Periplasmic Transport Systems Research Articles

Molecular characterization of the interaction of sialic acid with the periplasmic binding protein from Haemophilus ducreyi

The primary role of bacterial periplasmic binding proteins is sequestration of essential metabolites present at a low concentration in the periplasm and making them available for active transporters that transfer these ligands into the bacterial cell. The periplasmic binding proteins (SiaPs) from the tripartite ATP-independent periplasmic (TRAP) transport system that transports mammalian host-derived sialic acids have been well studied from different pathogenic bacteria, including Haemophilus influenzae, Fusobacterium nucleatum, Pasteurella multocida, and Vibrio cholerae SiaPs bind the sialic acid N-acetylneuraminic acid (Neu5Ac) with nanomolar affinity by forming electrostatic and hydrogen-bonding interactions. Here, we report the crystal structure of a periplasmic binding protein (SatA) of the ATP-binding cassette (ABC) transport system from the pathogenic bacterium Haemophilus ducreyi The structure of Hd-SatA in the native form and sialic acid-bound forms (with Neu5Ac and N-glycolylneuraminic acid (Neu5Gc)), determined to 2.2, 1.5, and 2.5 Å resolutions, respectively, revealed a ligand-binding site that is very different from those of the SiaPs of the TRAP transport system. A structural comparison along with thermodynamic studies suggested that similar affinities are achieved in the two classes of proteins through distinct mechanisms, one enthalpically driven and the other entropically driven. In summary, our structural and thermodynamic characterization of Hd-SatA reveals that it binds sialic acids with nanomolar affinity and that this binding is an entropically driven process. This information is important for future structure-based drug design against this pathogen and related bacteria.

Carbohydrate uptake in Advenella mimigardefordensis strain DPN7T is mediated by periplasmic sugar oxidation and a TRAP-transport system.

In this study, we investigated an SBP (DctPAm ) of a tripartite ATP-independent periplasmic transport system (TRAP) in Advenella mimigardefordensis strain DPN7T . Deletion of dctPAm as well as of the two transmembrane compounds of the tripartite transporter, dctQ and dctM, impaired growth of A. mimigardefordensis strain DPN7T , if cultivated on mineral salt medium supplemented with d-glucose, d-galactose, l-arabinose, d-fucose, d-xylose or d-gluconic acid, respectively. The wild type phenotype was restored during complementation studies of A. mimigardefordensis ΔdctPAm using the broad host vector pBBR1MCS-5::dctPAm . Furthermore, an uptake assay with radiolabeled [14 C(U)]-d-glucose clearly showed that the deletion of dctPAm , dctQ and dctM, respectively, disabled the uptake of this aldoses in cells of either mutant strain. Determination of KD performing thermal shift assays showed a shift in the melting temperature of DctPAm in the presence of d-gluconic acid (KD 11.76 ± 1.3 µM) and the corresponding aldonic acids to the above-mentioned carbohydrates d-galactonate (KD 10.72 ± 1.4 µM), d-fuconic acid (KD 13.50 ± 1.6 µM) and d-xylonic acid (KD 8.44 ± 1.0 µM). The sugar (glucose) dehydrogenase activity (E.C.1.1.5.2) in the membrane fraction was shown for all relevant sugars, proving oxidation of the molecules in the periplasm, prior to transport.

Identification of the Gene Cluster for the Anaerobic Degradation of 3,5-Dihydroxybenzoate (α-Resorcylate) in Thauera aromatica Strain AR-1.

Thauera aromatica strain AR-1 degrades 3,5-dihydroxybenzoate (3,5-DHB) with nitrate as an electron acceptor. Previous biochemical studies have shown that this strain converts 3,5-DHB to hydroxyhydroquinone (1,2,4-trihydroxybenzene) through water-dependent hydroxylation of the aromatic ring and subsequent decarboxylation, and they suggest a pathway homologous to that described for the anaerobic degradation of 1,3-dihydroxybenzene (resorcinol) by Azoarcus anaerobius. Southern hybridization of a T. aromatica strain AR-1 gene library identified a 25-kb chromosome region based on its homology with A. anaerobius main pathway genes. Sequence analysis defined 20 open reading frames. Knockout mutations of the most relevant genes in the pathway were generated by reverse genetics. Physiological and biochemical analyses identified the genes for the three main steps in the pathway which were homologous to those described in A. anaerobius and suggested the function of several auxiliary genes possibly involved in enzyme maturation and intermediate stabilization. However, T. aromatica strain AR-1 had an additional enzyme to metabolize hydroxyhydroquinone, a putative cytoplasmic quinone oxidoreductase. In addition, a specific tripartite ATP-independent periplasmic (TRAP) transport system was required for efficient growth on 3,5-DHB. Reverse transcription-PCR (RT-PCR) analysis showed that the pathway genes were organized in five 3,5-DHB-inducible operons, three of which have been shown to be under the control of a single LysR-type transcriptional regulator, DbdR. Despite sequence homology, the genetic organizations of the clusters in T. aromatica strain AR-1 and A. anaerobius differed substantially.

Sulfur-34S Stable Isotope Labeling of Amino Acids for Quantification (SULAQ34) of Proteomic Changes in Pseudomonas fluorescens during Naphthalene Degradation

The relative quantification of proteins is one of the major techniques used to elucidate physiological reactions. Because it allows one to avoid artifacts due to chemical labeling, the metabolic introduction of heavy isotopes into proteins and peptides is the preferred method for relative quantification. For eukaryotic cells, stable isotope labeling by amino acids in cell culture (SILAC) has become the gold standard and can be readily applied in a vast number of scenarios. In the microbial realm, with its highly versatile metabolic capabilities, SILAC is often not feasible, and the use of other (13)C or (15)N labeled substrates might not be practical. Here, the incorporation of heavy sulfur isotopes is shown to be a useful alternative. We introduce (34)S stable isotope labeling of amino acids for quantification and the corresponding tools required for spectra extraction and disintegration of the isotopic overlaps caused by the small mass shift. As proof of principle, we investigated the proteomic changes related to naphthalene degradation in P. fluorescens ATCC 17483 and uncovered a specific oxidative-stress-like response.

Biosynthesis of Cysteine.

The synthesis of L-cysteine from inorganic sulfur is the predominant mechanism by which reduced sulfur is incorporated into organic compounds. L-cysteineis used for protein and glutathione synthesis and serves as the primary source of reduced sulfur in L-methionine, lipoic acid, thiamin, coenzyme A (CoA), molybdopterin, and other organic molecules. Sulfate and thiosulfate uptake in E. coli and serovar Typhimurium are achieved through a single periplasmic transport system that utilizes two different but similar periplasmic binding proteins. Kinetic studies indicate that selenate and selenite share a single transporter with sulfate, but molybdate also has a separate transport system. During aerobic growth, the reduction of sulfite to sulfide is catalyzed by NADPH-sulfite reductase (SiR), and serovar Typhimurium mutants lacking this enzyme accumulate sulfite from sulfate, implying that sulfite is a normal intermediate in assimilatory sulfate reduction. L-Cysteine biosynthesis in serovar Typhimurium and E. coli ceases almost entirely when cells are grown on L-cysteine or L-cystine, owing to a combination of end product inhibition of serine transacetylase by L-cysteine and a gene regulatory system known as the cysteine regulon, wherein genes for sulfate assimilation and alkanesulfonate utilization are expressed only when sulfur is limiting. In vitro studies with the cysJIH, cysK, and cysP promoters have confirmed that they are inefficient at forming transcription initiation complexes without CysB and N-acetyl-L-serine. Activation of the tauA and ssuE promoters requires Cbl. It has been proposed that the three serovar Typhimurium anaerobic reductases for sulfite, thiosulfate, and tetrathionate may function primarily in anaerobic respiration.

Crystal Structures of two Bordetella pertussis Periplasmic Receptors Contribute to Defining a Novel Pyroglutamic Acid Binding DctP Subfamily

Gram-negative bacteria have developed several different transport systems for solute uptake. One of these, the tripartite ATP independent periplasmic transport system (TRAP-T), makes use of an extracytoplasmic solute receptor (ESR) which captures specific solutes with high affinity and transfers them to their partner permease complex located in the bacterial inner membrane. We hereby report the structures of DctP6 and DctP7, two such ESRs from Bordetella pertussis. These two proteins display a high degree of sequence and structural similarity and possess the “Venus flytrap” fold characteristic of ESRs, comprising two globular α/β domains hinged together to form a ligand binding cleft. DctP6 and DctP7 both show a closed conformation due to the presence of one pyroglutamic acid molecule bound by highly conserved residues in their respective ligand binding sites. BLAST analyses have revealed that the DctP6 and DctP7 residues involved in ligand binding are strictly present in a number of predicted TRAP-T ESRs from other bacteria. In most cases, the genes encoding these TRAP-T systems are located in the vicinity of a gene coding for a pyroglutamic acid metabolising enzyme. Both the high degree of conservation of these ligand binding residues and the genomic context of these TRAP-T-coding operons in a number of bacterial species, suggest that DctP6 and DctP7 constitute the prototypes of a novel TRAP-T DctP subfamily involved in pyroglutamic acid transport.

The response of Mannheimia haemolytica to iron limitation: Implications for the acquisition of iron in the bovine lung

Mannheimia haemolytica is the major causative agent of shipping fever, a severe pneumonia in cattle causing high morbidity and mortality. A prerequisite of successful lung colonization by M. haemolytica is the necessity to adapt to the paucity of iron. The lack of genome information has precluded an assessment of the genetic repertoire available to M. haemolytica to adapt to low iron environments. To close this knowledge-gap, we have determined 90% of a virulent M. haemolytica serotype A1 genome sequence and produced a microarray in order to study gene expression under iron-limiting growth for 15, 30 and 60 min. M. haemolytica responded to iron limitation by the up-regulation of transcripts coding for receptors and ABC-type transporters of transferrin, haemoglobin, haem and siderophores. Real time PCR analysis of lung tissue from Mannheimia-infected calves demonstrated the in vivo transcription of two potential haemoglobin receptors, hmbR1 and hmbR2. The relative hmbR1 and hmbR2 transcript levels in the infected lung tissue were comparable to the induced levels observed under iron-limiting growth, demonstrating in vivo induction of receptor transcription in the context of an infection. When the iron response of M. haemolytica was compared to the iron response of Pasteurella multocida, another pathogen colonizing the bovine lung, only few homologous genes were induced in both organisms. These included the haemoglobin receptor hmbR2 and the periplasmic transport systems yfeABCD and fbpABC. The comparative analysis suggests that the two pathogens use different strategies to adapt to the iron-limiting environment in the bovine host.

A Periplasmic Glutamate/Aspartate Binding Protein from Shigella flexneri: Gene Cloning, Over-Expression, Purification and Preliminary Crystallographic Studies of the Recombinant Protein

Periplasmic substrate binding proteins (PSBPs) are essential components of the bacterial periplasmic transport system, which transports a wide variety of nutrients from the periplasmic space to the cytoplasm. The glutamate/aspartate binding protein SfGlu/AspBP is a unique PSBP consisting of 279 amino acid residues. The SfGlu/AspBP gene was cloned, over-expressed, and purified by immobilized metal ion affinity chromatography and size-exclusion chromatography. The recombinant protein SfGlu/AspBP has been crystallized by the hanging-drop vapor-diffusion method and its X-ray diffraction data were collected at an atomic resolution of 1.15 A. The crystals belong to the space group P2(1) with unit cell parameters: a = 48.41 A, b = 68.18 A, c = 80.21 A and beta = 98.78 degrees. There are two molecules per asymmetric unit.

Structure of the Clade 1 catalase, CatF of Pseudomonas syringae, at 1.8 A resolution.

Catalase CatF of Pseudomonas syringae has been identified phylogenetically as a clade 1 catalase, closely related to plant catalases, a group from which no structure has been determined. The structure of CatF has been refined at 1.8 A resolution by using X-ray synchrotron data collected from a crystal flash-cooled with liquid nitrogen. The crystallographic agreement factors R and R(free) are, respectively, 18.3% and 24.0%. The asymmetric unit of the crystal contains a whole molecule that shows accurate 222-point group symmetry. The crystallized enzyme is a homotetramer of subunits with 484 residues, some 26 residues shorter than predicted from the DNA sequence. Mass spectrometry analysis confirmed the absence of 26 N-terminal residues, possibly removed by a periplasmic transport system. The core structure of the CatF subunit was closely related to seven other catalases with root-mean-square deviations (RMSDs) of 368 core Calpha atoms of 0.99-1.30 A. The heme component of CatF is heme b in the same orientation that is found in Escherichia coli hydroperoxidase II, an orientation that is flipped 180 degrees with respect the orientation of the heme in bovine liver catalase. NADPH is not found in the structure of CatF because key residues required for nucleotide binding are missing; 2129 water molecules were refined into the model. Water occupancy in the main or perpendicular channel of CatF varied among the four subunits from two to five in the region between the heme and the conserved Asp150. A comparison of the water occupancy in this region with the same region in other catalases reveals significant differences among the catalases.

Overexpression, purification, and characterization of the periplasmic space thiamin-binding protein of the thiamin traffic ATPase in Escherichia coli

Thiamin (Vitamin B 1) transport in Escherichia coli occurs by the superfamily of traffic ATPases in which the initial receptor is the periplasmic binding protein. We have cloned the periplasmic thiamin-binding protein (TBP) of the E. coli periplasmic thiamin transport system and purified the overexpressed protein to apparent homogeneity. A subsequent biochemical characterization demonstrates that TBP is a 34.205 kDa monomer. TBP also contains one tightly bound thiamin species [thiamin, thiamin monophosphate (TMP), or thiamin diphosphate (TDP)] per monomer ( K D =0.8 μM ) when isolated under conditions that would remove any loosely bound ligands. We also demonstrate that thiamin is readily exchangeable in the presence of exogenous thiamin with a k off =0.12 s −1 . The biochemical characteristics of the overexpressed, plasmid-derived TBP are indistinguishable from those determined for endogenous TBP purified from E. coli. The overexpression and purification of TBP that we present here allows the rapid isolation of large amounts of pure protein that are required for further mechanistic and structural studies and demonstrates a vast improvement over previously reported purifications.

Identification of the Vibrio cholerae enterobactin receptors VctA and IrgA: IrgA is not required for virulence.

The gram-negative enteric pathogen Vibrio cholerae requires iron for growth. V. cholerae has multiple iron acquisition systems, including utilization of heme and hemoglobin, synthesis and transport of the catechol siderophore vibriobactin, and transport of several siderophores that it does not itself make. One siderophore that V. cholerae transports, but does not make, is enterobactin. Enterobactin transport requires TonB and is independent of the vibriobactin receptor ViuA. In this study, two candidate enterobactin receptor genes, irgA (VC0475) and vctA (VCA0232), were identified by analysis of the V. cholerae genomic sequence. A single mutation in either of these genes did not significantly impair enterobactin utilization, but a strain defective in both genes did not use enterobactin. When either irgA or vctA was supplied on a plasmid, the ability of the irgA vctA double mutant to use enterobactin was restored. This indicates that both VctA and IrgA transport enterobactin. We also identify the genes vctPDGC, which are linked to vctA and encode a periplasmic binding protein-dependent ABC transport system that functions in the utilization of both enterobactin and vibriobactin (VCA0227-0230). An irgA::TnphoA mutant strain, MBG40, was shown in a previous study to be highly attenuated and to have a strong colonization defect in an infant mouse model of V. cholerae infection (M. B. Goldberg, V. J. DiRita, and S. B. Calderwood, Infect. Immun. 58:55-60, 1990). In this work, a new irgA mutation was constructed, and this mutant strain was not significantly impaired in its ability to compete with the parental strain in infant mice and was not attenuated for virulence in an assay of 50% lethal dose. These data indicate that the virulence defect in MBG40 is not due to the loss of irgA function and that irgA is unlikely to be an important virulence factor.

Pathogenesis of enteric Campylobacter infection.

Campylobacter jejuni and related species are important human pathogens, causing acute human enterocolitis, and they are the most common cause of food-borne diarrhoea in many industrialized countries. Previous infection with certain strains of C. jejuni is also linked with the development of the neurological disorder Guillain-Barre syndrome (GBS). Despite its importance as a human pathogen, relatively little is understood of the mechanisms of C. jejuni-associated disease. The recent release of the complete genome sequence of C. jejuni strain NCTC 11168, together with new strategies for random and directed mutagenesis, have allowed a better insight into some of the genetic determinants of C. jejuni virulence. In this review current knowledge on the pathogenesis of C. jejuni infection is summarized, and possible directions of future research indicated.

TRAP transporters: a new family of periplasmic solute transport systems encoded by the dctPQM genes of Rhodobacter capsulatus and by homologs in diverse gram-negative bacteria.

The dct locus of Rhodobacter capsulatus encodes a high-affinity transport system for the C4-dicarboxylates malate, succinate, and fumarate. The nucleotide sequence of the region downstream of the previously sequenced dctP gene (encoding a periplasmic C4-dicarboxylate-binding protein) was determined. Two open reading frames (ORFs) of 681 bp (dctQ) and 1,320 bp (dctM) were identified as additional dct genes by insertional mutagenesis and complementation studies. DctQ (24,763 Da) and DctM (46,827 Da) had hydropathic profiles consistent with the presence of 4 and 12 potential transmembrane segments, respectively, and were localized in the cytoplasmic membrane fraction after heterologous expression of the dctQM ORFs in Escherichia coli. DctP, DctQ, and DctM were found to be unrelated to known transport proteins in the ABC (ATP-binding cassette) superfamily but were shown to be homologous with the products of previously unidentified ORFs in a number of gram-negative bacteria, including Bordetella pertussis, E. coli, Salmonella typhimurium, Haemophilus influenzae, and Synechocystis sp. strain PCC6803. An additional ORF (rypA) downstream of dctM encodes a protein with sequence similarity to eukaryotic protein-tyrosine phosphatases, but interposon mutagenesis of this ORF did not result in a Dct- phenotype. Complementation of a Rhizobium meliloti dctABD deletion mutant by heterologous expression of the dctPQM genes from R. capsulatus demonstrated that no additional structural genes were required to form a functional transport system. Transport via the Dct system was vanadate insensitive, and in uncoupler titrations with intact cells, the decrease in the rate of succinate transport correlated closely with the fall in membrane potential but not with the cellular ATP concentration, implying that the proton motive force, rather than ATP hydrolysis, drives uptake. It is concluded that the R. capsulatus Dct system is a new type of periplasmic secondary transporter and that similar, hitherto-unrecognized systems are widespread in gram-negative bacteria. The name TRAP (for tripartite ATP-independent periplasmic) transporters is proposed for this new group.

Characterization of the Adenosine Triphosphatase Activity of the Periplasmic Histidine Permease, a Traffic ATPase (ABC Transporter)

The superfamily of traffic ATPases (ABC transporters) includes bacterial periplasmic transport systems (permeases) and eukaryotic transporters. The histidine permease of Salmonella typhimurium is composed of a membrane-bound complex (HisQMP2) containing four subunits, and of a soluble receptor, the histidine-binding protein (HisJ). Transport is energized by ATP. In this article the ATPase activity of HisQMP2 has been characterized, using a novel assay that is independent of transport. The assay uses Mg2+ ions to permeabilize membrane vesicles or proteoliposomes, thus allowing access of ATP to both sides of the bilayer. HisQMP2 displays a low level of intrinsic ATPase activity in the absence of HisJ; unliganded HisJ stimulates the activity and liganded HisJ stimulates to an even higher level. All three levels of activity display positive cooperativity for ATP with a Hill coefficient of 2 and a K0. 5 value of 0.6 mM. The activity has been characterized with respect to pH, salt, phospholipids, substrate, and inhibitor specificity. Free histidine has no effect. The activity is inhibited by orthovanadate, but not by N-ethylmaleimide, bafilomycin A1, or ouabain. Several nucleotide analogs, ADP, 5'-adenylyl-beta, gamma-imidodiphosphate, adenosine 5'-(beta,gammaimino)triphosphate, and adenosine 5'-O-(3-thio)triphosphate, inhibit the activity. Unliganded HisJ does not compete with liganded HisJ for the stimulation of the ATPase activity of HisQMP2.

Characterization of Transport through the Periplasmic Histidine Permease Using Proteoliposomes Reconstituted by Dialysis

The superfamily of traffic ATPases (ABC transporters) includes bacterial periplasmic transport systems (permeases) and various eukaryotic transporters. The histidine permease of Salmonella typhimurium and Escherichia coli is composed of a membrane-bound complex containing four subunits and of a soluble receptor, the substrate-binding protein (HisJ), and is energized by ATP. The permease was previously reconstituted into proteoliposomes by a detergent dilution method (1). Here we extensively characterize the properties of this permease after reconstitution into proteoliposomes by dialysis and encapsulation of ATP or other reagents by freeze-thawing. We show that histidine transport depends entirely on both ATP and liganded HisJ, with apparent Km values of 8 mM and 8 microM, respectively, and is affected by pH, temperature, and salt concentration. Transport is irreversible and accumulation reaches a plateau at which point transport ceases. The permease is inhibited by ADP and by high concentrations of internal histidine. The inhibition by histidine implies that the membrane-bound complex HisQ/M/P carries a substrate-binding site. The reconstituted permease activity corresponds to about 40-70% turnover rate of the in vivo rate of transport.

Identification of a locus involved in the utilization of iron by Actinobacillus pleuropneumoniae

The cloned afu locus of Actinobacillus pleuropneumoniae restored the ability of an Escherichia coli K-12 mutant ( aroB) to grow on iron-limited media. DNA sequence analysis of the fragment showed that there are three genes designated afuA, afuB and afuC (Actinobacillus ferric uptake) that encode products similar to the SfuABC proteins of Serratia marcescens, the HitABC proteins of Haemophilia influenzae, the FbpABC proteins of Neisseria gonorrhoeae and the YfuABC proteins of Yersinia enterocolitica. The three genes encode a periplasmic iron-binding protein (AfuA), a highly hydrophobic integral cytoplasmic membrane protein with two consensus permease motifs (AfuB) and one hydrophilic peripheral cytoplasmic membrane protein with Walker ATP-binding motifs (AfuC), respectively. This system has been shown to constitute a periplasmic binding protein-dependent iron transport system in these organisms. The afuABC operon is locating approximately 200 bp upstream of apxIC gene, but transcribed in opposite direction to the ApxI-toxin genes.

Identification of a locus involved in the utilization of iron by Actinobacillus pleuropneumoniae.

The cloned afu locus of Actinobacillus pleuropneumoniae restored the ability of an Escherichia coli K-12 mutant (aroB) to grow on iron-limited media. DNA sequence analysis of the fragment showed that there are three genes designated afuA, afuB and afuC (Actinobacillus ferric uptake) that encode products similar to the SfuABC proteins of Serratia marcescens, the HitABC proteins of Haemophilus influenzae, the FbpABC proteins of Neisseria gonorrhoeae and the YfuABC proteins of Yersinia enterocolitica. The three genes encode a periplasmic iron-binding protein (AfuA), a highly hydrophobic integral cytoplasmic membrane protein with two consensus permease motifs (AfuB) and one hydrophilic peripheral cytoplasmic membrane protein with Walker ATP-binding motifs (AfuC), respectively. This system has been shown to constitute a periplasmic binding protein-dependent iron transport system in these organisms. The afuABC operon is locating approximately 200 bp upstream of apxIC gene, but transcribed in opposite direction to the ApxI-toxin genes.

Binding-protein-dependent arginine transport in Pasteurella haemolytica.

A periplasmic arginine transport system that is a member of the ATP -dependent transport superfamily was identified in Pasteurella haemolytica. The gene encoding the periplasmic binding protein (lapT) was cloned and the protein overexpressed in Escherichia coli. LapT was purified to homogeneity using a modified osmotic shock procedure and anion-exchange column chromatography. Filter-binding assays established that LapT is an L-arginine-binding protein. Various amino acids were tested for their ability to inhibit L-arginine binding to LapT. When present in 100-fold excess, only L-arginine, D-arginine and citrulline competed with L-arginine for binding. Arginine transport in P. haemolytica whole cells was competitively inhibited by the same amino acids, suggesting that the LapT permease specifically transports L-arginine. The dissociation constant for the L-arginine-LapT complex was 170 nM and the stoichiometry of binding was approximately 0.8 mol L-arginine (mol LapT)-1. A polyclonal antibody raised against the purified protein permitted detection of LapT in P. haemolytica periplasmic fractions.

Polyamine transport inEscherichia coli

The polyamine content in cells is regulated by both polyamine biosynthesis and its transport. We recently obtained and characterized three clones of polyamine transport genes (pPT104, pPT79 and pPT71) inEscherichia coli. The system encoded by pPT104 was the spermidine-preferential uptake system and that encoded by pPT79 the putrescine-specific uptake system. Furthermore, these two systems were periplasmic transport systems consisting of four kinds of proteins: pPT104 clone encoded potA, -B,-C, and -D proteins and pPT79 clone encoded potF, -G, -H, and -I proteins, judging from the deduced amino acid sequences of the nucleotide sequences of these clones. PotD and -F proteins were periplasmic substrate binding proteins and potA and -G proteins membrane associated proteins having the nucleotide binding site. PotB and -C proteins, and potH and -I proteins were transmembrane proteins probably forming channels for spermidine and putrescine, respectively. Their amino acid sequences in the corresponding proteins were similar to each other. The functions of potA and -D proteins in the spermidine-preferential uptake system encoded by pPT104 clone were studied in detail through a combined biochemical and genetic approach. In contrast, the putrescine transport system encoded by pPT71 consisted of one membrane protein (potE protein) haveing twelve transmembrane segments, and was active in both the uptake and excretion of putrescine. The uptake was dependent on membrane potential, and the excretion was due to the exchange reaction between putrescine and ornithine.

Cloning and sequencing of a 23-kb region of the Bacillus subtilis genome between the iol and hut operons.

Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, a 23-kb chromosomal segment, which covers the region between the iol and hut operons, has been cloned and sequenced, creating a 99-kb contig from the gnt operon to the wapA locus. This region (23351 bp) contains 25 complete open reading frames (ORFs; genes) including deoR, dra, nupC and pdp and two partial ones. The region (5140 bp) containing these four genes, being also sequenced by H. H. Saxild et al., was sequenced by subjecting a long polymerase chain reaction product to random sequencing using phage M13mp19. However, we could detect no conflict, between two independently determined sequences, which could be attributed to our sequencing method. A homology search for the 24 newly identified gene products revealed significant homology to known proteins in 14 of them. It was notable that three proteins, encoded by the successive genes (yxeMNO), exhibited meaningful homology to the E. coli GlnHPQ products constituting a periplasmic ATP-dependent transport system for glutamine.