Novel Phosphotransferase Systems Revealed by Bacterial Genome Analysis: The Complete Repertoire of pts Genes in Pseudomonas aeruginosa

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1 J. Mol. Microbiol. Biotechnol. (1999) 1(2): JMMB Minireview pts Genes in P. aeruginosa 289 Novel Phosphotransferase Systems Revealed by Bacterial Genome Analysis: The Complete Repertoire of pts Genes in Pseudomonas aeruginosa Jonathan Reizer 1, Aiala Reizer 1, Michelle J. Lagrou 2, Kim R. Folger 2, C. Kendall Stover 2, and Milton H. Saier, Jr. 1 * 1 Department of Biology, University of California at San Diego, La Jolla, CA , USA 2 PathoGenesis Corp., 201 Elliott Ave., Seattle, WA 98119, USA Abstract We herein describe all genes encoding constituents of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in the 6Mbp genome of the opportunistic human pathogen, Pseudomonas aeruginosa. Only four gene clusters were found to encode identifiable PTS homologues. These genes clusters encode novel multidomain proteins, two complete sugar-specific PTS phosphoryl transfer chains for the metabolism of fructose and N- acetylglucosamine, and a complex regulatory system that may function to coordinate carbon and nitrogen metabolism. No previously characterized organism has been shown to exhibit such a novel and restricted complement of PTS proteins. recognition and transmembrane transport (Saier and Reizer, 1992). The domains of an Enzyme II complex may be detached or fused in variable but non-random orders, and different Enzyme II complexes therefore consist of one, two, three, or four polypeptide chains depending on the system and organism. Regardless, the phosphoryltransfer pathway from PEP to the incoming sugar includes five phosphoryltransfer reactions and four phosphoprotein intermediates. Enzyme I, HPr and Enzyme IIA (IIA) are each phosphorylated on a unique histidyl residue, whereas Enzyme IIB (IIB) is phosphorylated on a conserved cysteyl or histidyl residue, depending on the particular Enzyme II complex (Postma et al., 1993). Recently, we have undertaken the task of analyzing the nearly completely sequenced genome of Pseudomonas aeruginosa strain PA01, identifying all genes that code for proteins homologous to established constituents of the PTS in other bacteria. Although P. aeruginosa has the largest genome of any bacterium sequenced to date (approximately 6Mbp), it has the smallest percentage of pts genes of any bacterium with a fully sequenced genome that exhibits a functional PTS. Only four gene clusters encoding clearly identifiable PTS Introduction The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a complex enzyme system that couples sugar transport to sugar phosphorylation (group translocation) in both Gram-negative and Gram-positive bacteria (Postma et al., 1993). In addition to the primary roles of the PTS in sugar chemotaxis, transport and phosphorylation, it secondarily regulates various physiological processes by virtue of its function as a protein kinase system (Saier and Reizer, 1994). The unique features of this system include the use of phosphoenolpyruvate (PEP) as the phosphoryl donor for sugar phosphorylation and three essential catalytic entities, named Enzyme I, HPr (heat stable, histidine-phosphorylatable protein) and Enzyme II. Enzyme I and HPr are cytoplasmic, soluble, energy-coupling proteins, whereas Enzymes II are membranal proteins that serve as the sugar-specific permeases/kinases. A sugar-specific Enzyme II complex is comprised of two hydrophilic peripheral membrane proteins or domains (IIA and IIB), as well as one or two integral membrane proteins or domains (IIC and sometimes IID) that function in sugar Received September 23, 1999; revised October 3, 1999; accepted October 4, *For correspondence. msaier@ucsd.edu; Tel. (858) ; Fax. (858) Figure 1. Genetic map of the Pseudomonas aeruginosa fru operon (A), nag operon (B), rpon operon (C), and the ptsp gene cluster (D). The genetic map shows the proposed gene designations (in italic), the transcriptional orientation, and the functional assignments of the encoded proteins when known, based on homology to previously characterized proteins. The relative lengths of the orf s and intergenic regions shown are proportional to the numbers of bp Horizon Scientific Press

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3 290 Reizer et al. Figure 2. The three PTS phosphoryl transfer chains encoded within the genome of Pseudomonas aeruginosa. See text for protein designations and discussion. protein homologues are present in the P. aeruginosa genome, and these pts genes represent only about 0.1% of the total gene complement. This is to be contrasted with the E. coli genome which includes about five dozens genes encoding PTS proteins representing about 1.3% of the total gene complement (Reizer and Reizer, 1996, and unpublished data). Data reported herein reveal several unique features that have not been observed in PTS proteins of other organisms. These include hitherto unrecognized multidomain PTS proteins, two complete sugar-specific PTS phosphoryl transfer chains, and a potentially new metabolic pathway for PTS-dependent utilization of N-acetylglucosamine. Panel A of Figure 1 shows the fru operon of P. aeruginosa encoding enzymes involved in fructose (Fru) catabolism; Panel B shows the nag operon encoding enzymes of N-acetylglucosamine (NAG) metabolism; Panel C shows the rpon operon encoding the sigma factor, σ N, (σ 54 ) and two putative PTS regulatory proteins, IIA Ntr and NPr, and Panel D shows the gene cluster that includes the ptsp gene encoding Enzyme I Ntr. Although there are few PTS-related genes in P. aeruginosa, the genome encodes three pairs of the PTS energy coupling proteins, Enzyme I and HPr, each providing a distinct function (see Figure 2). The fru operon The fru operon of P. aeruginosa (Figure 1A) resembles the fru operon of Rhodobacter capsulatus (Wu et al., 1990). It includes three genes encoding proteins similar to those of R. capsulatus and in the same gene order. These genes are: (1) frub encoding an orthologue of the R. capsulatus IIA Fru /FPr/Enzyme I-multiphosphoryl transfer protein (MTP) (SwissProt identifier (sp) P23388; 41% identity, 92 Standard Deviations (SD)); (2) fruk encoding an orthologue of the R. capsulatus fructose 1-phosphate kinase (sp P23386; 40% identity, 36 SD), and (3) frua encoding an orthologue of the R. capsulatus fructose-1-phosphate-forming fructose-specific Enzyme IIB'BC (sp P23387; 55% identity, 79 SD). It is noteworthy that unlike the homologous E. coli protein, the N-terminal IIB' domain and the middle IIB domain of the P. aeruginosa Enzyme II Fru both contain catalytic cysteine residues that serve as sites of phosphorylation (Reizer et al., 1995). Nevertheless, only the middle IIB domain of the P. aeruginosa protein contains the three residues AlaHisThr which are conserved four residues downstream of this cysteine in all catalytic IIB Fru domains. We therefore suggest that in P. aeruginosa, the IIB' domain probably plays a regulatory role as has been demonstrated in E. coli (Charbit et al., 1996). The fru operon of P. aeruginosa differs from that in E. coli in two respects. First, the P. aeruginosa fructose repressor, FruR, an orthologue of the E. coli FruR protein (sp P21168; 45% identity; 83 SD) is encoded by a gene upstream of and divergently transcribed from frub while the E. coli frur maps distantly from the fru operon (Berlyn, 1998). The map position of this gene suggests that in contrast to the E. coli FruR which is a pleiotropic transcriptional regulator controlling the flux of carbon through metabolic pathways (Saier and Ramseier, 1996), the P. aeruginosa protein serves as a specific regulator of fru operon expression. This is likely to have been the primordial function of the E. coli protein. Second, a novel gene, FruB, encodes an orthologue of the MTP of Rhodobacter capsulatus (sp P23388; 41% identity, 92 SD) (Figure 1A). Due to the location of this gene and because an additional Enzyme I-like protein domain is encoded within the nag operon (see below), we suggest that frub encodes a fructose-inducible Enzyme I that functions exclusively in fructose catabolism. The nag Operon The putative nag operon of P. aeruginosa includes five genes (Figure 1B). The first two of these genes potentially encode a complete N-acetylglucosamine-specific PTS. The first gene, nage, encodes an Enzyme II with an unprecedented domain organization. It contains an N- terminal IIC domain fused to twin C-terminal IIB domains, IIB and IIB'. Only IIB (not IIB') has the active site cysteine residue. NagE of P. aeruginosa exhibits high similarity to the NagE protein of E. coli (sp P09323; 49% identity, 103 SD). The second nag operon gene, nagf, encodes a unique multidomain protein which we designate the NAGtriphosphoryl transfer protein, TTP Nag. It includes three PTS phosphoryl transfer domains homologous to (1) the IIA Nag protein domain of the E. coli Enzyme II Nag (P45604; 33 SD), (2) HPr (17-23 SD) and (3) Enzyme I (72-89 SD), respectively, that occur in that order. While triphosphoryl transfer proteins of the PTS have been identified in other organisms (Wu et al., 1990; for review see Reizer and Saier, 1997), the P. aeruginosa TTP Nag is the first recognized protein of this class that possesses a IIA Glc -like domain. The third gene, nags, encodes a protein that is homologous to the C-terminal isomerase domain of E. coli GlmS, the glutamine-fructose-6-phosphate amidotransferase (glucosamine-6-phosphate synthase; sp P17169; 30% identity; 31 SD). Interestingly, P. aeruginosa NagS (340 residues) is of about the same length as and is homologous throughout its length to AgaS (384 residues; sp P42907; 28% identity; 8 SD) encoded within the putative N-acetylgalactosamine (aga) catabolic operon of E. coli (Reizer et al., 1996a). A tandem pair of SIS (sugar isomerase) domains (each approximately 130 residues) is present in NagS and in AgaS. SIS domains are found in a variety of isomerases and regulatory proteins that bind sugar-phosphates (Bateman, 1999). SIS-containing proteins include members of the glucosamine-6-phosphate

4 pts Genes in P. aeruginosa 291 synthase family (GlmS, NodM and GfaT), the characterized phosphoheptose isomerase of E. coli (LpcA or GmhA), the transcriptional regulator (RpiR) of the E. coli ribose 5- phosphate isomerase (RpiB), regulators (GckR) of human, rat and Xenopus glucokinases, and the repressor (HexR) of the hex regulon of P. aeruginosa. The fourth gene in the P. aeruginosa nag operon, naga, encodes an orthologue of the N-acetyl glucosamine-6-phosphate deacetylase (NagA) of E. coli (sp P15300; 32% identity, 34 SD). No orthologue of the transcriptional regulator of the E. coli nag operon, NagC, was found in or near the nag operon of P. aeruginosa. Instead, a putative transcriptional regulator encoded by the fifth gene in this operon, nagr, may control nag operon expression. NagR is a member of the GntR family of transcriptional regulators which include the trehalose repressor, TreR of Bacillus subtilis (sp P39796; 28% identify, 26 SD), and the fatty acyl responsive regulator, FarR of E. coli (sp P13669; 30% identity; 33 SD). It is noteworthy that no orthologue of the E. coli glucosamine-6-phosphate deaminating isomerase (NagB) was found in the nag operon of P. aeruginosa although this organism utilizes NAG as the sole source of carbon and energy (unpublished observation). The rpon Operon The rpon operon of P. aeruginosa (Figure 1C) resembles the corresponding operon in E. col in all essential respects (Powell et al., 1995). Thus, the rpon genes in the two organisms are followed by four homologous genes of similar size and with the same gene order. Two of these genes encode PTS proteins: ptsn encodes IIA Ntr, and ptso encodes NPr (Powell et al., 1995). Upstream of both rpon operons are three genes which in E. coli are designated yhbg, yhbn and yrbk. YhbG is a putative cytoplasmic ATPhydrolyzing protein, but YhbN and YrbK possess N-terminal putative transmembrane signal sequences. Well conserved homologues of these proteins have been identified only in H. influenzae (E. Dassa, M. Hofnung, I.T. Paulsen, and M.H. Saier, Jr., unpublished observations). The ptsp Gene Cluster The ptsp gene of P. aeruginosa (Figure 1D) encodes a protein that is most similar to the Enzyme I Ntr proteins of Azotobacter vinelandii (NCBI identifier gi ; 87% identity; 236 SD) and E. coli (sp P37177; 43% identity, 121 SD). All three enzymes are believed to function in regulation rather than sugar translocation, and they possess N-terminal domains homologous to the N-terminal sensing domains of NifA proteins (Reizer et al., 1996b). Recent in silico analyses have revealed that the N-terminal domains of the NifA and PtsP proteins resemble GAF domains (Aravind and Pontig, 1997). GAF domains are found in functionally diverse proteins such as cgmp phosphodiesterases, adenylate cyclases, phytochromes, sensory transduction histidine kinases, and transcriptional regulatory proteins. While the precise functions of GAF domains are not known, finding these modules in proteins that respond to signaling ligands reaffirms a possible sensory transduction function (Reizer et al., 1996b). Most surprisingly, a recent elegant study has demonstrated that a clinical isolate of P. aeruginosa (strain PA14) bearing a TnphoA insertion in the ptsp gene exhibits significantly reduced virulence when compared to the isogenic parent strain in model systems that include Caenorhabditis elegans, Arabidopsis thaliana and mice (Tan et al., 1999). The mode of action of ptsp in this capacity has yet to be determined. ptsp of P. aeruginosa is preceded by a gene (orf167) encoding a homologue of YgdP of E. coli (sp Q46930; 65% identity; 64 SD), a protein of 176 residues that is encoded in the E. coli ptsp operon (Reizer et al., 1996b). While the functions of ORF167 of P. aeruginosa and YgdP of E. coli are not known, it is interesting to note that the two proteins possess core MutT-like domains, and they exhibit high degrees of sequence similarity to the InvA invasion protein of Bartonella bacilliformis (sp P35640; 34-39% identity; SD) and diadenosine 5',5'''-P1,P4-tetraphosphate hydrolases of plants (NCBI identifiers gi and gi ; Maksel et al., 1998; Churin et al., 1998). We anticipate that ORF167 is a pyrophosphate hydrolase. Conclusions and Perspectives The reported in silico analyses provide guidelines for future biochemical, physiological, and molecular genetic studies: 1) frub encodes a fructose-inducible Enzyme I domain. Since TTP Nag contains an Enzyme I-like domain, and because previously characterized IIA Glc -like proteins or domains do not serve as phosphoryl donors to IIB Fru, it is likely that P. aeruginosa mutants impaired in the Enzyme I domain of MTP will be specifically impaired in fructose utilization. 2) Due to the position of the frur gene adjacent to the P. aeruginosa fru operon, we anticipate that this protein functions exclusively in fru operon regulation rather than as a global regulator (Saier et al., 1996). 3) Because no orthologue of the E. coli glucosamine-6- phosphate deaminating isomerase (NagB) was found in the nag operon of P. aeruginosa, it is possible that the primary function of the proteins encoded within the nag operon is biosynthesis of UDP-N-acetylglucosamine, the first dedicated precursor of peptidoglycan and lipopolysaccharide synthesis (Raetz, 1996; van Heijenoort, 1996). Alternatively, catabolism of glucosamine-6- phosphate may proceed by conversion of this phosphorylated sugar to glucosaminate-6-phosphate which, in turn, may be converted to ammonia and the Entner-Doudoroff intermediate, 2-keto-3-deoxy-Dgluconate-6-phosphate. This proposal stems from the elucidated route of glucosamine metabolism in Pseudomonas fluorescens that involves conversion of intracellular glucosamine to glucosaminate by glucose dehydrogenase-catalyzed oxidation and subsequent conversion of glucosaminate to 2-keto-3-deoxy-Dgluconate by glucosaminate dehydratase (Iwamoto and Imanaga, 1991). Indeed, a gene, pgl (NCBI identifier gi ), present between the zwf and eda genes of P. aeruginosa, was shown to encode an orthologue (243 residues) of the E. coli NagB. While the protein encoded by pgl, 6-phosphogluconolactonase, functions primarily in the conversion of 6-phospho-D-glucono-1,5-lactone to 6- phospho-d-gluconate, it is possible that it can secondarily catalyze deamination of glucosaminate-6-phosphate, generated from glucosamine-6-phosphate by glucose-6- phosphate dehydrogenase-catalyzed oxidation. In spite of

5 292 Reizer et al. this consideration we cannot eliminate the possibility that P. aeruginosa possesses a non-orthologous but functionally analogous protein of E. coli NagB. 4) The synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine, and the degradation of glucosamine-6-phosphate to fructose-6-phosphate and NH 3, catalyzed in E. coli respectively by the glms and nagb gene products, represent a potential futile cycle. Simultaneous function of both enzymes can lead to the dissipation of ATP due to the continuous need for glutamine which is synthesized from glutamate and NH 3 in the glutamine synthetase reaction (Vogler et al., 1989). Since NagB could not be found in the P. aeruginosa nag operon, it is possible that, in contrast to E. coli NagC, the P. aeruginosa NagR does not play a role in regulation of the distant glmus operon concerned with amino sugar biosynthesis (Plumbridge, 1995). 5) Previous studies have indicated that camp does not mediate catabolite repression in P. aeruginosa (Siegel et al., 1977; Phillips and Mulfinger, 1981). This suggestion is consistent with differences between P. aeruginosa and enteric bacteria in (1) the mechanism of glucose transport (Lessie and Phibbs, 1984) and (2) the nature of the PTS proteins encoded within the P. aeruginosa genome. 6) Recently it has been shown that Enzyme I Ntr of E. coli functions in the phosphorylation of NPr (but not HPr) whereas Enzyme I phosphorylates HPr (but not NPr) (Powell et al., 1995; Rabus et al., 1999). We therefore suggest that, as in E. coli, Enzyme I Ntr, NPr and IIA Ntr of P. aeruginosa exclusively play a regulatory role and do not function in sugar transport. The recent evidence regarding a potential regulatory role for Enzyme I Ntr in poly-ßhydroxybutyrate metabolism in Azotobacter vinelandii (Segura and Espin, 1998) should provide a guide for studies aimed at determining the function of this protein in P. aeruginosa. The data presented clearly indicate that regulatory constituents of the P. aeruginosa PTS (Figure 1C and 1D) resemble those in E. coli but that the sugar phosphorylating constituents of this system exhibit different features (Figs. 1A and 1B). Of particular note is the fact that the P. aeruginosa genome encodes two complete sugarspecific PTS phosphoryl transfer chains as well as a regulatory phosphoryl transfer cascade as shown in Figure 2. No other organism to date has been shown to exhibit such a complement of PTS proteins. Many Gram-negative and Gram-positive bacteria, including Gram-negative species of Rhodobacter, Rhodospirillum, Xanthomonas, Pseudomonas, Alcaligenes, and Azospirillum, have been shown to utilize fructose (but no other sugar) via the PTS (Saier et al., 1971; Conrad and Schlegel, 1977; Sawyer et al, 1977; Durham and Phibbs, 1982; Goebel and Krieg, 1984; Gupta and Ghosh, 1984; Lessie and Phibbs, 1984; de Crécy-Legard et al., 1991; Romano and Saier, 1992). In all of these bacteria, the PTS phosphorylates fructose on the 1-hydroxyl, and fructose-1-p is converted to fructose- 1,6-bisphosphate (FBP), the key glycolytic intermediate, in a reaction catalyzed by fructose-1-p kinase. Pseudomonads lack 6-phosphofructokinase (PFK), a key enzyme for the entry of sugar-6-phosphates into the Embden-Meyerhof-Parnas (EMP) glycolytic pathway (Lessie and Phibbs, 1984). Consequently, all other sugars, phosphorylated on the 6-position, can not be converted to FBP and must be metabolized through alternative pathways such as the Entner-Doudoroff glycolytic pathway and/or the pentose-phosphate pathway. P. aeruginosa is the first organism that theoretically might use the EMP pathway specifically for fructose catabolism to have its genome sequenced. The availability of its complete gene complement allows recognition of the genetic basis for this proposal. Thus, all of the enzymes required for the metabolism of fructose via the EMP pathway were found, but the absence of PFK clearly suggests that other pathways must be used for the metabolism of other sugars. Previously published results, however, suggest that the major pathway of fructose dissimilation in P. aeruginosa involves (1) transport and phosphorylation to fructose-1-p via the PTS, (2) fructose 1-P kinase-mediated conversion to fructose 1,6-bis phosphate (FBP), (3) subsequent cleavage of phosphate from FBP to form fructose 6-P, and (4) metabolism via the Entner-Doudoroff pathway (Lessie and Phibbs, 1984). This conclusion is consistent with our unpublished observation that fructose cannot be utilized by P. aeruginosa under anaerobic conditions. It remains unexplained why this organism (and possibly other organisms) use the PTS for the energetically expensive uptake of fructose. The minimal complement of sugar-specific PTSencoding genes in P. aeruginosa correlates with (1) the lack of PFK, (2) the aerobic lifestyle of this bacterium, and (3) the use of the EMP pathway mainly for gluconeogenic purposes. Other aerobic bacteria such as Rickettsia prowazekii (Andersson et al., 1998) and Mycobacterium tuberculosis (Cole et al., 1998) do not encode enzymes of the PTS within their genomes. It is surprising that the PTS Enzyme II Man, which in E. coli takes up fructose to yield fructose-6-p, is not present in P. aeruginosa since its use would have been more economical from an energetic standpoint. P. aeruginosa encodes a complete Nag PTS as well as all of the expected Nag catabolic enzymes except the glucosamine-6-phosphate deaminating isomerase that converts glucosamine-6-p to fructose-6-p in other bacteria. The absence of this isomerase correlates with the absence of PFK. While Nag can be utilized for biosynthetic purposes, i.e., for peptidoglycan and lipopolysaccharide synthesis, its efficient metabolism as a sole source of carbon and energy may require oxidative phosphorylation in the presence of an electron acceptor. 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