Source: http://smart.embl.de/smart/show_secondary.cgi?domain=FeoA
Timestamp: 2019-04-25 10:11:45+00:00

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Lon-mediated proteolysis of the FeoC protein prevents Salmonella enterica from accumulating the Fe(II) transporter FeoB under high-oxygen conditions.
The Salmonella Feo system consists of the FeoA, FeoB, and FeoC proteins and mediates ferrous iron [Fe(II)] import. FeoB is an inner membrane protein that, along with contributions from two small hydrophilic proteins, FeoA and FeoC, transports Fe(II). We previously reported that FeoC binds to and protects the FeoB transporter from FtsH-mediated proteolysis. In the present study, we report proteolytic regulation of FeoC that occurs in an oxygen-dependent fashion. While relatively stable under low-oxygen conditions, FeoC was rapidly degraded by the Lon protease under high-oxygen conditions. The putative Fe-S cluster of FeoC seemed to function as an oxygen sensor to control FeoC stability, as evidenced by the finding that mutation of the putative Fe-S cluster-binding site greatly increased FeoC stability under high-oxygen conditions. Salmonella ectopically expressing the feoB and feoC genes was able to accumulate FeoB and FeoC only under low-oxygen conditions, suggesting that FeoC proteolysis prevents Salmonella from accumulating the FeoB transporter under high-oxygen conditions. Finally, we propose that Lon-mediated FeoC proteolysis followed by FtsH-mediated FeoB proteolysis helps Salmonella to avoid uncontrolled Fe(II) uptake during the radical environmental changes encountered when shifting from low-iron anaerobic conditions to high-iron aerobic conditions.
The role of transition metal transporters for iron, zinc, manganese, and copper in the pathogenesis of Yersinia pestis.
Yersinia pestis, the causative agent of bubonic, septicemic and pneumonic plague, encodes a multitude of Fe transport systems. Some of these are defective due to frameshift or IS element insertions, while others are functional in vitro but have no established role in causing infections. Indeed only 3 Fe transporters (Ybt, Yfe and Feo) have been shown to be important in at least one form of plague. The yersiniabactin (Ybt) system is essential in the early dermal/lymphatic stages of bubonic plague, irrelevant in the septicemic stage, and critical in pneumonic plague. Two Mn transporters have been characterized (Yfe and MntH). These two systems play a role in bubonic plague but the double yfe mntH mutant is fully virulent in a mouse model of pneumonic plague. The same in vivo phenotype occurs with a mutant lacking two (Yfe and Feo) of four ferrous transporters. A role for the Ybt siderophore in Zn acquisition has been revealed. Ybt-dependent Zn acquisition uses a transport system completely independent of the Fe-Ybt uptake system. Together Ybt components and ZnuABC play a critical role in Zn acquisition in vivo. Single mutants in either system retain high virulence in a mouse model of septicemic plague while the double mutant is completely avirulent.
Global identification of genes affecting iron-sulfur cluster biogenesis and iron homeostasis.
Iron-sulfur (Fe-S) clusters are ubiquitous cofactors that are crucial for many physiological processes in all organisms. In Escherichia coli, assembly of Fe-S clusters depends on the activity of the iron-sulfur cluster (ISC) assembly and sulfur mobilization (SUF) apparatus. However, the underlying molecular mechanisms and the mechanisms that control Fe-S cluster biogenesis and iron homeostasis are still poorly defined. In this study, we performed a global screen to identify the factors affecting Fe-S cluster biogenesis and iron homeostasis using the Keio collection, which is a library of 3,815 single-gene E. coli knockout mutants. The approach was based on radiolabeling of the cells with [2-(14)C]dihydrouracil, which entirely depends on the activity of an Fe-S enzyme, dihydropyrimidine dehydrogenase. We identified 49 genes affecting Fe-S cluster biogenesis and/or iron homeostasis, including 23 genes important only under microaerobic/anaerobic conditions. This study defines key proteins associated with Fe-S cluster biogenesis and iron homeostasis, which will aid further understanding of the cellular mechanisms that coordinate the processes. In addition, we applied the [2-(14)C]dihydrouracil-labeling method to analyze the role of amino acid residues of an Fe-S cluster assembly scaffold (IscU) as a model of the Fe-S cluster assembly apparatus. The analysis showed that Cys37, Cys63, His105, and Cys106 are essential for the function of IscU in vivo, demonstrating the potential of the method to investigate in vivo function of proteins involved in Fe-S cluster assembly.
Salmonella acquires ferrous iron from haemophagocytic macrophages.
Bacteria harbour both ferrous and ferric iron transporters. We now report that infection of macrophages and mice with a Salmonella enterica Typhimurium strain containing an inactivated feoB-encoded ferrous iron transporter results in increased bacterial replication, compared to infection with wild type. Inactivation of other cation transporters, SitABCD or MntH, did not increase bacterial replication. The feoB mutant strain does not have an intrinsically faster growth rate. Instead, increased replication correlated with increased expression in macrophages of the fepB-encoded bacterial ferric iron transporter and also required siderophores, which capture ferric iron. Co-infection of mice with wild type and a feoB mutant strain yielded a different outcome: FeoB is clearly required for tissue colonization. In co-infected primary mouse macrophages, FeoB is required for S. Typhimurium replication if the macrophages were IFNgamma treated and contain phagocytosed erythrocytes, a model for haemophagocytosis. Haemophagocytes are macrophages that have engulfed erythrocytes and/or leucocytes and can harbour Salmonella in mice. These observations suggest that Salmonella acquires ferrous iron from haemophagocytic macrophages.
Comparative genomics of freshwater Fe-oxidizing bacteria: implications for physiology, ecology, and systematics.
The two microaerophilic, Fe-oxidizing bacteria (FeOB) Sideroxydans ES-1 and Gallionella ES-2 have single circular chromosomes of 3.00 and 3.16 Mb that encode 3049 and 3006 genes, respectively. Multi-locus sequence analysis (MLSA) confirmed the relationship of these two organisms to one another, and indicated they may form a novel order, the Gallionellalaes, within the Betaproteobacteria. Both are adapted for chemolithoautotropy, including pathways for CO2-fixation, and electron transport pathways adapted for growth at low O2-levels, an important adaptation for growing on Fe(II). Both genomes contain Mto-genes implicated in iron-oxidation, as well as other genes that could be involved in Fe-oxidation. Nearly 10% of their genomes are devoted to environmental sensing, signal transduction, and chemotaxis, consistent with their requirement for growing in narrow redox gradients of Fe(II) and O2. There are important differences as well. Sideroxydans ES-1 is more metabolically flexible, and can utilize reduced S-compounds, including thiosulfate, for lithotrophic growth. It has a suite of genes for nitrogen fixation. Gallionella ES-2 contains additional gene clusters for exopolysaccharide production, and has more capacity to resist heavy metals. Both strains contain genes for hemerythrins and globins, but ES-1 has an especially high numbers of these genes that may be involved in oxygen homeostasis, or storage. The two strains share homology with the marine FeOB Mariprofundus ferrooxydans PV-1 in CO2 fixation genes, and respiratory genes. In addition, ES-1 shares a suite of 20 potentially redox active genes with PV-1, as well as a large prophage. Combined these genetic, morphological, and physiological differences indicate that these are two novel species, Sideroxydans lithotrophicus ES-1(T) (ATCC 700298(T); JCM 14762; DSMZ 22444; NCMA B100), and Gallionella capsiferriformans ES-2(T) (ATCC 700299(T); JCM 14763; DSMZ 22445; NCMA B101).
Catecholate siderophores protect bacteria from pyochelin toxicity.
BACKGROUND: Bacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition. METHODS AND PRINCIPAL FINDINGS: Here we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast, addition of the carboxylate-type siderophore, citrate, did not prevent pyochelin-induced ROS increases and their associated toxicity. CONCLUSIONS: We have shown that the catecholate siderophore enterobactin protects E. coli against the toxic effects of pyochelin by reducing ROS. Thus, it appears that catecholate siderophores can behave as protectors of oxidative stress. These results support the idea that siderophores can have physiological roles aside from those in iron acquisition.
BACKGROUND: Despite the availability of numerous complete genome sequences from E. coli strains, published genome-scale metabolic models exist only for two commensal E. coli strains. These models have proven useful for many applications, such as engineering strains for desired product formation, and we sought to explore how constructing and evaluating additional metabolic models for E. coli strains could enhance these efforts. RESULTS: We used the genomic information from 16 E. coli strains to generate an E. coli pangenome metabolic network by evaluating their collective 76,990 ORFs. Each of these ORFs was assigned to one of 17,647 ortholog groups including ORFs associated with reactions in the most recent metabolic model for E. coli K-12. For orthologous groups that contain an ORF already represented in the MG1655 model, the gene to protein to reaction associations represented in this model could then be easily propagated to other E. coli strain models. All remaining orthologous groups were evaluated to see if new metabolic reactions could be added to generate a pangenome-scale metabolic model (iEco1712_pan). The pangenome model included reactions from a metabolic model update for E. coli K-12 MG1655 (iEco1339_MG1655) and enabled development of five additional strain-specific genome-scale metabolic models. These additional models include a second K-12 strain (iEco1335_W3110) and four pathogenic strains (two enterohemorrhagic E. coli O157:H7 and two uropathogens). When compared to the E. coli K-12 models, the metabolic models for the enterohemorrhagic (iEco1344_EDL933 and iEco1345_Sakai) and uropathogenic strains (iEco1288_CFT073 and iEco1301_UTI89) contained numerous lineage-specific gene and reaction differences. All six E. coli models were evaluated by comparing model predictions to carbon source utilization measurements under aerobic and anaerobic conditions, and to batch growth profiles in minimal media with 0.2% (w/v) glucose. An ancestral genome-scale metabolic model based on conserved ortholog groups in all 16 E. coli genomes was also constructed, reflecting the conserved ancestral core of E. coli metabolism (iEco1053_core). Comparative analysis of all six strain-specific E. coli models revealed that some of the pathogenic E. coli strains possess reactions in their metabolic networks enabling higher biomass yields on glucose. Finally the lineage-specific metabolic traits were compared to the ancestral core model predictions to derive new insight into the evolution of metabolism within this species. CONCLUSION: Our findings demonstrate that a pangenome-scale metabolic model can be used to rapidly construct additional E. coli strain-specific models, and that quantitative models of different strains of E. coli can accurately predict strain-specific phenotypes. Such pangenome and strain-specific models can be further used to engineer metabolic phenotypes of interest, such as designing new industrial E. coli strains.
More than 200 genes required for methane formation from H(2) and CO(2) and energy conservation are present in Methanothermobacter marburgensis and Methanothermobacter thermautotrophicus.
The hydrogenotrophic methanogens Methanothermobacter marburgensis and Methanothermobacter thermautotrophicus can easily be mass cultured. They have therefore been used almost exclusively to study the biochemistry of methanogenesis from H(2) and CO(2), and the genomes of these two model organisms have been sequenced. The close relationship of the two organisms is reflected in their genomic architecture and coding potential. Within the 1,607 protein coding sequences (CDS) in common, we identified approximately 200 CDS required for the synthesis of the enzymes, coenzymes, and prosthetic groups involved in CO(2) reduction to methane and in coupling this process with the phosphorylation of ADP. Approximately 20 additional genes, such as those for the biosynthesis of F(430) and methanofuran and for the posttranslational modifications of the two methyl-coenzyme M reductases, remain to be identified.
The initiation of GTP hydrolysis by the G-domain of FeoB: insights from a transition-state complex structure.
The polytopic membrane protein FeoB is a ferrous iron transporter in prokaryotes. The protein contains a potassium-activated GTPase domain that is essential in regulating the import of iron and conferring virulence to many disease-causing bacteria. However, the mechanism by which the G-domain of FeoB hydrolyzes GTP is not well understood. In particular, it is not yet known how the pivotal step in GTP hydrolysis is achieved: alignment of a catalytic water molecule. In the current study, the crystal structure of the soluble domains from Streptococcus thermophilus FeoB (NFeoB(St)) in complex with the activating potassium ion and a transition-state analogue, GDPAlF(4) (-), reveals a novel mode of water alignment involving contacts with the protein backbone only. In parallel to the structural studies, a series of seven mutant proteins were constructed that targeted conserved residues at the active site of NFeoB(St), and the nucleotide binding and hydrolysis properties of these were measured and compared to the wild-type protein. The results show that mutations in Thr35 abolish GTPase activity of the protein, while other conserved residues (Tyr58, Ser64, Glu66 and Glu67) are not required for water alignment by NFeoB(St). Together with the crystal structure, the findings suggest a new mechanism for hydrolysis initiation in small G-proteins, in which the attacking water molecule is aligned by contacts with the protein backbone only.
Role of the FeoB protein and siderophore in promoting virulence of Xanthomonas oryzae pv. oryzae on rice.
Xanthomonas oryzae pv. oryzae causes bacterial blight, a serious disease of rice. Our analysis revealed that the X. oryzae pv. oryzae genome encodes genes responsible for iron uptake through FeoB (homolog of the major bacterial ferrous iron transporter) and a siderophore. A mutation in the X. oryzae pv. oryzae feoB gene causes severe virulence deficiency, growth deficiency in iron-limiting medium, and constitutive production of a siderophore. We identified an iron regulated xss gene cluster, in which xssABCDE (Xanthomonas siderophore synthesis) and xsuA (Xanthomonas siderophore utilization) genes encode proteins involved in biosynthesis and utilization of X. oryzae pv. oryzae siderophore. Mutations in the xssA, xssB, and xssE genes cause siderophore deficiency and growth restriction under iron-limiting conditions but are virulence proficient. An xsuA mutant displayed impairment in utilization of native siderophore, suggesting that XsuA acts as a specific receptor for a ferric-siderophore complex. Histochemical and fluorimetric assays with gusA fusions indicate that, during in planta growth, the feoB gene is expressed and that the xss operon is not expressed. This study represents the first report describing a role for feoB in virulence of any plant-pathogenic bacterium and the first functional characterization of a siderophore-biosynthetic gene cluster in any xanthomonad.
Metal uptake in host-pathogen interactions: role of iron in Porphyromonas gingivalis interactions with host organisms.
Crystallization and preliminary X-ray diffraction analysis of the truncated cytosolic domain of the iron transporter FeoB.
FeoB-family proteins are widely distributed in bacteria and archaea and are involved in high-affinity Fe(2+) uptake through the plasma membrane. FeoB consists of an N-terminal cytosolic region followed by a C-terminal transmembrane region. The cytosolic region contains small GTPase and GDP dissociation inhibitor-like domains, which serve a regulatory function. The truncated cytosolic region of the iron transporter FeoB from Thermotoga maritima was overexpressed, purified and crystallized. Four native or SeMet crystal forms in a nucleotide-free state or in complex with either GDP or GMPPNP diffracted to resolutions of between 1.5 and 2.1 A.
Bactericidal effect of zero-valent iron nanoparticles on Escherichia coli.
Zero-valent iron nanoparticles (nano-Fe0) in aqueous solution rapidly inactivated Escherichia coli. A strong bactericidal effect of nano-Fe0 was found under deaerated conditions, with a linear correlation between log inactivation and nano-Fe0 dose (0.82 log inactivation/mg/L nano-Fe0 x h). The inactivation of E. coli under air saturation required much higher nano-Fe0 doses due to the corrosion and surface oxidation of nano-Fe0 by dissolved oxygen. Significant physical disruption of the cell membranes was observed in E. coli exposed to nano-Fe0, which may have caused the inactivation or enhanced the biocidal effects of dissolved iron. The reaction of Fe(II) with intracellular oxygen or hydrogen peroxide also may have induced oxidative stress by producing reactive oxygen species. The bactericidal effect of nano-Fe0 was a unique property of nano-Fe0, which was not observed in other types of iron-based compounds.
Discovery of Fur binding site clusters in Escherichia coli by information theory models.
Fur is a DNA binding protein that represses bacterial iron uptake systems. Eleven footprinted Escherichia coli Fur binding sites were used to create an initial information theory model of Fur binding, which was then refined by adding 13 experimentally confirmed sites. When the refined model was scanned across all available footprinted sequences, sequence walkers, which are visual depictions of predicted binding sites, frequently appeared in clusters that fit the footprints ( approximately 83% coverage). This indicated that the model can accurately predict Fur binding. Within the clusters, individual walkers were separated from their neighbors by exactly 3 or 6 bases, consistent with models in which Fur dimers bind on different faces of the DNA helix. When the E. coli genome was scanned, we found 363 unique clusters, which includes all known Fur-repressed genes that are involved in iron metabolism. In contrast, only a few of the known Fur-activated genes have predicted Fur binding sites at their promoters. These observations suggest that Fur is either a direct repressor or an indirect activator. The Pseudomonas aeruginosa and Bacillus subtilis Fur models are highly similar to the E. coli Fur model, suggesting that the Fur-DNA recognition mechanism may be conserved for even distantly related bacteria.
When grown in divalent cation-limited medium, Pseudomonas aeruginosa becomes resistant to cationic antimicrobial peptides and polymyxin B. This resistance is regulated by the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems. To further characterize Mg(2+) regulation in P. aeruginosa, microarray transcriptional profiling was conducted to compare wild-type P. aeruginosa grown under Mg(2+)-limited and Mg(2+)-replete conditions to isogenic phoP and pmrA mutants grown under Mg(2+)-limited conditions. Under Mg(2+)-limited conditions (0.02 mM Mg(2+)), approximately 3% of the P. aeruginosa genes were differentially expressed compared to the expression in bacteria grown under Mg(2+)-replete conditions (2 mM Mg(2+)). Only a modest subset of the Mg(2+)-regulated genes were regulated through either PhoP or PmrA. To determine which genes were directly regulated, a bioinformatic search for conserved binding motifs was combined with confirmatory reverse transcriptase PCR and gel shift promoter binding assays, and the results indicated that very few genes were directly regulated by these response regulators. It was found that in addition to the previously known oprH-phoP-phoQ operon and the pmrHFIJKLM-ugd operon, the PA0921 and PA1343 genes, encoding small basic proteins, were regulated by Mg(2+) in a PhoP-dependent manner. The number of known PmrA-regulated genes was expanded to include the PA1559-PA1560, PA4782-PA4781, and feoAB operons, in addition to the previously known PA4773-PA4775-pmrAB and pmrHFIJKLM-ugd operons.
Temporal transcriptomic analysis as Desulfovibrio vulgaris Hildenborough transitions into stationary phase during electron donor depletion.
Desulfovibrio vulgaris was cultivated in a defined medium, and biomass was sampled for approximately 70 h to characterize the shifts in gene expression as cells transitioned from the exponential to the stationary phase during electron donor depletion. In addition to temporal transcriptomics, total protein, carbohydrate, lactate, acetate, and sulfate levels were measured. The microarray data were examined for statistically significant expression changes, hierarchical cluster analysis, and promoter element prediction and were validated by quantitative PCR. As the cells transitioned from the exponential phase to the stationary phase, a majority of the down-expressed genes were involved in translation and transcription, and this trend continued at the remaining times. There were general increases in relative expression for intracellular trafficking and secretion, ion transport, and coenzyme metabolism as the cells entered the stationary phase. As expected, the DNA replication machinery was down-expressed, and the expression of genes involved in DNA repair increased during the stationary phase. Genes involved in amino acid acquisition, carbohydrate metabolism, energy production, and cell envelope biogenesis did not exhibit uniform transcriptional responses. Interestingly, most phage-related genes were up-expressed at the onset of the stationary phase. This result suggested that nutrient depletion may affect community dynamics and DNA transfer mechanisms of sulfate-reducing bacteria via the phage cycle. The putative feoAB system (in addition to other presumptive iron metabolism genes) was significantly up-expressed, and this suggested the possible importance of Fe2+ acquisition under metal-reducing conditions. The expression of a large subset of carbohydrate-related genes was altered, and the total cellular carbohydrate levels declined during the growth phase transition. Interestingly, the D. vulgaris genome does not contain a putative rpoS gene, a common attribute of the delta-Proteobacteria genomes sequenced to date, and the transcription profiles of other putative rpo genes were not significantly altered. Our results indicated that in addition to expected changes (e.g., energy conversion, protein turnover, translation, transcription, and DNA replication and repair), genes related to phage, stress response, carbohydrate flux, the outer envelope, and iron homeostasis played important roles as D. vulgaris cells experienced electron donor depletion.
Group A streptococcus (GAS) is a common pathogen of the human skin and mucosal surfaces capable of producing a variety of diseases. In this study, we investigated regulation of iron uptake in GAS and the role of a putative transcriptional regulator named MtsR (for Mts repressor) with homology to the DtxR family of metal-dependent regulatory proteins. An mtsR mutant was constructed in NZ131 (M49 serotype) and analyzed. Western blot and RNA analysis showed that mtsR inactivation results in constitutive transcription of the sia (streptococcal iron acquisition) operon, which was negatively regulated by iron in the parent strain. A recombinant MtsR with C-terminal His(6) tag fusion (rMtsR) was cloned and purified. Electrophoretic mobility gel shift assays demonstrated that rMtsR specifically binds to the sia promoter region in an iron- and manganese-dependent manner. Together, these observations indicate that MtsR directly represses the sia operon during cell growth under conditions of high metal levels. Consistent with deregulation of iron uptake, the mtsR mutant is hypersensitive to streptonigrin and hydrogen peroxide, and (55)Fe uptake assays demonstrate that it accumulates 80% +/- 22.5% more iron than the wild-type strain during growth in complete medium. Studies with a zebrafish infection model revealed that the mtsR mutant is attenuated for virulence in both the intramuscular and the intraperitoneal routes. In conclusion, MtsR, a new regulatory protein in GAS, controls iron homeostasis and has a role in disease production.
Shigella dysenteriae ShuS promotes utilization of heme as an iron source and protects against heme toxicity.
Shigella dysenteriae serotype 1, a major cause of bacillary dysentery in humans, can use heme as a source of iron. Genes for the transport of heme into the bacterial cell have been identified, but little is known about proteins that control the fate of the heme molecule after it has entered the cell. The shuS gene is located within the heme transport locus, downstream of the heme receptor gene shuA. ShuS is a heme binding protein, but its role in heme utilization is poorly understood. In this work, we report the construction of a chromosomal shuS mutant. The shuS mutant was defective in utilizing heme as an iron source. At low heme concentrations, the shuS mutant grew slowly and its growth was stimulated by either increasing the heme concentration or by providing extra copies of the heme receptor shuA on a plasmid. At intermediate heme concentrations, the growth of the shuS mutant was moderately impaired, and at high heme concentrations, shuS was required for growth on heme. The shuS mutant did not show increased sensitivity to hydrogen peroxide, even at high heme concentrations. ShuS was also required for optimal utilization of heme under microaerobic and anaerobic conditions. These data are consistent with the model in which ShuS binds heme in a soluble, nontoxic form and potentially transfers the heme from the transport proteins in the membrane to either heme-containing or heme-degrading proteins. ShuS did not appear to store heme for future use.
Cloning, sequencing and partial characterisation of sorbitol transporter (srlT) gene encoding phosphotransferase system, glucitol/sorbitol-specific IIBC components of Erwinia herbicola ATCC 21998.
A DNA fragment of approximately 1500 bp, harbouring the sorbitol transport gene (srlT), was amplified from the chromosomal DNA of Erwinia herbicola ATCC 21998 by PCR and cloned in Escherichia coli JM109. Degenerate oligonucleotide primers used were designed based on the conserved regions in the gene sequences within the gut operon of E. coli (Gene Bank accession no. J02708) and the srl operon of Erwinia amylovora (Gene Bank accession no. Y14603). The cloned DNA fragment was sequenced and found to contain an open reading frame of 1473 nucleotides coding for a protein of 491 amino acids, corresponding to a mass of 52410 Da. The nucleotide sequence of this ORF was highly homologous to that of the gutA gene of Escherichia coli gut operon, the srlE gene of Shigella flexrni and the sorbitol transporter gene sequence of Escherichia coli K12 (Gene Bank accession nos. J02708, AE016987 and D90892 respectively). The protein sequence showed significant homology to that of the phosphotransferase system i.e. the glucitol/sorbitol-specific IIBC components of Escherichia coli and Erwinia amylovora (P56580, O32522). The cloned DNA fragment was introduced into a pRA90 vector and the recombinant was used for developing srlT mutants of Erwinia herbicola, by homologous recombination. Mutants obtained were unable to grow on minimal medium with sorbitol. The insertion of the pRA90 vector inside the srlT gene sequence of the mutants was confirmed by DNA-DNA hybridisation.
Discovery of a haem uptake system in the soil bacterium Bradyrhizobium japonicum.
In Bradyrhizobium japonicum, the nitrogen-fixing symbiont of soybeans, we have identified a haem uptake system, Hmu, that comprises a cluster of nine open reading frames. Predicted products of these genes include: HmuR, a TonB-dependent haem receptor in the outer membrane; HmuT, a periplasmic haem-binding protein; and HmuUV, an ABC transporter in the inner membrane. Furthermore, we identified homologues of ExbBD and TonB, that are required for energy transduction from the inner to the outer membrane. Mutant analysis and complementation tests indicated that HmuR and the ExbBD-TonB system, but not the HmuTUV transporter, are essential for haem uptake or haem acquisition from haemoglobin and leghaemoglobin. The TonB system seems to be specific for haem uptake as it is dispensable for siderophore uptake. Therefore, we propose the existence of a second TonB homologue functioning in the uptake of Fe-chelates. When tested on soybean host plants, hmuT-hmuR and exbD-tonB mutants exhibited wild-type symbiotic properties. Thus, haem uptake is not essential for symbiotic nitrogen fixation but it may enable B. japonicum to have access to alternative iron sources in its non-symbiotic state. Transcript analysis and expression studies with lacZ fusions showed that expression of hmuT and hmuR is induced under low iron supply. The same was observed in fur and irr mutant backgrounds although maximal induction levels were decreased. We conclude either that both regulators, Fur and Irr, independently mediate transcriptional control by iron or that a yet unknown iron regulatory system activates gene expression under iron deprivation. An A/T-rich cis-acting element, located in the promoter region of the divergently transcribed hmuTUV and hmuR genes, is possibly required for this type of iron control.
Identification and characterization of the sodA genes encoding manganese superoxide dismutases in Vibrio parahaemolyticus, Vibrio mimicus, and Vibrio vulnificus.
Sequencing of Fur titration assay-positive clones obtained from genomic DNA libraries of Vibrio parahaemolyticus, V. mimicus and V. vulnificus revealed open reading frames encoding proteins of 202, 205 and 202 amino acid residues, respectively. Each open reading frame was preceded by a predicted Fur box which overlaps a likely promoter with similarity to the -10 and -35 consensus sequence of Escherichia coli. The deduced amino acid sequences shared considerable homology with bacterial Mn-containing superoxide dismutases (MnSODs). Consistent with this, these Vibrio strains produced proteins with SOD activity resistant to inhibition by H2O2 and KCN only when grown under iron-limiting conditions. Primer extension analysis of the total RNA from these vibrios revealed iron-repressible expression of the genes. Furthermore, when grown under iron-limiting conditions, E. coli carrying a plasmid with each cloned gene overexpressed protein with the same electrophoretic mobility and insensitivity of SOD activity to H2O2 and KCN. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing revealed that proteins (MnSODs) having N-terminal amino acid sequences consistent with those deduced from the corresponding genes were present in cell lysates of the vibrios grown under these iron-limited conditions. These results demonstrate that the genes cloned in this study are sodA homologs encoding MnSODs, whose expression is regulated by the iron status of the growth medium. PCR using a primer set based on the V. parahaemolyticus sodA sequence revealed the presence of homologous genes in certain other Vibrio species.
Requirement of the Pseudomonas aeruginosa tonB gene for high-affinity iron acquisition and infection.
To investigate the contribution of the TonB protein to high-affinity iron acquisition in Pseudomonas aeruginosa, we constructed tonB-inactivated mutants from strain PAO1 and its derivative deficient in producing the siderophores pyoverdin and pyochelin. The tonB mutants could not grow in a free-iron-restricted medium prepared by apotransferrin addition, even though the medium was supplemented with each purified siderophore or with a heme source (hemoglobin or hemin). The tonB inactivation was shown to make P. aeruginosa unable to acquire iron from the transferrin with either siderophore. Introduction of a plasmid carrying the intact tonB gene restored growth of the tonB mutant of PAO1 in the free-iron-restricted medium without any supplements and restored growth of the tonB mutant of the siderophore-deficient derivative in the medium supplemented with pyoverdin, pyochelin, hemoglobin, or hemin. In addition, animal experiments showed that, in contrast to PAO1, the tonB mutant of PAO1 could not grow in vivo, such as in the muscles and lungs of immunosuppressed mice, and could not kill any of the animals. The in vivo growth ability and lethal virulence were also restored by introduction of the tonB-carrying plasmid in the tonB mutant. These results indicate clearly that the intact tonB gene-and, therefore, the TonB protein encoded by it-is essential for iron acquisition mediated by pyoverdin and pyochelin and via heme uptake in P. aeruginosa and suggest that the TonB-dependent iron acquisition may be essential for P. aeruginosa to infect the animal host.
Cloning and expression of the secA gene of a marine bacterium, Vibrio alginolyticus, and analysis of its function in Escherichia coli.
We report the cloning, sequencing and functional characterization of the secA gene of a marine bacterium, Vibrio alginolyticus, which has been suggested to utilize ATP and the sodium motive force for protein translocation. Oligodeoxynucleotides corresponding to highly conserved regions of Escherichia coli secA located in the high affinity ATP binding site were utilized as PCR primers to clone the secA gene of V. alginolyticus. It was shown to encode a 103.3-kDa protein. The deduced amino acid sequence of V. alginolyticus SecA (VaSecA) exhibits a high degree of identity (72.7%) to SecA of E. coli (EcSecA). The secA gene of E. coli forms an operon with upstream orfX, whereas no counterpart is present upstream of V. alginolyticus secA. Azide derepresses the EcSecA translation, whereas the level of VaSecA was unaffected by azide. Expression of VaSecA in E. coli carrying a temperature-sensitive secA mutation restored both growth and protein translocation at a non-permissive temperature. VaSecA was thus able to substitute for EcSecA despite the fact that the energy requirement for protein translocation differs between the two organisms. VaSecA was overproduced in V. alginolyticus and purified to homogeneity for N-terminal sequencing. The endogenous ATPase activity of the purified VaSecA was comparable with that of EcSecA. 1998 Elsevier Science B.V.
Cloning, overexpression, and characterization of glutaredoxin 2, an atypical glutaredoxin from Escherichia coli.
Glutaredoxin 2 (Grx2) from Escherichia coli catalyzes GSH-disulfide oxidoreductions via two redox-active cysteine residues, but in contrast to glutaredoxin 1 (Grx1) and glutaredoxin 3 (Grx3), is not a hydrogen donor for ribonucleotide reductase. To characterize Grx2, a chromosomal fragment containing the E. coli Grx2 gene (grxB) was cloned and sequenced. grxB (645 base pairs) is located between the rimJ and pyrC genes while an open reading frame immediately upstream grxB encodes a novel transmembrane protein of 402 amino acids potentially belonging to class II of substrate export transporters. The deduced amino acid sequence for Grx2 comprises 215 residues with a molecular mass of 24.3 kDa. There is almost no similarity between the amino acid sequence of Grx2 and Grx1 or Grx3 (both 9-kDa proteins) with the exception of the active site which is identical in all three glutaredoxins (C9PYC12 for Grx2). Only limited similarities were noted to glutathione S-transferases (Grx2 amino acids 16-72), and protein disulfide isomerases from different organisms (Grx2 amino acids 70-180). Grx2 was overexpressed and purified to homogeneity and its activity was compared with those of Grx1 and Grx3 using GSH, NADPH, and glutathione reductase in the reduction of 0.7 mM beta-hydroxyethyl disulfide. The three glutaredoxins had similar apparent Km values for GSH (2-3 mM) but Grx2 had the highest apparent kcat (554 s-1). Expression of two truncated forms of Grx2 (1-114 and 1-133) which have predicted secondary structures similar to Grx1 (betaalphabetaalphabetabetaalpha) gave rise to inclusion bodies. The mutant proteins were resolubilized and purified but lacked GSH-disulfide oxidoreductase activity. The latter should therefore require the participation of amino acid residues from the COOH-terminal half of the molecule and is probably not confined to a Grx1-like NH2-terminal subdomain. Grx2 being radically different from the presently known glutaredoxins in terms of molecular weight, amino acid sequence, catalytic activity, and lack of a consensus GSH-binding site is the first member of a novel class of glutaredoxins.
Bacteriocin 28b from Serratia marcescens binds to Escherichia coli outer membrane proteins OmpA and OmpF and to lipopolysaccharide (LPS) core (J. Enfedaque, S. Ferrer, J. F. Guasch, J. Tomas, and M. Reque, Can. J. Microbiol. 42:19-26, 1996). A cosmid-based genomic library of S. marcescens was introduced into E. coli NM554, and clones were screened for bacteriocin 28b resistance phenotype. One clone conferring resistance to bacteriocin 28b and showing an altered LPS core mobility in polyacrylamide gel electrophoresis was found. Southern blot experiments using DNA fragments containing E. coli rfa genes as probes suggested that the recombinant cosmid contained S. marcescens genes involved in LPS core biosynthesis. Subcloning, isolation of subclones and Tn5tac1 insertion mutants, and sequencing allowed identification of two apparently cotranscribed genes. The deduced amino acid sequence from the upstream gene showed 80% amino acid identity to the KdtA protein from E. coli, suggesting that this gene codes for the 3-deoxy-manno-octulosonic acid transferase of S. marcescens. The downstream gene (kdtX) codes for a protein showing 20% amino acid identity to the Haemophilus influenzae kdtB gene product. The S. marcescens KdtX protein is unrelated to the KdtB protein of E. coli K-12. Expression of the kdtX gene from S. marcescens in E. coli confers resistance to bacteriocin 28b.
Characterization of the aegA locus of Escherichia coli: control of gene expression in response to anaerobiosis and nitrate.
Analysis of the DNA sequence upstream of the narQ gene, which encodes the second nitrate-responsive sensor-transmitter protein in Escherichia coli, revealed an open reading frame (ORF) whose product shows a high degree of similarity to a number of iron-sulfur proteins as well as to the beta subunit of glutamate synthase (gltD) of E. coli. This ORF, located at 53.0 min on the E. coli chromosome, is divergently transcribed and is separated by 206 bp from the narQ gene. Because of the small size of the intergenic region, we reasoned that the genes may be of related function and/or regulated in a similar fashion. An aegA-lacZ gene fusion was constructed and examined in vivo; aegA expression was induced 11-fold by anaerobiosis and repressed 5-fold by nitrate. This control was mediated by the fnr, narX, narQ, and narL gene products. Analysis of an aegA mutant indicated that the aegA gene product is not essential for cell respiration or fermentation or for the utilization of ammonium or the amino acids L-alanine, L-arginine, L-glutamic acid, glycine, and DL-serine as sole nitrogen sources. The ORF was designated aegA to reflect that it is an anaerobically expressed gene. The structural properties of the predicted AegA amino acid sequence and the regulation of aegA are discussed with regard to the possible function of aegA in E. coli.
Reactivity of the N-terminal cysteine residues in active and inactive forms of FNR, and O2-responsive, Fe containing transcriptional regulator of Escherichia coli.
FNR, the O2-responsive gene regulator of anaerobic respiratory genes in Escherichia coli, contains an N-terminal cluster of four cysteine residues (Cys16-X3-Cys20-X2-Cys23-X5-Cys29), three of which are thought to be involved in the binding of an iron cofactor. The accessibility of the cysteine residues for iodoacetate is known to increase upon switch from the active (anaerobic) to the inactive (aerobic or metal depleted) state. It was analyzed which residues become accessible under either condition. Up to four modified forms, FNR-I, FNR-II, FNR-III, and FNR-IV, containing approximately 1, 2, 3.5, and 5 carboxymethyl groups, were obtained either by reaction in vivo and in vitro under conditions of aerobiosis, anaerobiosis, or iron limitation. By N-terminal sequencing, the carboxymethylated cysteine residues were identified. The amount of label in each of the four cysteine residues increased rather uniformly and gradually from FNR-I to FNR-IV irrespective of the condition of labeling; only Cys16 was preferentially labeled to some extent. It is concluded that the four essential cysteine residues change their accessibility in a similar way in the switch from active to inactive (aerobic or metal depleted) FNR, without specific differences in their reaction or function. Potential modes of Fe-binding by the cysteine residues are discussed. In addition, a different type of interaction of Fe(II) with FNR is described. The interaction occurred also in FNR carboxymethylated at approximately three cysteine residues.
The mobile ferrous iron pool in Escherichia coli is bound to a phosphorylated sugar derivative.
Based on in vivo Mossbauer spectroscopy it has previously been demonstrated that the intracellular iron pool of Escherichia coli, grown in iron deficient media supplemented with siderophores as the sole iron source, is dominated by a single Fe2+ and a single Fe3+ species. We have isolated the ferrous ion species and have purified it employing native column PAGE, chromatography and ultrafiltration. The purified compound displays an Mapp of 2.2 kDa and an extremely low isoelectric point (pI) of 1.05. It is shown that this ferrous ion binding compound is neither a protein nor a nucleotide, rather it is composed mainly of phosphorylated sugar derivatives. This compound binds approximately 40% of the cytoplasmic iron. Therefore it is proposed that this oligomeric ferrous carbohydrate phosphate represents the long sought after mobile, low molecular mass iron pool.
Sequencing and characterization of the sdaB gene from Escherichia coli K-12.
The sdaB gene which codes for the second L-serine deaminase (L-SD) of Escherichia coli K-12 has been sequenced and shown to be very similar to the sdaA gene which codes for the first L-serine deaminase. sdaB is transcribed in rich medium, particularly in the absence of glucose, and is under the control of catabolite activator protein. A mutation which established expression of the sdaB gene and synthesis of L-serine deaminase 2 in minimal medium has been demonstrated to result in a change in the ribosome-binding site of the sdaB gene.
Structural model of the nucleotide-binding conserved component of periplasmic permeases.
The amino acid sequences of 17 bacterial membrane proteins that are components of periplasmic permeases and function in the uptake of a variety of small molecules and ions are highly homologous to each other and contain sequence motifs characteristic of nucleotide-binding proteins. These proteins are known to bind ATP and are postulated to be the energy-coupling components of the permeases. Several medically important eukaryotic proteins, including the multidrug-resistance transporters and the protein encoded by the cystic fibrosis gene, are also homologous to this family. By multiple sequence alignment of these 17 proteins, the consensus sequence, secondary structure, and surface exposure were predicted. The secondary structural motifs that are conserved among nucleotide-binding proteins were identified in adenylate kinase, p21ras, and elongation factor Tu by superposition of their known tertiary structures. The equivalent secondary structural elements in the predicted conserved component were located. These, together with sequence information, served as guides for alignment with adenylate kinase. A model for the structure of the ATP-binding domain of the permease proteins is proposed by analogy to the adenylate kinase structure. The characteristics of several permease mutations and biochemical data lend support to the model.
Escherichia coli orfE (upstream of pyrE) encodes RNase PH.
RNase PH from extracts of Escherichia coli was purified to homogeneity and subjected to NH2-terminal sequencing. Comparison of this sequence with all open reading frames in the GenBank data base revealed at least 95% identity to an unidentified open reading frame (orfE) upstream of pyrE at 81.7 min on the E. coli chromosome. Clones of orfE overexpress RNase PH activity, verifying that orfE encodes this ribonuclease. We suggest that orfE be renamed rph.
Molecular biology of carbon-phosphorus bond cleavage. Cloning and sequencing of the phn (psiD) genes involved in alkylphosphonate uptake and C-P lyase activity in Escherichia coli B.
Whereas bacteria such as Escherichia coli have been known for some time to cleave carbon-phosphorus (C-P) bonds in unactivated alkylphosphonates, the enzymes responsible for C-P lyase activity have resisted detection or purification. Genes from E. coli B that support growth on alkylphosphonates as the sole phosphorus source have now been cloned (B. L. Wanner and J. A. Boline, unpublished data). Deletion analysis demonstrated that at least 13 kilobases of DNA information is required for E. coli to express the phosphonate utilization phenotype (Phn+). The complete nucleotide sequence of 15,611 bases has been determined, and the gene structures were examined. Seventeen open reading frames (phnA to phnQ) were identified in one transcriptional direction and five open reading frames in the divergent direction. Sequence homology searches identify PhnC, PhnK, PhnL, and, possibly, PhnN proteins as members of nucleotide-binding proteins of the binding protein-dependent transport systems. Candidates for other membrane components and regulatory proteins are also identified. A Pho box-like promoter sequence is also found upstream of the gene cluster starting at phnA, which is consistent with the observation of phosphate regulation of the Phn+ response. Fourteen repetitive extragenic palindromic sequences are found in the phn DNA: 10 exist in the extragenic region between phnA and phnB, two between phnD and phnE, and two between phnK and phnL. An unusual finding is that one of the repetitive extragenic palindromic sequences actually overlaps with the reading frame of the phnE gene.
Nucleotide sequences of the fecBCDE genes and locations of the proteins suggest a periplasmic-binding-protein-dependent transport mechanism for iron(III) dicitrate in Escherichia coli.
The fec region of the Escherichia coli chromosome determines a citrate-dependent iron(III) transport system. The nucleotide sequence of fec revealed five genes, fecABCDE, which are transcribed from fecA to fecE. The fecA gene encodes a previously described outer membrane receptor protein. The fecB gene product is formed as a precursor protein with a signal peptide of 21 amino acids; the mature form, with a molecular weight of 30,815, was previously found in the periplasm. The fecB genes of E. coli B and E. coli K-12 differed in 3 nucleotides, of which 2 gave rise to conservative amino acid exchanges. The fecC and fecD genes were found to encode very hydrophobic polypeptides with molecular weights of 35,367 and 34,148, respectively, both of which are localized in the cytoplasmic membrane. The fecE product was a rather hydrophilic but cytoplasmic membrane-bound protein of Mr 28,189 and contained regions of extensive homology to ATP-binding proteins. The number, structural characteristics, and locations of the FecBCDE proteins were typical for a periplasmic-binding-protein-dependent transport system. It is proposed that after FecA- and TonB-dependent transport of iron(III) dicitrate across the outer membrane, uptake through the cytoplasmic membrane follows the binding-protein-dependent transport mechanism. FecC and FecD exhibited homologies to each other, to the N- and C-terminal halves of FhuB of the iron(III) hydroxamate transport system, and to BtuC of the vitamin B12 transport system. FecB showed some homology to FhuD, suggesting that the latter may function in the same manner as a binding protein in iron(III) hydroxamate transport. The close homology between the proteins of the two iron transport systems and of the vitamin B12 transport system indicates a common evolution for all three systems.
Iron and virulence in Shigella.
Iron limitation, a condition encountered within mammalian hosts, induces the synthesis of a number of proteins in pathogenic Shigella species. These include several outer membrane proteins, Shiga toxin, and proteins involved in the biosynthesis and transport of high-affinity iron-binding compounds or siderophores. Although siderophores have been shown to play a major role in the virulence of some bacterial pathogens, these compounds do not appear to be essential for the virulence of Shigella species. Unlike those pathogens which are restricted to the extracellular compartments of the host, the Shigella species invade and multiply within host cells. Alternative iron-acquisition systems, such as the ability to utilize haem-iron, permit growth of the intracellular bacteria. Virulent shigellae also possess a cell-surface haem-binding protein, and synthesis of this protein correlates with infectivity and virulence. This protein does not appear to be involved in iron acquisition. Rather, it may allow the bacteria to coat themselves with haem compounds, thus enhancing their ability to interact with target host cells.
Nucleotide sequences of fic and fic-1 genes involved in cell filamentation induced by cyclic AMP in Escherichia coli.
The nucleotide sequences of fic-1 involved in the cell filamentation induced by cyclic AMP in Escherichia coli and its normal counterpart fic were analyzed. The open reading frame of both fic-1 and fic coded for 200 amino acids. The Gly at position 55 in the Fic protein was changed to Arg in the Fic-1 protein. The promoter activity of fic was confirmed by fusing fic and lacZ. The gene downstream from fic was found to be pabA (p-aminobenzoate). There is an open reading frame (ORF190) coding for 190 amino acids upstream from the fic gene. Computer-assisted analysis showed that Fic has sequence similarity with part of CDC28 of Saccharomyces cerevisiae, CDC2 of Schizosaccharomyces pombe, and FtsA of E. coli. In addition, ORF190 has sequence similarity with the cyclosporin A-binding protein cyclophilin.
The toxR gene of Vibrio cholerae encodes a transmembrane, DNA-binding protein that activates transcription of the cholera toxin operon and a gene (tcpA) for the major subunit of a pilus colonization factor. We constructed site-directed insertion mutations in the toxR gene by a novel method employing the chromosomal integration of a mobilizable suicide plasmid containing a portion of the toxR coding sequence. Mutants containing these new toxR alleles had an altered outer membrane protein profile, suggesting that two major outer membrane proteins (OmpT and OmpU) might be under the control of toxR. Physiological studies indicated that varying the concentration of the amino acids asparagine, arginine, glutamate, and serine caused coordinate changes in the expression of cholera toxin, TcpA, OmpT, and OmpU. Changes in the osmolarity of a tryptone-based medium also produced coordinate changes in the expression of these proteins. Other environmental signals (temperature and pH) had a more pronounced effect on the expression of cholera toxin and TcpA than they did on the outer membrane proteins. These results suggest that certain environmental signals (i.e., osmolarity and the presence of amino acids) are tightly coupled to the expression of toxR-regulated proteins and therefore may be signals that are directly sensed by the ToxR protein.
The nucleotide sequence of the ugp genes of Escherichia coli K-12, which encode a phosphate-limitation inducible uptake system for sn-glycerol-3-phosphate and glycerophosphoryl diesters, was determined. The genetic organization of the operon differed from previously published results. A single promoter, containing a putative pho box, was detected by S1-nuclease mapping. The promoter is followed by four open reading frames, designated ugpB, A, E and C, which encode a periplasmic binding protein, two hydrophobic membrane proteins and a protein that is likely to couple energy to the transport system, respectively. The sequences of the proteins contain the characteristics of several other binding protein-dependent transport systems, but they seem to be particularly closely related to the maltose system.
Membrane-bound phosphatases in Escherichia coli: sequence of the pgpB gene and dual subcellular localization of the pgpB product.
The phosphatidyl glycerophosphate B phosphatase of Escherichia coli has a multiple substrate specificity and a peculiar dual subcellular localization in the envelope. Its phosphatidyl glycerophosphate phosphatase activity is higher in the cytoplasmic membrane, while phosphatidic acid and lysophosphatidic acid phosphatase activities are higher in the outer membrane. The DNA sequencing of the pgpB gene revealed a protein of 251 amino acids which had at least five hydrophobic membrane-spanning regions. About 37 hydrophilic residues in the middle of the sequence had considerable homology with the C-terminal conserved region of the ras family genes in eucaryotes. A protein of 28,000 daltons was expressed from the pgpB gene under a tac promoter in a runaway replication plasmid. This overproduced protein also revealed the dual subcellular localization.
Evaluation with an iuc::Tn10 mutant of the role of aerobactin production in the virulence of Shigella flexneri.
To evaluate the role of aerobactin production in the virulence of Shigella flexneri, a iuc::Tn10 insertion mutant was obtained from strain M90T, a serotype 5 isolate. This mutant was tested for its ability to invade and kill HeLa cells in monolayers, to elicit keratoconjunctivitis in guinea pigs and to infect ligated segments of rabbit ileal loops. Although this mutant did not grow in iron-depleted media, its ability to grow intracellulalry and eventually kill HeLa cells was unchanged from that of the wild-type strain. On the other hand, an inoculum-dependent effect was observed in the Sereny test, as well as in the rabbit ligated ileal loop model, which was monitored for fluid production and for both gross and microscopical alterations of the mucosa. Transduction of the mutation within a noninvasive plasmidless derivative of the parental strain did not alter growth within the intestinal lumen. We conclude that aerobactin production most probably provides invasive strains with a selective advantage for growth within tissues when located in extracellular compartments.
Cloning in Escherichia coli K-12 of a Na+-dependent transport system from a marine bacterium.
The transport of D-alanine by Escherichia coli K-12 neither requires nor is stimulated by Na+. The transport of D-alanine by the marine bacterium Alteromonas haloplanktis 214 requires Na+ specifically. Mutants of E. coli which were unable to transport D-alanine were isolated by enrichment for D-cycloserine resistance. One of the mutants was transformed with a gene bank of A. haloplanktis chromosomal DNA. Two transformants, E. coli RM1(pPM1) and E. coli RM1(pPM2) were able to transport D-alanine by a Na+-dependent mechanism. Li+ and K+ were unable to replace Na+. Both transformants contained chimeric plasmids with inserts which hybridized with A. haloplanktis but not E. coli chromosomal DNA or each other. Despite the lack of homology between the inserts, Na+-dependent D-alanine transport in the two transformants could not be distinguished either by kinetic studies or by differences in the capacity of various amino acids to compete for D-alanine uptake.
Iron hydroxamate transport of Escherichia coli: nucleotide sequence of the fhuB gene and identification of the protein.
The genes fhuA, fhuC and fhuD encode products involved in the transport of ferrichrome into cells of Escherichia coli. The nucleotide sequence of an additional locus contiguous to fhuD and formerly designated fhuB was determined. It contains an open reading frame for a polypeptide that consists of 659 amino acids. Expression of plasmids containing the fhuB region in an in vitro transcription/translation system and in E. coli minicells revealed a polypeptide with an electrophoretic mobility on SDS-polyacrylamide gels extremely dependent on the experimental conditions employed. This property could be explained by the very hydrophobic nature of the protein and it was concluded that the fhuB locus consists of a structural gene that encodes a membrane protein with a molecular weight of 82,181. The fhuB gene is required for all iron transport systems which operate via hydroxamate compounds. The order of the genes in the fhu operon is fhuA fhuC fhuD fhuB, and transcription proceeds from fhuA to fhuB.
Requirement of Fnr and NarL functions for nitrate reductase expression in Escherichia coli K-12.
I used a chlC-lac operon fusion to study regulatory mutations which affect nitrate reductase expression in Escherichia coli. A NarL- mutant apparently lacks a nitrate-specific positive regulatory component. Furthermore, an fnr (nirR) mutation prevented enzyme induction under any conditions. These data are consistent with a two-step, positive control model for nitrate reductase regulation.
Citrate-dependent iron transport system in Escherichia coli K-12.
Induction of the citrate-dependent iron transport system of Escherichia coli K-12 required 0.1 mM citrate and 0.1 micrometer iron in the growth medium. Five--ten-times more iron than citrate was taken up into the cells which suggests that citrate was largely excluded from the transport. Fluorocitrate and phosphocitrate induced the citrate-dependent iron transport system although they supported iron uptake only very poorly. An outer membrane protein (FecA), belonging to the transport system, was induced in fecB mutants which were devoid of citrate-dependent iron transport. The intracellular citrate and iron concentrations were 10--100-times higher than the external concentrations required for induction of the transport system. It is concluded that only exogenous ferric citrate induced the transport system, and that citrate did not have to enter the cytoplasm. The Tn10 transposon, conferring tetracycline resistance, was inserted near the fec gene region which controls the expression of the citrate-dependent iron transport system. The determination of the cotransduction frequencies of Tn10 with the fecA and fecB markers suggested the gene order fecA fecB Tn10.
The nature of the uptake systems for nucleic acid bases in Escherichia coli.
Transport of iron into bacterial cells.
Iron transport in Escherichia coli K-12. 2,3-Dihydroxybenzoate-promoted iron uptake.
Energy-dependent zinc transport by escherichia coli.

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