Bioinformatics classification and functional analysis of PhoH homologs

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Abstract

PhoH protein is a putative ATPase belonging to the phosphate regulon in Escherichia coli. EC-PhoH homologs are present in different organisms, but it is not clear if they are functionally related, besides nothing is known about their regulation. To distinguish true functional orthologs of EC-PhoH in different classes of bacteria and to identify their functional role in bacterial metabolic network we performed phylogenetic analysis of these proteins and comparative study of position and regulation of the related genes. Three groups of proteins were identified. Proteins of the first group (BS-PhoH orthologs) are present in most of bacteria and are proposed to be functionally linked to phospholipid metabolism and RNA modification. Proteins of the second group (BS-YlaK orthologs) are present in most of aerobes and Actinobacterial YlaK orthologs are shown to be members of a fatty acid beta-oxidation regulons. EC-PhoH orthologs are classified in a third group, specific for Enterobacteria. Functional role of PhoH homologs in the lipid and RNA metabolism and proposed interrelation of PhoH paralogs in one organism are discussed.

Key words: PhoH, regulon, Mycobacterium, phylogenetic analysis, gene clusters, phospholipid

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Introduction

Phosphate starvation triggers crucial metabolic perturbations and adaptive response in bacteria. It is known to cause oxidative stress in Escherichia coli [1] and involves rebuilding of different metabolic subsystems [2, 3]. Phosphate starvation was shown to be a regulator of the polyhydroxybutyrate biosynthesis in Acinetobacter [4]. In Mycobacterium, phosphate starvation is known to trigger the development of bacterial virulence, where lipid metabolism was shown to be one of the important factors [5, 6, 7].

Expression of a majority of genes belonging to the phosphate regulon in Escherichia coli is controlled by the PhoR/PhoB system in response to inorganic phosphate levels [1, 8, 9]. This regulation involves activation of sn-glycerol-3-phosphate membrane transport [10, 11] and activity of glycerophosphate dehydrogenase during phosphate limitation [12].

The function of one member of the Escherichia coli phosphate regulon, PhoH, is still undefined, though it was shown to express phosphatase activity [8] and is considered as a putative RNA helicase. PhoH paralog in E. coli, PhoL (formerly Ybez), and EC-PhoH homologs in other bacteria are often being referred to as phosphate starvation regulated proteins and commonly are annotated s have PhoH. In Bacillus subtilis, phoH (BS-phoH) is located in a locus with diacylglycerol kinase gene (dgkA) [13]. According to our data this coupling is highly conserved in Gram-positive organisms. Moreover, closest homologs of BS-phoH conservatively cluster with several genes related to the phospholipid turnover and RNA modification in a majority of Gram-positive and Gram-negative bacteria. It is not clear whether PhoH homologs in different organisms are functionally related, nothing is known about their regulation and role in the bacterial metabolic network. BS-phoH, for instance, was not proven to be a member of the phosphate regulon in Bacillus subtilis, as its expression is not affected in the phoB mutant as shown by DNA microarray analysis [14].

Position of a gene in similar gene clusters in non-closely related bacteria suggests existence of a functional link between the products of the co-localized genes [15]. In this paper we present the results of phylogenetic classification and positional analysis of E. coli and B. subtilis PhoH homologs. Additional information was produced by comparative analysis of phoH regulation [16]. Paralogs of EC-PhoH and BS-PhoH in Mycobacterium tuberculosis and Thermomonospora fusca are shown to belong to the fatty acid beta-oxidation regulon. PhoH homologs, belonging to the BS-PhoH/EC-PhoL branch of the phylogenetic tree are proposed to be functionally linked to the phospholipid turnover and modification of the translation machinery in different classes of bacteria.

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Materials and methods

Genome comparative analysis was performed using the ERGO (previously WIT [17]) genomic database and a set of tools therein (available by subscription from Integrated Genomics, Inc, IL). Not all mentioned protein sequences are available from public genome databases and thus their sequences are attached as a supplementary file (proteins.aa).

Multiple sequence alignment was constructed using the CLUSTALX program [18]. A phylogenetic tree (attached in a supplementary file Tree.gif) was constructed using the program PROML from the PHYLIP package (maximum likelihood method) [19].

A simple iterative procedure implemented in the software package Genome Explorer is performed in order to construct a profile from a set of upstream gene fragments and to search for possible regulatory sites in genomic sequences [20].

Positional nucleotide weights in these profiles were defined as follows [21]:

where N(b,k) denoted the count of nucleotide b at position k. The score of a L-mer candidate site was calculated as the sum of the respective positional nucleotide weights:

The comparative approach to the analysis of transcriptional regulation in bacterial genomes is based on the assumption that sets of genes regulated by orthologous transcription factors are conserved in related genomes. Thus the candidate sites occurring upstream of orthologous genes are true, whereas false positives are scattered at random [16].

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Results

Positional analysis of PhoH orthologs

Using the ERGO database we found PhoH homologs in 130 genomes of both Gram-negative and Gram-positive bacteria. More than one PhoH homolog per organism was found in many cases. Thus, we aligned protein sequences and constructed a phylogenetic tree to identify true PhoH orthologs (The tree is attached as a supplementing file Tree.gif, with BS-PhoH, BS-YlaK and EC-PhoH branches colored, respectively, blue, red and black).

Two main branches were observed in the tree: one containing BS-PhoH orthologs and the other containing orthologs of BS-YlaK.

BS-PhoH orthologs, including EC-PhoL, were found in 76 genomes, whereas BS-YlaK orthologs were found in 33 genomes. The third branch of the tree includes EC-PhoH and its orthologs in four other gamma-proteobacteria. Genomes containing orthologs of BS-PhoH, BS-YlaK and EC-PhoH are shown in Table 1.

Table 1: Genomes containing orthologs of BS-PhoH, BS-YlaK and EC-PhoH.

Positional analysis of genes belonging to the BS-ylaK and BS-phoH branches of the tree shows that they belong to different gene loci: genes encoding BS-PhoH orthologs are in semi-conserved gene clusters in various bacteria, whereas genes encoding BS-YlaK orthologs and EC-PhoH orthologs are in nonconserved, distinct loci.

The BS-phoH gene orthologs in different groups of bacteria, including EC-phoL, belong to gene clusters of one through six genes from the following list (Tab. 2):

1. miaB encoding a protein involved in methylthiolation of isopentenylated A37 derivatives in tRNA (in 30 Gram-negative organisms). This gene is almost specific for proteobacteria.
2. yqfF encoding possible metal-dependent phosphohydrolase (in 9 organisms, mainly Gram-positive bacteria). Absent in actinobacteria, most of cocci, alpha, beta and gamma proteobacteria.
3. yqfG encoding a conserved protein with unknown function (in 55 organisms). Individually present in all studied genomes.
4. dgkA encoding diacylglycerol kinase (in 11 organisms, mainly Gram-positive bacteria). Proteobacteria and actinobacteria have a non-orthologous enzyme.
5. ybeX/corC encoding CBS domain-containing protein, putative Cobalt/Magnesium efflux protein, also involved in establishing of cobalt resistance and magnesium homeostasis (in 33 organisms, including Gram-negative bacteria and Actinobacteria). Individually present in almost all genomes, that contain phoH homolog.
6. era encoding GTP-binding protein ERA, involved in RNA adaptive modification (in 13 organisms). Present only in Gram-positive bacteria and in Spirochaetales.
7. lnt encoding apolipoprotein N-acyltransferase (in 24 Gram-negative organisms). Present in alpha, gamma and, sporadically, beta proteobacteria, actinobacteria, clostridia.

Table 2: Occurrence of genes, forming the phoH gene cluster in phoH loci of genomes containing BS-PhoH homologs (* - incomplete contig).

Fig. 1 demonstrates chromosome loci around BS-phoH from selected bacteria representing different systematic groups.

Absence of BS-phoH homolog clustering with at least one of the listed genes (Tab. 2) in a genome correlates with the absence of ybeX, ygfF and miaB from the genomes of Streptococci and Aptobium minutum), absence of ybeX, miaB, lnt and era from the genomes of all Cyanobacteria, bacteria of the Neisseria group, Cytophaga hutchinsonii and Deinococcus radiodurans.

In Brucella melitensis, Pasteurella multocida, Haemophilus influenzae and Haemophilus ducreyi, the BS-phoH loci contain genes encoding 1-acyl-sn-glycerol-3-phosphate acyltransferase and undecaprenyl pyrophosphate synthase. Several stress regulators are also associated with the phoH locus in a number of bacteria: bolA (general stress response), sprT, involved in the bolA expression in the stationary phase, htpG (heat shock).

Genes encoding BS-YlaK orthologs occur in non-conserved gene loci, containing bacterioferritin comigratory protein (in beta and gamma proteobacteria) and glycine cleavage system regulator (in gamma proteobacteria) encoding genes. The difference between gene clusters around BS-phoH and BS-ylaK orthologs clearly correlates with position of the corresponding protein in the phylogenetic tree. Co-localization and possible coexpression of BS-ylaK with alkyl hydroperoxide peroxidase-encoding gene, and its sporadic clustering with three other above-mentioned enzymes, points to its functional connection to the oxidative stress. Accordingly, BS-YlaK orthologs were found only in organisms with aerobic metabolism. EC-phoH positional coupling is also not conserved, even within genera of Enterobacteria (Fig. 2). In Escherichia coli it is colocalized with high affinity iron permease (Fig. 2).

Search for potential regulatory sites upstream of BS-ylaK orthologs in Actinobacteria

Search for possible regulatory sites upstream of genes encoding BS-YlaK orthologs revealed a conserved 18-bp pseudopalindrome in Mycobacterium tuberculosis, Mycobacterium bovis and Thermomonospora fusca. An iterative signal search procedure was applied to the genomic sequences of M. tuberculosis and T. fusca using the PSI-SITE program from the software package Genome Explorer [20, 21]. First, a recognition rule was generated using all three initially identified sites. Second, ten best sites were selected in each genome and used for generation of new genome-specific recognition rules. Third, these recognition rules were applied to the respective genomes and sites scoring at worst 10% below the highest possible value were selected (Tab. 3).

Table 3: Members of the BS-ylaK regulon with strong candidate sites.

All identified genes in M. tuberculosis and M. bovis seem to be orthologous. Among genes identified in T. fusca, only RTFU00810 and RTFU00852 have orthologs in M. tuberculosis and M. bovis. RMB00348 and RMT05630 (orthologs of RTFU00852) probably are not co-regulated with BS-ylaK orthologs.

Some of the identified genes seem to be co-transcribed with other genes. Thus, RMB00929 and RMT05592 are probably co-transcribed with RMB00928 and RMT04295 respectively, the latter encoding the alpha subunit of the fatty oxidation complex. RTFU02009 probably forms an operon with RTFU02062 that encodes the short-chain precursor of Acyl-CoA dehydrogenase.

Next, potential regulatory sites were identified in M. tuberculosis and T. fusca using the same recognition rule with a lower cutoff. Genes having candidate sites upstream of orthologous genes were selected. Six pairs of genes (in addition to the BS-ylaK orthologs) were identified (Tab. 4).

Table 4: Members of the BS-ylaK regulon with conserved candidate sites.

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Discussion

Phylogenetic analysis of PhoH homologs reveals three distinct groups of proteins. The most numerous group includes BS-PhoH and its orthologs. They are located in a conservative locus containing conserved metal dependent hydrolase of unknown function, genes encoding diacylglycerol kinase and GTP-binding protein ERA in most of Gram-positive bacteria, and apolipoprotein N-acyltransferase, tRNA-modification protein MIAB and divalent metal efflux related protein in Gram-negative organisms. This group includes EC-PhoL. The second group, specific for most, but not all aerobes, includes orthologs of BS-YlaK. Candidate regulatory sites were found upstream of BS-ylaK in M. tuberculosis, M. bovis and T. fusca. Similar regulatory sites were found upstream of several genes involved in the fatty-acid metabolism. The third group includes EC-phoH and its orthologs in four Enterobacteria. Conserved PHO-boxes of the gamma-proteobacteria type were found upstream of all these genes (data not shown).

We performed search for orthologs of BS-PhoH, EC-PhoH and BS-YlaK in clusters of orthologous groups of proteins (http://www.ncbi.nlm.nih.gov/COG). EC-PhoH, BS-PhoH and their orthologs consitute COG1702 (24 proteins from 21 organisms), while BS-YlaK and its orthologs constitute COG1875 (11 proteins from 10 organisms). Our phylogenetic tree contains all members of these two COGs except four proteins from Caulobacter crescentus and Escherichia coli O157:H7 as these two genomes were not sequenced at the moment of our study. On the other hand, COG1702 contains EC-PhoH from Escherichia coli and Escherichia coli O157:H7, which, according to our analysis, is not orthologous to other members of this family.

In aerobic bacteria, where existence of BS-PhoH homologs is not supported by the presence of other members of the putative functional cluster, such as in Cyanobacteria, Neisseria and Steptococci, PhoH is likely to be a functional homolog of BS-YlaK.

Thus we observe clustering of various acyl-transfer-related functions to BS-phoH homologs and to other genes of the phoH locus in bacterial genomes. Diacylglycerol kinase and apolipoprotein N-acyltransferase are directly coupled to BS-phoH homologs. The latter requires phosphatidylglycerol and cardiolipin as donors of acyl-groups [22, 23, 24]. We can speculate that coupling of diacylglycerol kinase to phoH, observed only in Gram-positive bacteria, reflects the specifics of diacylglycerol utilization in the cell wall biosynthesis and, probably, in informational processes in this group of organisms. The lipid modification of prolipoprotein involves the transfer of the diacylglyceryl moiety from phosphatidylglycerol with the concomitant formation of sn-glycerol 1-phosphate in Gram-negative bacteria [23]. In Gram-positive bacteria, diacylglycerol moiety of phosphatidylglycerol is freed during the sn-glycerol 1-phosphate transfer to prolipoprotein and during biosynthesis of lipoteichoic acids [24].

Taking into account the acyl-transfer-related positional coupling of BS-phoH we can also speculate that non-annotated members of the BS-phoH gene cluster may represent functions related to mono-, diacylglycerol and diacylglycerol pyrophosphate [25] metabolism. Several genes in the related pathways are still not identified in bacteria. The gene responsible for the turnover of the most important anchor of acyl-groups, undecaprenyl-pyrophosphate phosphatase, is also missing in all bacterial genomes.

The regulatory link of BS-ylaK to fatty acids/alkane oxidation and the coupling of BS-phoH to acyl-transfer related functions both point to possible involvement of PhoH orthologs in the fatty acid-related metabolism. BS-YlaK, EC-PhoH and BS-PhoH orthologs may represent alternative pathways in relation to oxidative conditions of cell growth. Indeed, BS-YlaK homologs are positionally linked to a number of oxidative stress related genes, which may be inducible under specific stress conditions.

BS-phoH positional coupling points to its involvement in putative metal dependent RNA-modification. Metals were shown to be involved in the transfer RNAs modification in E. coli [26]. Coupling of BS-phoH to miaB and corC and coupling of EC-phoH to iron permease can be important in this respect. Magnesium and cobalt modify function of acyl-transferase rybozymes [27, 28], and, in direct relation to our findings, are involved in regulation of the fatty-acid desaturation in Mycobacterium [29]. They are also known modulators of enzymatic functions of phospholipid related phosphatases.

Transport of divalent metals may be commonly associated in the bacterial world with the transport of non-organic phosphorus in the metal-phosphate form, as it is known for the phosphate inorganic transport system of E. coli [30]. The latter observation provides the final link from a metal-transport dependent function to the phosphate starvation. Known elements of regulation of metal homeostasis [31] can provide new data relevant for the functional meaning of BS-phoH/EC-phoL gene clusters and functions of proteins involved in some yet unknown metabolic/regulatory pathway.

One should mention, that a temperature-sensitive mutant of Salmonella typhimurium, defective in apolipoprotein N-acyltransferase, expressed increased sensitivity to divalent cations [32], which can be a confirmation of the functional meaning of the observed gene clustering. Modification of transfer RNAs is a sensitive cell response to oxidative conditions [33] which itself can be provoked by starvation, intensified fatty-acid oxidation, disturbances in iron, magnesium, cobalt homeostasis [1, 3, 26, 33].

Association of the YlaK function with fatty-acid beta-oxidation regulon in Mycobacteria is a new and important observation. There are very limited data on phosphate starvation and regulation of lipid metabolism in this group of bacteria. It was shown that phosphate starvation induces expression of several mycobacterial pathogenicity determinants [5, 6]. On the other hand, changes in the phospholipid and fatty acid composition of the cellular membrane can be directly involved in bacterial survival in macrophages and are subject of regulation by the oxidative and heat stresses [34, 35, 36, 37, 38, 39]. Moreover, fatty acids seem to be a crucial catabolic source during the second stage of the pathogenic growth of Mycobacterium tuberculosis [7]. The possibility of the relation of these changes to the metal homeostasis and RNA modification can bring more light to understanding of pathogenicity of these bacteria.

We hope that our results will bring new questions for further research and will be helpful in solving already existing problems concerning the role of the PhoH protein. We hope that functional links, proposed in this paper, will be experimentally verified.

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