ABC Transport System Research Articles

Functional Characteristics of TauA Binding Protein from TauABC Escherichia coli System

Although TauA shares few common characteristics with other known periplasmic binding protein, TauA is a putative periplasmic binding protein, part of tauABCD gene cluster involved in sulfonate transport in sulphate starvation condition. This protein was expressed in E. coli BL 21 and purified before to assess its binding functionalities. Measurement of K (d) value (mean 11.3 nM) by binding/dialysis studies revealed high affinity and specificity with taurine and also indicated that TauA possessed a unique binding site for its ligand. Comparisons with other periplasmic binding proteins suggests TauA plays a major role in ABC transport system and could be ideal candidate to serve as taurine catcher in biological fluids.

The Structure of a Cyanobacterial Bicarbonate Transport Protein, CmpA

Cyanobacteria, blue-green algae, are the most abundant autotrophs in aquatic environments and form the base of the food chain by fixing carbon and nitrogen into cellular biomass. To compensate for the low selectivity of Rubisco for CO2 over O2, cyanobacteria have developed highly efficient CO2-concentrating machinery of which the ABC transport system CmpABCD from Synechocystis PCC 6803 is one component. Here, we have described the structure of the bicarbonate-binding protein CmpA in the absence and presence of bicarbonate and carbonic acid. CmpA is highly homologous to the nitrate transport protein NrtA. CmpA binds carbonic acid at the entrance to the ligand-binding pocket, whereas bicarbonate binds in nearly an identical location compared with nitrate binding to NrtA. Unexpectedly, bicarbonate binding is accompanied by a metal ion, identified as Ca2+ via inductively coupled plasma optical emission spectrometry. The binding of bicarbonate and metal appears to be highly cooperative and suggests that CmpA may co-transport bicarbonate and calcium or that calcium acts a cofactor in bicarbonate transport.

The Phn system of Mycobacterium smegmatis: a second high-affinity ABC-transporter for phosphate

Uptake of inorganic phosphate, an essential but often limiting nutrient, in bacteria is usually accomplished by the high-affinity ABC-transport system Pst. Pathogenic species of mycobacteria contain several copies of the genes encoding the Pst system (pstSCAB), and two of the encoded proteins, PstS1 and PstS2, have been shown to be virulence factors in Mycobacterium tuberculosis. The fast-growing Mycobacterium smegmatis contains only a single copy of the pst operon. This study reports the biochemical and molecular characterization of a second high-affinity phosphate transport system, designated Phn. The Phn system is encoded by a three-gene operon that constitutes the components of a putative ABC-type phosphonate/phosphate transport system. Expression studies using phnD- and pstS-lacZ transcriptional fusions showed that both operons were induced when the culture entered phosphate limitation, indicating a role for both systems in phosphate uptake at low extracellular concentrations. Deletion mutants in either phnD or pstS failed to grow in minimal medium with a 10 mM phosphate concentration, while the isogenic wild-type strain mc(2)155 grew at micromolar phosphate concentrations. Analysis of the kinetics of phosphate transport in the wild-type and mutant strains led to the proposal that the Phn and Pst systems are both high-affinity phosphate transporters with similar affinities for phosphate (i.e. apparent K(m) values between 40 and 90 muM P(i)). The Phn system of M. smegmatis appears to be unique in that, unlike previously identified Phn systems, it does not recognize phosphonates or phosphite as substrates.

Hierarchy of iron uptake systems: Yfu and Yiu are functional in Yersinia pestis.

In addition to the yersiniabactin (Ybt) siderophore-dependent system, two inorganic iron ABC transport systems of Yersinia pestis, Yfe and Yfu, have been characterized. Here we show that the Yfu system functions in Y. pestis: a Ybt- Yfe- Yfu- mutant exhibited a greater growth defect under iron-deficient conditions than its Ybt- Yfe- parental strain. We also demonstrate that another putative Y. pestis iron uptake system, Yiu, which potentially encodes an outer membrane receptor, YiuR, and an ABC iron transport cassette, YiuABC, is functional. The cloned yiuABC operon restored growth of an enterobactin-deficient mutant Escherichia coli strain, 1017, under iron-chelated conditions. Iron uptake by the Yiu system in Y. pestis was demonstrated only when the Ybt, Yfe, and Yfu systems were mutated. Using a yiuA::lacZ fusion, we show that the yiuABC promoter is repressed by iron through Fur. A mouse model of bubonic plague failed to show a significant role for the Yiu system in the disease process. These results demonstrate that two additional iron transporters are functional in Y. pestis and indicate that there is a hierarchy of iron transporters, with Ybt being most effective and Yiu being the least effective of those systems which have been characterized.

Tungsten Transport Protein A (WtpA) inPyrococcus furiosus: the First Member of a New Class of Tungstate and Molybdate Transporters

A novel tungstate and molybdate binding protein has been discovered from the hyperthermophilic archaeon Pyrococcus furiosus. This tungstate transport protein A (WtpA) is part of a new ABC transporter system selective for tungstate and molybdate. WtpA has very low sequence similarity with the earlier-characterized transport proteins ModA for molybdate and TupA for tungstate. Its structural gene is present in the genome of numerous archaea and some bacteria. The identification of this new tungstate and molybdate binding protein clarifies the mechanism of tungstate and molybdate transport in organisms that lack the known uptake systems associated with the ModA and TupA proteins, like many archaea. The periplasmic protein of this ABC transporter, WtpA (PF0080), was cloned and expressed in Escherichia coli. Using isothermal titration calorimetry, WtpA was observed to bind tungstate (dissociation constant [K(D)] of 17 +/- 7 pM) and molybdate (K(D) of 11 +/- 5 nM) with a stoichiometry of 1.0 mol oxoanion per mole of protein. These low K(D) values indicate that WtpA has a higher affinity for tungstate than do ModA and TupA and an affinity for molybdate similar to that of ModA. A displacement titration of molybdate-saturated WtpA with tungstate showed that the tungstate effectively replaced the molybdate in the binding site of the protein.

Mlc of Thermus thermophilus : a Glucose-Specific Regulator for a Glucose/Mannose ABC Transporter in the Absence of the Phosphotransferase System

We report the presence of Mlc in a thermophilic bacterium. Mlc is known as a global regulator of sugar metabolism in gram-negative enteric bacteria that is controlled by sequestration to a glucose-transporting EII(Glc) of the phosphotransferase system (PTS). Since thermophilic bacteria do not possess PTS, Mlc in Thermus thermophilus must be differently controlled. DNA sequence alignments between Mlc from T. thermophilus (Mlc(Tth)) and Mlc from E. coli (Mlc(Eco)) revealed that Mlc(Tth) conserved five residues of the glucose-binding motif of glucokinases. Here we show that Mlc(Tth) is not a glucokinase but is indeed able to bind glucose (K(D) = 20 microM), unlike Mlc(Eco). We found that mlc of T. thermophilus is the first gene within an operon encoding an ABC transporter for glucose and mannose, including a glucose/mannose-binding protein and two permeases. malK1, encoding the cognate ATP-hydrolyzing subunit, is located elsewhere on the chromosome. The system transports glucose at 70 degrees C with a K(m) of 0.15 microM and a V(max) of 4.22 nmol per min per ml at an optical density (OD) of 1. Mlc(Tth) negatively regulates itself and the entire glucose/mannose ABC transport system operon but not malK1, with glucose acting as an inducer. MalK1 is shared with the ABC transporter for trehalose, maltose, sucrose, and palatinose (TMSP). Mutants lacking malK1 do not transport either glucose or maltose. The TMSP transporter is also able to transport glucose with a K(m) of 1.4 microM and a V(max) of 7.6 nmol per min per ml at an OD of 1, but it does not transport mannose.

Salt Stress in Desulfovibrio vulgaris Hildenborough: an Integrated Genomics Approach

The ability of Desulfovibrio vulgaris Hildenborough to reduce, and therefore contain, toxic and radioactive metal waste has made all factors that affect the physiology of this organism of great interest. Increased salinity is an important and frequent fluctuation faced by D. vulgaris in its natural habitat. In liquid culture, exposure to excess salt resulted in striking elongation of D. vulgaris cells. Using data from transcriptomics, proteomics, metabolite assays, phospholipid fatty acid profiling, and electron microscopy, we used a systems approach to explore the effects of excess NaCl on D. vulgaris. In this study we demonstrated that import of osmoprotectants, such as glycine betaine and ectoine, is the primary mechanism used by D. vulgaris to counter hyperionic stress. Several efflux systems were also highly up-regulated, as was the ATP synthesis pathway. Increases in the levels of both RNA and DNA helicases suggested that salt stress affected the stability of nucleic acid base pairing. An overall increase in the level of branched fatty acids indicated that there were changes in cell wall fluidity. The immediate response to salt stress included up-regulation of chemotaxis genes, although flagellar biosynthesis was down-regulated. Other down-regulated systems included lactate uptake permeases and ABC transport systems. The results of an extensive NaCl stress analysis were compared with microarray data from a KCl stress analysis, and unlike many other bacteria, D. vulgaris responded similarly to the two stresses. Integration of data from multiple methods allowed us to develop a conceptual model for the salt stress response in D. vulgaris that can be compared to those in other microorganisms.

Study of nitrate stress in Desulfovibrio vulgaris Hildenborough using iTRAQ proteomics

The response of Desulfovibrio vulgaris Hildenborough (DvH), a sulphate-reducing bacterium, to nitrate stress was examined using quantitative proteomic analysis. DvH was stressed with 105 mM sodium nitrate (NaNO(3)), a level that caused a 50% inhibition in growth. The protein profile of stressed cells was compared with that of cells grown in the absence of nitrate using the iTRAQ peptide labelling strategy and tandem liquid chromatography separation coupled with mass spectrometry (quadrupole time-of-flight) detection. A total of 737 unique proteins were identified by two or more peptides, representing 22% of the total DvH proteome and spanning every functional category. The results indicate that this was a mild stress, as proteins involved in central metabolism and the sulphate reduction pathway were unperturbed. Proteins involved in the nitrate reduction pathway increased. Increases seen in transport systems for proline, glycine-betaine and glutamate indicate that the NaNO(3) exposure led to both salt stress and nitrate stress. Up-regulation observed in oxidative stress response proteins (Rbr, RbO, etc.) and a large number of ABC transport systems as well as in iron-sulphur-cluster-containing proteins, however, appear to be specific to nitrate exposure. Finally, a number of hypothetical proteins were among the most significant changers, indicating that there may be unknown mechanisms initiated upon nitrate stress in DvH.

A biosystem for alginate metabolism in Agrobacterium tumefaciens strain C58: Molecular identification of Atu3025 as an exotype family PL-15 alginate lyase

The Gram-negative bacterium Sphingomonas sp. strain A1 (strain A1) has a peculiar biosystem to directly import and depolymerize a macromolecule, alginate, which is encoded by a cluster of genes on the genome. We identified five clustered ORFs homologous to some genes of the strain A1 cluster in the genome of Agrobacterium tumefaciens strain C58 (strain C58). These ORFs are Atu3021, Atu3022, Atu3023, and Atu3024, encoding a putative sugar ABC transporter system and Atu3025, which encodes a putative alginate lyase. We analyzed the involvement of this gene cluster in alginate metabolism. Strain C58 cells grew significantly on low-molecular-weight (LMW) alginate (average molecular weight, 1000), and we detected specific alginate-induced expression of Atu3024 and Atu3025. This strain does not grow on alginate (average molecular weight, 25 600), suggesting that the strain C58 gene cluster is involved in importing and degrading LMW alginate. One protein, Atu3025, purified from strain C58, was identified as an alginate lyase, and the enzyme overexpressed in Escherichia coli was further characterized. Atu3025 released monosaccharides specifically from alginate most efficiently at pH 7.3 and 30 °C through a β-elimination reaction, indicating that Atu3025 is an exotype alginate lyase potentially involved in the assimilation of LMW alginate in strain C58.

Identification of a Meningococcall-Glutamate ABC Transporter Operon Essential for Growth in Low-Sodium Environments

GdhR is a meningococcal transcriptional regulator that was previously shown to positively control the expression of gdhA, encoding the NADP-specific L-glutamate dehydrogenase (NADP-GDH), in response to the growth phase and/or to the carbon source. In this study we used reverse transcriptase-PCR-differential display (to identify additional GdhR-regulated genes. The results indicated that GdhR, in addition to NADP-GDH, controls the expression of a number of genes involved in glucose catabolism by the Entner-Doudoroff pathway and in l-glutamate import by an unknown ABC transport system. The genes encoding the putative periplasmic substrate-binding protein (NMB1963) and the permease (NMB1965) of the ABC transporter were genetically inactivated. Uptake experiments demonstrated an impairment of L-glutamate import in the NMB1965-defective mutant in the absence or in the presence of a low sodium ion concentration. In contrast, at a sodium ion concentration above 60 mM, the uptake defect disappeared, possibly because the activity of a sodium-driven secondary transporter became predominant. Indeed, the NMB1965-defective mutant was unable to grow at a low sodium ion concentration (<20 mM) in a chemically defined medium containing L-glutamate and four other amino acids that supported meningococcal growth, but it grew when the sodium ion concentration was raised to higher values (>60 mM). The same growth phenotype was observed in the NMB1963-defective mutant. Cell invasion and intracellular persistence assays and expression data during cell invasion provided evidence that the l-glutamate ABC transporter, tentatively named GltT, was critical for meningococcal adaptation in the low-sodium intracellular environment.

Cloning and characterization of a Campylobacter jejuni 72Dz/92 gene encoding a 30 kDa immunopositive protein, component of the ABC transport system; expression of the gene in avirulent Salmonella typhimurium.

Three gene libraries of Campylobacter jejuni 72Dz/92 DNA were prepared using lambda gt11, pSupercos and pWSK129 cloning vectors. Screening of the libraries with Escherichia coli absorbed antiserum generated against whole C. jejuni revealed several immunoreactive clones of apparent molecular masses 19, 28, 30 and 50 kDa. The most commonly isolated clones expressed 30 kDa protein. The nucleotide sequence of the 1768 bp C. jejuni DNA yielded one complete (ORF2) and two partial open reading frames (ORF1 and ORF3). ORF2 encoded CjaA protein exhibits relevant overall homology to several prokaryotic solute binding proteins (family 3), components of the ABC transport system, while the product of the truncated ORF3 (CjaB protein) shows extensive homology to Gram-negative bacterial proteins, members of the sugar transporter family. The genetic organization of the putative cjaAB operon was studied. The cjaA gene fragment (616 bp) was amplified from three C. jejuni strains isolated from patients with acute bloody diarrhea, whereas it was not amplified from strains which caused acute diarrhea with no blood in the stools. The gene was introduced into avirulent Salmonella typhimurium vaccine strain where it is expressed at a reasonably high level.

Ectoine functions as an osmoprotectant in Bacillus subtilis and is accumulated via the ABC-transport system OpuC

We report here that the cyclic amino acid ectoine functions as an osmoprotectant for the soil bacterium Bacillus subtilis. Growth experiments with a set of B. subtilis strains that carry defined mutations in the glycine betaine transport systems OpuA, OpuC and OpuD and the choline transport system OpuB revealed that ectoine was specifically accumulated via the ABC-transport system OpuC. Competition experiments employing unlabeled ectoine and radiolabeled glycine betaine showed that the OpuC transport system has a low affinity for ectoine with a Ki value of approximately 1.5 mM. Ectoine was identified by 1H NMR spectroscopy in the solute pool of cells grown in the presence of ectoine. Ectoine could not be used by B. subtilis as sole carbon or nitrogen source. Our data thus characterise ectoine as a metabolically inert stress compound for B. subtilis and establish a crucial role for the ABC-transport system OpuC for the acquisition of the osmoprotectant ectoine from the environment.

Reciprocal Regulation of Pyoluteorin Production with Membrane Transporter Gene Expression in Pseudomonas fluorescens Pf-5

Pyoluteorin is a chlorinated polyketide antibiotic secreted by the rhizosphere bacterium Pseudomonas fluorescens Pf-5. Genes encoding enzymes and transcriptional regulators involved in pyoluteorin production are clustered in the genome of Pf-5. Sequence analysis of genes adjacent to the known pyoluteorin biosynthetic gene cluster revealed the presence of an ABC transporter system. We disrupted two putative ABC transporter genes by inserting transcriptional fusions to an ice nucleation reporter gene. Mutations in pltI and pltJ, which are predicted to encode a membrane fusion protein and an ATP-binding cassette of the ABC transporter, respectively, greatly reduced pyoluteorin production by Pf-5. During the transition from exponential growth to stationary phase, populations of a pltI mutant were lower than those of a pltI+ strain in a culture medium containing pyoluteorin, suggesting a role for the transport system in efflux and the resistance of Pf-5 to the antibiotic. Although pltI or pltJ mutant strains displayed low pyoluteorin production, they did not accumulate proportionately more of the antibiotic intracellularly, indicating that pltI and pltJ do not encode an exclusive exporter for pyoluteorin. Transcription of the putative pyoluteorin efflux genes pltI and pltJ was enhanced by exogenous pyoluteorin. These new observations parallel an earlier finding that pyoluteorin enhances the transcription of pyoluteorin biosynthesis genes and pyoluteorin production in Pf-5. This report provides evidence of a coordination of pyoluteorin production and the transcription of genes encoding a linked transport apparatus, wherein each requires the other for optimal expression.

A Salt-Bridge Motif Involved in Ligand Binding and Large-Scale Domain Motions of the Maltose-Binding Protein

The uptake of nutrients is essential for the survival of bacterial cells. Many specialized systems have evolved, such as the maltose-dependent ABC transport system that transfers oligosaccharides through the cytoplasmic membrane. The maltose/maltodextrin-binding protein (MBP) serves as an initial high-affinity binding component in the periplasm that delivers the bound sugar into the cognate ABC transporter MalFGK2. We have investigated the domain motions induced by the binding of the ligand maltotriose into the binding cleft using molecular dynamics simulations. We find that MBP is predominantly in the open state without ligand and in the closed state with ligand bound. Oligosaccharide binding induces a closure motion (30.0° rotation), whereas ligand removal leads to domain opening (32.6° rotation) around a well-defined hinge affecting key areas relevant for chemotaxis and transport. Our simulations suggest that a “hook-and-eye” motif is involved in the binding. A salt bridge between Glu-111 and Lys-15 forms that effectively locks the protein-ligand complex in a semiclosed conformation inhibiting any further opening and promoting complete closure. This previously unrecognized feature seems to secure the ligand in the binding site and keeps MBP in the closed conformation and suggests a role in the initial steps of substrate transport.

Nystatin Biosynthesis and Transport: nysH and nysG Genes Encoding a Putative ABC Transporter System in Streptomyces noursei ATCC 11455 Are Required for Efficient Conversion of 10-Deoxynystatin to Nystatin

The genes nysH and nysG, encoding putative ABC-type transporter proteins, are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. To assess the possible roles of these genes in nystatin biosynthesis, they were inactivated by gene replacements leading to in-frame deletions. Metabolite profile analysis of the nysH and nysG deletion mutants revealed that both of them synthesized nystatin at a reduced level and produced considerable amounts of a putative nystatin analogue. Liquid chromatography-mass spectrometry and nuclear magnetic resonance structural analyses of the latter metabolite confirmed its identity as 10-deoxynystatin, a nystatin precursor lacking a hydroxyl group at C-10. Washing experiments demonstrated that both nystatin and 10-deoxynystatin are transported out of cells, suggesting the existence of an alternative efflux system(s) for the transport of nystatin-related metabolites. This notion was further corroborated in experiments with the ATPase inhibitor sodium o-vanadate, which affected the production of nystatin and 10-deoxynystatin in the wild-type strain and transporter mutants in a different manner. The data obtained in this study suggest that the efflux of nystatin-related polyene macrolides occurs through several transporters and that the NysH-NysG efflux system provides conditions favorable for C-10 hydroxylation.

Sequence characterization of four putative membrane‐associated proteins from sweet potato little leaf strain V4 phytoplasma

To increase understanding of the genetic architecture of phytoplasmas, a random clone (pH80) of the sweet potato little leaf strain‐V4 (SPLL‐V4) phytoplasma and the glucose‐inhibited division protein A (gidA) gene were analysed. Clone pH80 and the gidA gene are located in a 100‐kb region near the putative origin of replication. Three ORFs were identified within the pH80 random clone, encoding the proteins PotB, PotC and PotD, which form part of an ABC transport system for polyamine import. Sequence data were supported by structural analysis which indicated that SPLL‐V4 PotB and PotC were integral membrane proteins, and PotD was a substrate‐binding, surface‐exposed membrane protein. The gidA gene in the SPLL‐V4 phytoplasma was also putatively identified as a membrane protein. The complete gidA gene sequences were characterized for the tomato big bud and SPLL‐V4 phytoplasmas. The proteins identified here were found to share high similarity with those recently identified in the onion yellows phytoplasma genome. Both the gidA and polyamine transport operons are believed to be associated with DNA replication.

Identification of four fimbria-encoding genomic islands that are highly specific for verocytotoxin-producing Escherichia coli serotype O157 strains.

Verocytotoxin-producing Escherichia coli causes zoonotic food- or waterborne infection that may be associated with massive outbreaks and with the serious complication of hemolytic uremic syndrome (HUS). Serotypes O157:H7 and O157:NM are more commonly associated with HUS and outbreaks than other serotypes, such as O26:H11. To determine whether a genetic basis exists for why serotype O157:H7/NM causes HUS and outbreaks more often than other serotypes, such as O26:H11, we conducted suppression subtractive hybridization (SSH) between the genomes of the sequenced O157:H7 strain EDL933 and CL1, a clinical serotype O26:H11 isolate. Genes from four EDL933 fimbria-encoding genomic O islands (OIs) (OI-1, -47, -141, and -154) were identified in the SSH library. OI-47 encodes several additional putative virulence factors, including secreted and signaling proteins, a hemolysin locus, a lipoprotein, an ABC transport system, and a lipid biosynthesis locus. The distribution of the OIs was investigated by PCR and Southern hybridization (when PCR was negative) with 69 VTEC strains belonging to 39 different serotypes corresponding to 5 seropathotypes that differ in their disease and epidemic potential. The four OIs described here were distributed almost exclusively in serotypes O157:H7 and O157:NM, which indicates that they may be associated with the ability of these strains to colonize human and/or animal intestinal tracts and to cause epidemic and serious disease more frequently than other serotypes. The occurrence of the four OIs in enteropathogenic E. coli O55:H7 strains is consistent with their vertical inheritance by VTEC O157:H7/NM from this clonally related ancestor.

Effects of growth phase and the developmentally significant bldA-specified tRNA on the membrane-associated proteome of Streptomyces coelicolor

Previous proteomic analyses of Streptomyces coelicolor by two-dimensional electrophoresis and protein mass fingerprinting focused on extracts from total cellular material. Here, the membrane-associated proteome of cultures grown in a liquid minimal medium was partially characterized. The products of some 120 genes were characterized from the membrane fraction, with 70 predicted to possess at least one transmembrane helix. A notably high proportion of ABC transporter systems was represented; the specific types detected provided a snapshot of the nutritional requirements of the mycelium. The membrane-associated proteins did not change very much in abundance in different phases of growth in liquid minimal medium. Identification of gene products not expected to be present in membrane protein extracts led to a reconsideration of the genome annotation in two cases, and supplemented scarce information on 11 hypothetical/conserved hypothetical proteins of unknown function. The wild-type membrane proteome was compared with that of a bldA mutant lacking the only tRNA capable of efficient translation of the rare UUA (leucine) codon. Such mutants are unaffected in vegetative growth but are defective in many aspects of secondary metabolism and morphological differentiation. There were a few clear changes in the membrane proteome of the mutant. In particular, two hypothetical proteins (SCO4244 and SCO4252) were completely absent from the bldA mutant, and this was associated with the TTA-containing regulatory gene SCO4263. Evidence for the control of a cluster of function-unknown genes by the SCO4263 regulator revealed a new aspect of the pleiotropic bldA phenotype.

Isolation and Characterization of a Genetically Tractable Photoautotrophic Fe(II)-Oxidizing Bacterium, Rhodopseudomonas palustris Strain TIE-1

We report the isolation and characterization of a phototrophic ferrous iron [Fe(II)]-oxidizing bacterium named TIE-1 that differs from other Fe(II)-oxidizing phototrophs in that it is genetically tractable. Under anaerobic conditions, TIE-1 grows photoautotrophically with Fe(II), H2, or thiosulfate as the electron donor and photoheterotrophically with a variety of organic carbon sources. TIE-1 also grows chemoheterotrophically in the dark. This isolate appears to be a new strain of the purple nonsulfur bacterial species Rhodopseudomonas palustris, based on physiological and phylogenetic analysis. Fe(II) oxidation is optimal at pH 6.5 to 6.9. The mineral products of Fe(II) oxidation are pH dependent: below pH 7.0 goethite (alpha-FeOOH) forms, and above pH 7.2 magnetite (Fe3O4) forms. TIE-1 forms colonies on agar plates and is sensitive to a variety of antibiotics. A hyperactive mariner transposon is capable of random insertion into the chromosome with a transposition frequency of approximately 10(-5). To identify components involved in phototrophic Fe(II) oxidation, mutants of TIE-1 were generated by transposon mutagenesis and screened for defects in Fe(II) oxidation in a cell suspension assay. Among approximately 12,000 mutants screened, 6 were identified that are specifically impaired in Fe(II) oxidation. Five of these mutants have independent disruptions in a gene that is predicted to encode an integral membrane protein that appears to be part of an ABC transport system; the sixth mutant has an insertion in a gene that is a homolog of CobS, an enzyme involved in cobalamin (vitamin B12) biosynthesis.

A Two-component Hydroxylase Involved in the Assimilation of 3-Hydroxyphenyl Acetate in Pseudomonas putida

The complete catabolic pathway involved in the assimilation of 3-hydroxyphenylacetic acid (3-OH-PhAc) in Pseudomonas putida U has been established. This pathway is integrated by the following: (i) a specific route (upper pathway), which catalyzes the conversion of 3-OH-PhAc into 2,5-dihydroxyphenylacetic acid (2,5-diOH-PhAc) (homogentisic acid, Hmg), and (ii) a central route (convergent route), which catalyzes the transformation of the Hmg generated from 3-OH-PhAc, l-Phe, and l-Tyr into fumarate and acetoacetate (HmgABC). Thus, in a first step the degradation of 3-OH-PhAc requires the uptake of 3-OH-PhAc by means of an active transport system that involves the participation of a permease (MhaC) together with phosphoenolpyruvate as the energy source. Once incorporated, 3-OH-PhAc is hydroxylated to 2,5-diOH-PhAc through an enzymatic reaction catalyzed by a novel two-component flavoprotein aromatic hydroxylase (MhaAB). The large component (MhaA, 62,719 Da) is a flavoprotein, and the small component (MhaB, 6,348 Da) is a coupling protein that is essential for the hydroxylation of 3-OH-PhAc to 2,5-diOH-PhAc. Sequence analyses and molecular biology studies revealed that homogentisic acid synthase (MhaAB) is different from the aromatic hydroxylases reported to date, accounting for its specific involvement in the catabolism of 3-OH-PhAc. Additionally, an ABC transport system (HmgDEFGHI) involved in the uptake of homogentisic acid and two regulatory elements (mhaSR and hmgR) have been identified. Furthermore, the cloning and the expression of some of the catabolic genes in different microbes presented them with the ability to synthesize Hmg (mhaAB) or allowed them to grow in chemically defined media containing 3-OH-PhAc as the sole carbon source (mhaAB and hmgABC).