analyses of gene contents and clusters, as well as synteny examination, indicates that it is possible to further classify this PTS family into three sub-groups, which are corresponding to the three sugar substrates. Furthermore, we proposed a model how these PTS systems are evolved in bacteria.
This work may explain the experimental result that Clostridium beijerinckii NCIMB 8052 can better utilize fructose as substrate, thus could lead to a better understanding of the ABE-producing mechanism in Clostridium beijerinckii and other microbial species. It may help to illustrate a higher butanol-productivity future.
Previous studies have revealed that enzyme I and the phosphocarrier protein (Hpr) of PTS generally has only one or two copies in clostridium strains, and they exist in the genomes monocistronically . However, the genes consisted of the enzyme II complex may have multiple copies, and often form operons together with their transcription regulatory units and the genes with related catalytical functions . Furthermore, the phosphotransferase systems in different types of microbial species have different mechanisms for regulation. For low GC Gram-positive bacteria like clostridium, Hpr is acting as an important switch. When carbon source is in short supply, Hpr will be phosphorylated on the serine-46 residue by certain unique Hpr kinase/phosphatase. In contrast, if carbon source is sufficient, the His-15 site of Hpr is phosphorylated, and the PTS-dependent sugar transportation and substrate-specific regulation are activated .
Although the study on phosphotransferase system has been going on for several decades, most of the previous reports are concentrated on limited number of model strains such as Escherichia coli and Bacillus subtilis. Since the beginning of this century, there are some researches on PTS in clostridium, mostly on Clostridium acetobutylicum ATCC 824, which is the first clostridium sequenced. The importance of PTS to the life process of various clostridium strains, and how the system is evolved, remain largely unknown. This work starts from the genomic data of Clostridium beijerinckii NCIMB 8052, and other sequenced clostridium strains as well as some important related model species, and analyzes the complexity of the phosphotransferase system, especially focusing on the fructose/mannose/sorbose family. We hope to explain why and how Clostridium beijerinckii NCIMB 8052 can utilize more types of sugar substrate, and propose an evolutionary model for the phosphotransferase system. It may help to illustrate the solvent-producing mechanism in Clostridium beijerinckii and other related industrial bacteria, which could have theoretical and practical importance for guiding to higher butanol production through certain optimizations.
Results and discussion
Large number of PTS II genes are identified in the Clostridium beijerinckii NCIMB 8052 genome
The bacterial strains studied and the sets of the PTS II genes in their genomes
Sets of PTS II genes
Clostridium beijerinckii NCIMB 8052
Clostridium botulinum A str. ATCC 3502
Clostridium acetobutylicum ATCC 824
Clostridium perfringens strain 13
Clostridium tetani E88
Clostridium difficile 630
Bacillus subtilis subsp. subtilis str. 168
Escherichia coli str. K-12 substr. DH10B
In contrast, the PTS enzyme I and the phosphocarrier protein Hpr, each have only one copy in the Clostridium beijerinckii NCIMB 8052 genome (Cbei0196 and Cbei1219, respectively). This is consistent with other clostridium species.
Analysis of GC content
The GC distribution of the fructose/mannose/sorbose PTS II gene sets in Clostridium beijerinckii NCIMB 8052. Three groups are indicated, with *, #, or nothing at all.
As we can see from the result, the GC contents of these sets of fructose/mannose/sorbose family PTS II genes can be divided into three groups, which are indicated in the Table 2 with three different denotations, with *, # or nothing at all.
For the two sets of Cbei3871/3872/3873/3874 and Cbei4911/4912/4913/4914, both their GC percentages are 35.1%. The GC% of Cbei0711/0712/0713 is over 37%. For the remaining sets, their GC percentages are ranging from 30.3% to 33.1%. Although that of Cbei2195/2196/2197/2199 is 33.9%, but if the inserted hypothetical protein Cbei2198 is considered altogether, it drops to 33.1%. The two orphan genes of this family, Cbei1840 and Cbei3344, are not necessarily conforming to this rule. It is noteworthy that the GC percentage of the Clostridium beijerinckii NCIMB 8052 whole genome is only 29.9%, lower than that of any PTS II genes sets. We will discuss this later.
According to Figure 1, the fructose/mannose/sorbose family PTS II genes in Clostridium beijerinckii NCIMB 8052 can also be classified into three groups based on their synteny features. One group has both sigma-54 factor and sugar isomerase (SIS) in the neighborhood, such as Cbei0955/0956/0957/0958, Cbei0963/0964/0965/0966 and Cbei4557/4558/4559/4560. Another group has sigma-54 factor only, including Cbei3871/3872/3873/3874 and Cbei4911/4912/4913/4914. The last group has neither sigma-54 factor nor sugar isomerase, like Cbei0711/0712/0713. We found these groupings are quite consistent with the ones discussed in the last section which are based on their GC percentages.
Sigma-54 is a kind of transcriptional factors, commonly existed in bacteria. It can interact with promoters to activate the transcription process. The first sigma-54 factor, rpoN, was found in 1985 through the sequencing of Klebsiella pneumoniae. According to previous report  , in Listeria monocytogenes, sigma-54 factor-dependent fructose/mannose/sorbose PTS II genes are found to be associated with the bacteria’s sensitivity of cytotoxin. Therefore, it is reasonable to suggest that their presence around the fructose/mannose/sorbose PTS II genes in Clostridium beijerinckii NCIMB 8052, could also have certain functional importance.
Construction of phylogenetic tree
Since IIB is the subunit which decides the PTS substrate specificity, in this part of study we use Cbei3873 as the query for blastp, and analyze the evolutionary relationship among the PTS fructose/mannose/sorbose family IIB genes in Clostridium beijerinckii NCIMB 8052 and their homologous genes identified from other representative bacteria species (selecting according to the e-value so that every e-value intervals have representations).
Among the group which contains Cbei1840, Cbei3873 and Cbei4913, Cbei3873 is 95% identical to Cbei4913, 75% identical to Cbei1840, and 39% identical to the fructose-specific IIB gene Bsu27060 in Bacillus subtilis over 160 aa (68% positive) (Figure 2). However, Cbei3873 has also been found to be 59% identical to the mannose-specific IIAB gene Ecoli_UTI89_C2014. On the other hand, although Cbei0711 is the closest to Ecoli_UTI89_C2014 in terms of gene organization since in both cases the IIA and IIB subunits are fused together, they are not grouped in the same cluster.
This result reveals the complexity of the PTS fructose/mannose/sorbose family. The reason that the transport systems for these three sugars can be classified into one family is because they are close to each other, highly homologous to each other, with few homologies to the genes in other families. But looking into it in more detail, we can find that PTS II genes for fructose and mannose transportation are closer than their relation with that of sorbose. As the result, both fructose and mannose PTS II genes phosphorylate fructose into phospho-6-fructose (F-6-P), while sorbose PTS II genes turns it into F-1-P. Furthermore, according to reference , the phosphorylation site on the fructose by the fructose-specific PTS genes is O-6, instead of the more common O-1 site. This is another proof that fructose-specific PTS genes are closer to the mannose-specific ones.
An evolutionary model is proposed
To get a clearer picture of why there are so many copies of fructose/mannose/sorbose family PTS II genes in the Clostridium beijerinckii NCIMB 8052 genome, we applied the same analytical approaches to the model strain of Bacillus subtilis, B. subtilis subsp. subtilis str. 168, by identifying all the PTS II genes in its genome, annotating and classifying each sets as well as calculating their GC percentages. The results are in Table S6 and showed that there are 12 sets of PTS II genes in B. subtilis subsp. subtilis str. 168. One of them belongs to the fructose/mannose/sorbose family, namely Bsu27040/27050/27050/27070, which phosphorylates fructose into F-6-P. The GC percentages of these PTS II gene sets in B. subtilis subsp. subtilis str. 168 are ranging from 44.4% to 50.8%, with that of Bsu27040/27050/27050/27070 is 44.4%, slightly higher than the GC percentage of the whole genome, which is 43.5%.
We did the same analysis to the model strain of E. coli, Escherichia coli str. K-12 substr. DH10B (Table S7). 25 sets of PTS II genes are identified, including one complete set of the fructose/mannose/sorbose family, i.e., ECDH10B 1955/1956/1957. Their GC contents are as high as 56.1%, and as low as 47.3%, while that of ECDH10B 1955/1956/1957 is 51.6%, close to the average GC percentage of the Escherichia coli str. K-12 substr. DH10B genome, 50.8%.
As the results indicate, in all these bacterial species we studied, the GC percentages of PTS II gene sets are generally higher than the overall GC values of the host genomes. This leads us to speculate that the phosphotransferase system may have originated from certain high GC percentage microbial species, and during the evolutionary process lower GC percentage species acquired the components of this system from higher GC percentage species through horizontal gene transfer (HGT). The transfer may occur at an earlier stage of the evolution, since it becomes clear that the GC percentages of PTS II gene sets are being drawing closer to the average values of the host species after the transfer. For example, in Escherichia coli str. K-12 substr. DH10B, they are generally between 50-60%. The values drop to the interval of 45-50% in B. subtilis subsp. subtilis str. 168. In the clostridium strains in which GC contents are even lower, the values further decrease. In the case of Clostridium beijerinckii NCIMB 8052, they are 30 to 38%. Of course, this makes sense. Understandably, the sugar transport system is of the utmost importance to bacteria, concerning the life or death of each organism. So this system must have existed in microbial species for quite a long time. However, as two strains highly representative of the fructose/mannose/sorbose family, both the fructose-specific PTS II genes in Bacillus subtilis, and the mannose-specific ones in E. coli, their GC percentages are closer to the overall GC contents of their genomes. Therefore, they may be ‘native’ to these species and later on transferred into Clostridium beijerinckii NCIMB 8052.
How the PTS II systems are evolved in Clostridium beijerinckii NCIMB 8052?
The genome size of Clostridium beijerinckii NCIMB 8052 is about 5.9MB, including 5104 ORFs. That is significantly larger than that of Clostridium acetobutylicum ATCC 824, which has a chromosome of 3.94MB plus a plasmid of 192KB , totaling 3917 ORFs (3739 on the chromosome and 178 on the plasmid pSol1). This is likely due to more diverse living environments enjoyed by Clostridium beijerinckii NCIMB 8052, which means more types of carbon sources it can utilize [1, 3]. Although they are in the same genus of clostridium, they separated relatively early in the evolutionary process . Clostridium acetobutylicum ATCC 824 has been shown acquiring large quantities of outside genes through horizontal gene transfer ; Clostridium difficile 630 also has been found with as high as 11% of mobile gene contents in its genome , which strongly indicates that it gained genes from other species. So, it is reasonable to expect similar events happened in C. beijerinckii NCIMB 8052 too. But in its distintive evolutionary process, C. beijerinckii NCIMB 8052 is able to keep large number of genes transferred from other species because it needs these genes to adapt to various soil environments. On the other hand, the living condition of C. acetobutylicum ATCC 824 is relatively simpler, so only fewer genes which are essential to its life activity are kept. More specifically, C. beijerinckii NCIMB 8052 is quite different from C. acetobutylicum ATCC 824 in terms of sugar needed. While C. acetobutylicum ATCC 824 is starch-based, C. beijerinckii NCIMB 8052 is molasses-based. Molasses contains 35% of sucrose, 7% of fructose and 5% of glucose (W. Jiang, personal communication). Also, C. beijerinckii NCIMB 8052 can ferment xylose degraded from agricultural wastes . Since C. beijerinckii NCIMB 8052 can utilize more sugar substrates than C. acetobutylicum ATCC 824, it makes senses that it needs more genes to break down these substrates. After querying the ISFinder database (http://www-is.biotoul.fr/), we identified 92 different types of insertion sequences in the C. beijerinckii NCIMB 8052 genome, among them 19 with e-score lower than 1e-5 (data not shown). A lot of these insertion sequences have multiple copies. For example, ISCb1 has 9 copies. In Clostridium perfringens, some PTS genes are found to be flanked by insertion sequences . In C. beijerinckii NCIMB 8052, we have similar findings too. This is in agreement with our hypothesis that C. beijerinckii NCIMB 8052 genome gained and kept large quantities of outside genes through HGT, including some PTS genes.
Another reason which leads to the fact that there are so many sets of the PTS II genes in the C. beijerinckii NCIMB 8052 genome is gene duplication. As shown in Figure 2, many PTS IIB genes have several close-related copies. For example, Cbei3873 and Cbei4913, they are 95% identical. Figure 1 shows that they share the same genetic organization and gene order. This strongly indicates that these genes are of great importance to the host bacteria, so they are duplicated in group, as functional backups for the original ones.
The correlation between sugar utilization and PTS II gene numbers
Clostridium beijerinckii NCIMB 8052 can better utilize fructose in the fermentation media than Clostridium acetobutylicum ATCC 824 (W. Jiang, personal communication). Our findings have shown that in the Clostridium beijerinckii NCIMB 8052 genome there are so many more gene sets of PTS II fructose/mannose/sorbose family than C. acetobutylicum ATCC 824. And with the consensus from previous reports  that all gene sets of this family can transport fructose, we established a likely correlation between these facts.
In addition to the PTS system, fructokinase  can also phosphorylate fructose into phospho-6-fructose. However, fructokinase may not have played a major role in determining the difference between the fructose utilization efficiencies of these two clostridium model strains. In both genomes, there are two copies of fructokinase genes, Cbei3497 and Cbei5009 ;CAC0424 and CAC1523.
It remains an unsolved question why these three sugars, fructose, mannose and sorbose, can form such a distinctive phosphotransferase family. In terms of their chemical structures, all three are hexose, but fructose and sorbose are ketoses while mannose is an aldose. Probably they have very similar 3D structures. The presence of sugar isomerases around certain types of the fructose/mannose/sorbose family PTS II genes in the C. beijerinckii NCIMB 8052 genome seems to suggest this too.
In summation of the analysis performed in this research , we found that the fructose/mannose/sorbose family of PTS II genes in Clostridium beijerinckii NCIMB 8052 can be further classified into three groups , possibly corresponding to the three different sugar substrates of this family. In addition to their respective specific sugars, all of them are capable of transport fructose. It is highly likely that this finding offers a valid explanation that Clostridium beijerinckii NCIMB 8052 can better utilize fructose as the sole carbon source than Clostridium acetobutylicum ATCC 824.
We also proposed an evolutionary model which suggests the horizontal gene transfer of the phosphotransferase system from higher GC microbial to lower GC ones. This may help to illustrate the mechanism why bacteria can survive in different environments through their variable carbon sources.
The amino acid sequences of the studied genes are used as query, run blastp search  against the non-redundant (nr) database ,keep all the default parameters values, and find their homologous genes in other sequenced microbial species. E-value should be less than 1e-5.
Annotation of the PTS II genes
Blastp: described above.
The domain structures of the PTS II genes are compared with those known variations of all the families from the literatures.
For each sets of the PTS II genes, we first visually inspect their downstream and upstream regions for possible existence of synteny. The amino acid sequences of the 4 surrounding genes in both directions are picked out to identify their domains through InterproScan. They are used as the blastp queries to identify their homologous genes in other microbial species (e-value less than 1e-5). We then check the locations of these homologous genes in their respective genomes to see whether they also appear in the surrounding areas of PTS II genes.
Multi-sequence alignment and construction of phylogenetic trees
We use the embedded ClustalW  function of software package MEGA4  for multisequence alignment, using all default parameter values. NJ (Neighbor-Joining) method  is applied to construct phylogenetic trees, with bootstrap values  calculated by repeating 500 times to evaluate the robustness of the nodes on the trees.
We would like to thank Professor Weihong Jiang and Mr. Cong Ren of the Institute of Plant Physiology & Ecology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences for their support and useful discussions. Also, we thank colleagues including Drs. Guohui Ding, Zhiqiang Ye, Hong Sun, Mr. Hong Yu, Mr. Xiao Chang, Dr. Fudong Yu, Mr. Rudong Li and Ms. Hui Yu for their help during the study. This work was supported by National High-Tech R&D Program (863) (2007AA02Z331).
This article has been published as part of BMC Bioinformatics Volume 11 Supplement 11, 2010: Proceedings of the 21st International Conference on Genome Informatics (GIW2010). The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/11?issue=S11.
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