Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the top three causes of opportunistic human infections. A major factor in its prominence as a pathogen is its intrinsic resistance to antibiotics and disinfectants. Here we report the complete sequence of P. aeruginosa strain PAO1. At 6.3 million base pairs, this is the largest bacterial genome sequenced, and the sequence provides insights into the basis of the versatility and intrinsic drug resistance of P. aeruginosa. Consistent with its larger genome size and environmental adaptability, P. aeruginosa contains the highest proportion of regulatory genes observed for a bacterial genome and a large number of genes involved in the catabolism, transport and efflux of organic compounds as well as four potential chemotaxis systems. We propose that the size and complexity of the P. aeruginosa genome reflect an evolutionary adaptation permitting it to thrive in diverse environments and resist the effects of a variety of antimicrobial substances.

Enzyme I(Ntr) from Escherichia coli. A novel enzyme of the phosphoenolpyruvate-dependent phosphotransferase system exhibiting strict specificity for its phosphoryl acceptor, NPr.

The phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) phosphorylates sugars and regulates cellular metabolic processes using a phosphoryl transfer chain including the general energy coupling proteins, Enzyme I (EI) and HPr as well as the sugar-specific Enzyme II complexes. Analysis of the Escherichia coli genome has revealed the presence of 5 paralogues of EI and 5 paralogues of HPr, most of unknown function. The ptsP gene encodes an EI paralogue designated Enzyme I(nitrogen) (EI(Ntr)), and two genes located in the rpoN operon encode PTS protein paralogues, NPr and IIA(Ntr), both implicated in the regulation of sigma(54) activity. The ptsP gene was polymerase chain reaction amplified from the E. coli chromosome and cloned into an overexpression vector allowing the overproduction and purification of EI(Ntr). EI(Ntr) was shown to phosphorylate NPr in vitro using either a [(32)P]PEP-dependent protein phosphorylation assay or a quantitative sugar phosphorylation assay. EI(Ntr) phosphorylated NPr but not HPr, whereas Enzyme I exhibited a strong preference for HPr. These two pairs of proteins (EI(Ntr)/NPr and EI/HPr) thus exhibit little cross-reactivity. Phosphoryl transfer from PEP to NPr catalyzed by EI(Ntr) has a pH optimum of 8.0, is dependent on Mg(2+), is stimulated by high ionic strength, and exhibits two K(m) values for NPr (2 and 10 microM) possibly because of negative cooperativity. The results suggest that E. coli possesses at least two distinct PTS phosphoryl transfer chains, EI(Ntr) --> NPr --> IIA(Ntr) and EI --> HPr --> IIA(sugar). Sequence comparisons allow prediction of residues likely to be important for specificity. This is the first report demonstrating specificity at the level of the energy coupling proteins of the PTS.

Novel phosphotransferase systems revealed by bacterial genome analysis: the complete repertoire of pts genes in Pseudomonas aeruginosa.

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.

Unique regulation of carbohydrate chemotaxis in Bacillus subtilis by the phosphoenolpyruvate-dependent phosphotransferase system and the methyl-accepting chemotaxis protein McpC.

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) plays a major role in the ability of Escherichia coli to migrate toward PTS carbohydrates. The present study establishes that chemotaxis toward PTS substrates in Bacillus subtilis is mediated by the PTS as well as by a methyl-accepting chemotaxis protein (MCP). As for E. coli, a B. subtilis ptsH null mutant is severely deficient in chemotaxis toward most PTS carbohydrates. Tethering analysis revealed that this mutant does respond normally to the stepwise addition of a PTS substrate (positive stimulus) but fails to respond normally to the stepwise removal of such a substrate (negative stimulus). An mcpC null mutant showed no response to the stepwise addition or removal of D-glucose or D-mannitol, both of which are PTS substrates. Therefore, in contrast to E. coli PTS carbohydrate chemotaxis, B. subtilis PTS carbohydrate chemotaxis is mediated by both MCPs and the PTS; the response to positive stimulus is primarily McpC mediated, while the duration or magnitude of the response to negative PTS carbohydrate stimulus is greatly influenced by components of the PTS and McpC. In the case of the PTS substrate D-glucose, the response to negative stimulus is also partially mediated by McpA. Finally, we show that B. subtilis EnzymeI-P has the ability to inhibit B. subtilis CheA autophosphorylation in vitro. We hypothesize that chemotaxis in the spatial gradient of the capillary assay may result from a combination of a transient increase in the intracellular concentration of EnzymeI-P and a decrease in the concentration of carbohydrate-associated McpC as the cell moves down the carbohydrate concentration gradient. Both events appear to contribute to inhibition of CheA activity that increases the tendency of the bacteria to tumble. In the case of D-glucose, a decrease in D-glucose-associated McpA may also contribute to the inhibition of CheA. This bias on the otherwise random walk allows net migration, or chemotaxis, to occur.

The three-dimensional structure of the nitrogen regulatory protein IIANtr from Escherichia coli.

The bacterial rpoN operon codes for sigma 54, which is the key sigma factor that, under nitrogen starvation conditions, activates the transcription of genes needed to assimilate ammonia and glutamate. The rpoN operon contains several other open reading frames that are cotranscribed with sigma 54. The product of one of these, the 17.9 kDa protein IIANtr, is homologous to IIA proteins of the phosphoenolpyruvate:sugar phosphotransferase (PTS) system. IIANtr influences the transcription of sigma 54-dependent genes through an unknown mechanism and may thereby provide a regulatory link between carbon and nitrogen metabolism. Here we describe the 2.35 A X-ray structure of Escherichia coli IIANtr. It is the first structure of a IIA enzyme from the fructose-mannitol family of the PTS. The enzyme displays a novel fold characterized by a central mixed parallel/anti-parallel beta-sheet surrounded by six alpha-helices. The active site His73 is situated in a shallow depression on the protein surface.

High-resolution solution structure of Bacillus subtilis IIAglc.

The high-resolution solution structure of the phosphocarrier protein IIAglc from Bacillus subtilis is determined using 3D and 4D heteronuclear NMR methods. B. subtilis IIAglc contains 162 amino acid residues and is one of the larger proteins for which high-resolution solution structure has been determined by NMR methods. The structures have been calculated from a total of 2,232 conformational constraints. Comparison with the X-ray crystal structure indicates that the overall fold is the same in solution and in crystalline environments, although some local structural differences are observed. These occur largely in turns and loops, and mostly correspond to regions with high-temperature factors in the crystal structure. The N-terminus of IIAglc is disordered in solution. The active site is located in a concave region of the protein surface. The histidine, which accepts the phosphoryl group (His 83), interacts with a neighboring histidine (His 68) and is surrounded by hydrophobic residues.

A novel protein kinase that controls carbon catabolite repression in bacteria.

HPr(Ser) kinase is the sensor in a multicomponent phosphorelay system that controls catabolite repression, sugar transport and carbon metabolism in gram-positive bacteria. Unlike most other protein kinases, it recognizes the tertiary structure in its target protein, HPr, a phosphocarrier protein of the bacterial phosphotransferase system and a transcriptional cofactor controlling the phenomenon of catabolite repression. We have identified the gene (ptsK) encoding this serine/threonine protein kinase and characterized the purified protein product. Orthologues of PtsK have been identified only in bacteria. These proteins constitute a novel family unrelated to other previously characterized protein phosphorylating enzymes. The Bacillus subtilis kinase is shown to be allosterically activated by metabolites such as fructose 1,6-bisphosphate and inhibited by inorganic phosphate. In contrast to wild-type B. subtilis, the ptsK mutant is insensitive to transcriptional regulation by catabolite repression. The reported results advance our understanding of phosphorylation-dependent carbon control mechanisms in Gram-positive bacteria.

6-phospho-alpha-D-glucosidase from Fusobacterium mortiferum: cloning, expression, and assignment to family 4 of the glycosylhydrolases.

The Fusobacterium mortiferum malH gene, encoding 6-phospho-alpha-glucosidase (maltose 6-phosphate hydrolase; EC 3.2.1.122), has been isolated, characterized, and expressed in Escherichia coli. The relative molecular weight of the polypeptide encoded by malH (441 residues; Mr of 49,718) was in agreement with the estimated value (approximately 49,000) obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the enzyme purified from F. mortiferum. The N-terminal sequence of the MalH protein obtained by Edman degradation corresponded to the first 32 amino acids deduced from the malH sequence. The enzyme produced by the strain carrying the cloned malH gene cleaved [U-14C]maltose 6-phosphate to glucose 6-phosphate (Glc6P) and glucose. The substrate analogs p-nitrophenyl-alpha-D-glucopyranoside 6-phosphate (pNP alphaGlc6P) and 4-methylumbelliferyl-alpha-D-glucopyranoside 6-phosphate (4MU alphaGlc6P) were hydrolyzed to yield Glc6P and the yellow p-nitrophenolate and fluorescent 4-methylumbelliferyl aglycons, respectively. The 6-phospho-alpha-glucosidase expressed in E. coli (like the enzyme purified from F. mortiferum) required Fe2+, Mn2+, Co2+, or Ni2+ for activity and was inhibited in air. Synthesis of maltose 6-phosphate hydrolase from the cloned malH gene in E. coli was modulated by addition of various sugars to the growth medium. Computer-based analyses of MalH and its homologs revealed that the phospho-alpha-glucosidase from F. mortiferum belongs to the seven-member family 4 of the glycosylhydrolase superfamily. The cloned 2.2-kb Sau3AI DNA fragment from F. mortiferum contained a second partial open reading frame of 83 residues (designated malB) that was located immediately upstream of malH. The high degree of sequence identity of MalB with IIB(Glc)-like proteins of the phosphoenol pyruvate dependent:sugar phosphotransferase system suggests participation of MalB in translocation of maltose and related alpha-glucosides in F. mortiferum.

Multiple phosphorylation of SacY, a Bacillus subtilis transcriptional antiterminator negatively controlled by the phosphotransferase system.

The Bacillus subtilis SacY transcriptional antiterminator is a regulator involved in sucrose-promoted induction of the sacB gene. SacY activity is negatively controlled by enzyme I and HPr, the general energy coupling proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and by SacX, a membranal protein homologous to SacP, the B. subtilis sucrose-specific PTS-permease. Previous studies suggested that the negative control exerted by the PTS on bacterial antiterminators of the SacY family involves phosphoenolpyruvate-dependent phosphorylation by the sugar-specific PTS-permeases. However, data reported herein show direct phosphorylation of SacY by HPr(His approximately P) with no requirement for SacX. Experiments were carried out to determine the phosphorylatable residues in SacY. In silico analyses of SacY and its homologues revealed the modular structure of these proteins as well as four conserved histidines within two homologous domains (here designated P1 and P2), present in 14 distinct mRNA- and DNA-binding bacterial transcriptional regulators. Single or multiple substitutions of these histidyl residues were introduced in SacY by site-directed mutagenesis, and their effects on phosphorylation and antitermination activity were examined. In vitro phosphorylation experiments showed that SacY was phosphorylated on three of the conserved histidines. Nevertheless, in vivo studies using cells bearing a sacB'-lacZ reporter fusion, as well as SacY mutants lacking the phosphorylatable histidyls, revealed that only His-99 is directly involved in regulation of SacY antitermination activity.

Modular multidomain phosphoryl transfer proteins of bacteria.

Recent phylogenetic and structural analyses of multidomain phosphoryl transfer proteins of bacteria have revealed that interdomain (but not intradomain) splicing and fusion, as well as domain duplication and deletion, have occurred frequently during evolution. These events have been found to be exceedingly rare in certain other protein families. Domain-shuffling events are illustrated by examples from the superfamilies of phosphoenolpyruvate-dependent sugar phosphotransferase systems, their transcriptional regulatory protein targets of phosphorylation, sensor autokinase/response regulator signal transduction systems, and permeases of the ATP-binding-cassette type.

The NMR side-chain assignments and solution structure of enzyme IIBcellobiose of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli.

The assignment of the side-chain NMR resonances and the determination of the three-dimensional solution structure of the C10S mutant of enzyme IIBcellobiose (IIBcel) of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli are presented. The side-chain resonances were assigned nearly completely using a variety of mostly heteronuclear NMR experiments, including HCCH-TOCSY, HCCH-COSY, and COCCH-TOCSY experiments as well as CBCACOHA, CBCA(CO)NH, and HBHA(CBCA)(CO)NH experiments. In order to obtain the three-dimensional structure, NOE data were collected from 15N-NOESY-HSQC, 13C-HSQC-NOESY, and 2D NOE experiments. The distance restraints derived from these NOE data were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In an iterative procedure, additional NOE assignments were derived from the calculated structures and new structures were calculated. The final set of structures, calculated with approximately 2000 unambiguous and ambiguous distance restraints, has an rms deviation of 1.1 A on C alpha atoms. IIBcel consists of a four stranded parallel beta-sheet, in the order 2134. The sheet is flanked with two and three alpha-helices on either side. Residue 10, a cysteine in the wild-type enzyme, which is phosphorylated during the catalytic cycle, is located at the end of the first beta-strand. A loop that is proposed to be involved in the binding of the phosphoryl-group follows the cysteine. The loop appears to be disordered in the unphosphorylated state.

The structure of an energy-coupling protein from bacteria, IIBcellobiose, reveals similarity to eukaryotic protein tyrosine phosphatases.

BACKGROUND: . The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates the energy-driven uptake of carbohydrates and their concomitant phosphorylation. In addition, the PTS is intimately involved in the regulation of a variety of metabolic and transcriptional processes in the bacterium. The multiprotein PTS consists of a membrane channel and at least four cytoplasmic proteins or protein domains that sequentially transfer a phosphoryl group from phosphoenolpyruvate to the transported carbohydrate. Determination of the three-dimensional structure of the IIB enzymes within the multiprotein complex would provide insights into the mechanisms by which they promote efficient transport by the membrane channel IIC protein and phosphorylate the transported carbohydrate on the inside of the cell. RESULTS: . The crystal structure of the IIB enzyme specific for cellobiose, IIBcellobiose (molecular weight 11.4 kDa), has been determined to a resolution of 1.8 and refined to an R factor of 18.7% (Rfree of 24. 1%). The enzyme consists of a single four-stranded parallel beta sheet flanked by helices on both sides. The phosphorylation site (Cys 10) is located at the C-terminal end of the first beta strand. No positively charged residues, which could assist in phosphoryl-transfer, can be found in or near the active site. The fold of IIBcellobiose is remarkably similar to that of the mammalian low molecular weight protein tyrosine phosphatases. CONCLUSIONS: . A comparison between IIBcellobiose and the structurally similar low molecular weight protein tyrosine phosphatases provides insight into the mechanism of the phosphoryltransfer reactions in which IIBcellobiose is involved. The differences in tertiary structure and active-site composition between IIBcellobiose and the glucose-specific IIBglucose give a structural explanation why the carbo-hydrate-specific components of different families cannot complement each other.

Characterization of a novel transporter family that includes multiple Escherichia coli gluconate transporters and their homologues.

The nucleotide sequences of seven Escherichia coli genes that encode members of the gluconate permease (GntP) family have recently become available. These genes include gntP, gntU, gntW, ORf449, dsdX, and ORFo454. The deduced amino acid sequences of all seven E. coli genes are homologous to the gntP gene products from Bacillus subtilis and B. licheniformis as well as two additional gene products from Haemophilus influenzae. These 11 proteins are not demonstrably homologous to members of the major facilitator superfamily or other recognized permease families. Four of the E. coli gluconate transporter genes have been cloned and shown to encode gluconate transporters with apparent affinities ranging from 6 to 212 microM. These studies serve to characterize a novel family of bacterial permeases.

Characterization of a family of bacterial response regulator aspartyl-phosphate (RAP) phosphatases.

We have characterized a novel family of response regulator aspartyl-phosphate (RAP) phosphatases found exclusively in gram-positive bacteria. The family consists of 15 members, 12 of which are from Bacillus subtilis. The N-terminal domains proved to be more highly conserved than the C-terminal domains, and a signature sequence for the family was derived from the former domains. Phylogenetic analyses revealed clustering patterns showing that all Bacillus proteins are closely related. Most of the Bacillus RAP phosphatase genes are followed by and are translationally coupled to small nonhomologous phosphatase regulator (phr) genes that encode exported peptides with regulatory functions. Most of the paralogous RAP phosphatases of B. subtilis may serve related functions in signal transduction systems. They appear to have arisen by relatively recent gene duplication events that occurred after the divergence of major groups within the gram-positive bacterial kingdom. We suggest that the N-terminal domains of the RAP phosphatases function in catalysis, whereas the C-terminal domains function in regulation.

Demonstration of protein-protein interaction specificity by NMR chemical shift mapping.

Chemical shift mapping is becoming a popular method for studying protein-protein interactions in solution. The technique is used to identify putative sites of interaction on a protein surface by detecting chemical shift perturbations in simple (1H, 15N)-HSQC NMR spectra of a uniformly labeled protein as a function of added (unlabeled) target protein. The high concentrations required for these experiments raise questions concerning the possibility for non-specific interactions being detected, thereby compromising the information obtained. We demonstrate here that the simple chemical shift mapping approach faithfully reproduces the known functional specificities among pairs of closely related proteins from the phosphoenolpyruvate:sugar phosphotransferase systems of Escherichia coli and Bacillus subtilis.

Novel phosphotransferase-encoding genes revealed by analysis of the Escherichia coli genome: a chimeric gene encoding an Enzyme I homologue that possesses a putative sensory transduction domain.

Two genes (ptsI and ptsA) that encode homologues of the energy coupling Enzyme I of the phosphoenolpyruvate-dependent sugar-transporting phosphotransferase system (PTS) have previously been identified on the Escherichia coli chromosome. We here report the presence of a third E. coli gene, designated ptsP, that encodes an Enzyme I homologue, here designated Enzyme INtr. Enzyme INtr possesses an N-terminal domain homologous to the N-terminal domains of NifA proteins [(127 amino acids (aa)] joined via two tandem flexible linkers to the C-terminal Enzyme I-like domain (578 aa). Structural features of the putative ptsP operon, including transcriptional regulatory signals, are characterized. We suggest that Enzyme INtr functions in transcriptional regulation of nitrogen-related operons together with previously described PTS proteins encoded within the rpoN operon. It may thereby provide a link between carbon and nitrogen assimilatory pathways.

Involvement of the central loop of the lactose permease of Escherichia coli in its allosteric regulation by the glucose-specific enzyme IIA of the phosphoenolpyruvate-dependent phosphotransferase system.

Allosteric regulation of several sugar transport systems such as those specific for lactose, maltose and melibiose in Escherichia coli (inducer exclusion) is mediated by the glucose-specific enzyme IIA (IIAGlc) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Deletion mutations in the cytoplasmic N and C termini of the lactose permease protein, LacY, and replacement of all cysteine residues in LacY with other residues did not prevent IIAGlc-mediated inhibition of lactose uptake, but several point and insertional mutations in the central cytoplasmic loop of this permease abolished transport regulation and IIAGlc binding. The results substantiate the conclusion that regulation of the lactose permease in E. coli by the PTS is mediated by a primary interaction of IIAGlc with the central cytoplasmic loop of the permease.