Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture

The arabinose-inducible promoter P(BAD) is subject to all-or-none induction, in which intermediate concentrations of arabinose give rise to subpopulations of cells that are fully induced and uninduced. To construct a host-vector expression system with regulatable control in a homogeneous population of cells, the araE gene of Escherichia coli was cloned into an RSF1010-derived plasmid under control of the isopropyl-beta-D-thiogalactopyranoside-inducible P(tac) and P(taclac) promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. To detect the effect of arabinose-independent araE expression on population homogeneity and cell-specific expression, the gfpuv gene was placed under control of the arabinose-inducible araBAD promoter (P(BAD)) on the pMB1-derived plasmid pBAD24. The transporter and reporter plasmids were transformed into E. coli strains with native arabinose transport systems and strains deficient in one or both of the arabinose transport systems (araE and/or araFGH). The effects of the arabinose concentration and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The araE, and araE araFGH mutant strains harboring the transporter and reporter plasmids were uniformly induced across the population at all inducer concentrations, and the level of gene expression in individual cells varied with arabinose concentration. In contrast, the parent strain, which expressed the native araE and araFGH genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.     

Energization of the transport systems for arabinose and comparison with galactose transport in Escherichia coli.

1. Strains of Escherichia coli were obtained containing either the AraE or the AraF transport system for arabinose. AraE+,AraF- strains effected energized accumulation and displayed an arabinose-evoked alkaline pH change indicative of arabinose-H+ symport. In contrast, AraE-,AraF+ strains accumulated arabinose but did not display H+ symport. 2. The ability of different sugars and their derivatives to elicit sugar-H+ symport in AraE+ strains was examined. Only L-arabinose and D-fucose were good substrates, and arabinose was the only inducer. 3. Membrane vesicles prepared from an AraE+,AraF+ strain accumulated the sugar, energized most efficiently by the respiratory substrates ascorbate + phenazine methosulphate. Addition of arabinose or fucose to an anaerobic suspension of membrane vesicles caused an alkaline pH change indicative or sugar-H+ symport on the membrane-bound transport system. 4. Kinetic studies and the effects of arsenate and uncoupling agents in intact cells and membrane vesicles gave further evidence that AraE is a low-affinity membrane-bound sugar-H+ symport system and that AraF is a binding-protein-dependent high-affinity system that does not require a transmembrane protonmotive force for energization. 5. The interpretation of these results is that arabinose transport into E. coli is energized by an electrochemical gradient of protons (AraE system) or by phosphate bond energy (AraF system). 6. In batch cultures the rates of growth and carbon cell yields on arabinose were lower in AraE-,AraF+ strains than in AraE+,AraF- or AraE+,AraF+ strains. The AraF system was more susceptible to catabolite repression than was the AraE system. 7. The properties of the two transport systems for arabinose are compared with those of the genetically and biochemically distinct transport systems for galactose, GalP and MglP. It appears that AraE is analogous to GalP, and AraF to MglP.     

The cloning, DNA sequence, and overexpression of the gene araE coding for arabinose-proton symport in Escherichia coli K12

A lambda placMu1 insertion was made into araE, the gene for arabinose-proton symport in Escherichia coli. A phage containing an araE'-'lacZ fusion was recovered from the lysogen and its restriction map compared with that of the 61-min region of the E. coli genome to establish the gene order thyA araE orf lysR lysA galR; araE was transcribed toward orf. A 4.8-kilobase SalI-EcoRI DNA fragment containing araE was subcloned from the phage lambda d(lysA+ galR+ araE+) into the plasmid vector pBR322. From this plasmid a 2.8-kilobase HincII-PvuII DNA fragment including araE was sequenced and also subcloned into the expression vector pAD284. The araE gene was 1416-base pairs long, encoding a hydrophobic protein of 472 amino acids with a calculated Mr of 51,683. The amino acid sequence was homologous with the xylose-proton symporter of E. coli and the glucose transporters from a human hepatoma HepG2 cell line, human erythrocytes, and rat brain. The overexpressed araE gene product was identified in Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of cell membranes as a protein of apparent Mr 35,000 +/- 1,150. Arabinose protected this protein against reaction with N-ethylmaleimide.     

Cloning, sequencing, and expression of the araE gene of Klebsiella oxytoca 8017, which encodes arabinose-H+ symport activity.

Southern analysis of the genomic DNA from species of the family Enterobacteriaceae, using a probe derived from the Escherichia coli araE gene, which encodes an arabinose-H+ symporter, detected araE in Salmonella, Citrobacter, Klebsiella, and Enterobacter spp. The Klebsiella oxytoca araE gene was cloned, sequenced, and expressed to compare its properties with those of araE from E. coli.     

L-arabinose transport systems in Escherichia coli K-12.

Mutations in the arabinose transport operons of Escherichia coli K-12 were isolated with the Mu lac phage by screening for cells in which beta-galactosidase is induced in the presence of L-arabinose. Standard genetic techniques were then used to isolate numerous mutations in either of the two transport systems. Complementation tests revealed only one gene, araE, in the low-affinity arabinose uptake system. P1 transduction placed araE between lysA (60.9 min) and thyA (60.5 min) and closer to lysA. The operon of the high-affinity transport system was found to contain two genes: araF, which codes for the arabinose-binding protein, and a new gene, araG. The newly identified gene, araG, was shown by two-dimensional gel electrophoresis to encode a protein which is located in the membrane. Only defects in araG could abolish uptake by the high-affinity system under the conditions we used.     

Pyruvate formate lyase and acetate kinase are essential for anaerobic growth of Escherichia coli on xylose

During anaerobic growth of bacteria, organic intermediates of metabolism, such as pyruvate or its derivatives, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate-level phosphorylation. In Escherichia coli, conversion of glucose to pyruvate yields 2 net ATPs, while metabolism of a pentose, such as xylose, to pyruvate only yields 0.67 net ATP per xylose due to the need for one (each) ATP for xylose transport and xylulose phosphorylation. During fermentative growth, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP from two pyruvates (one hexose equivalent) while still maintaining the overall redox balance. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose. An E. coli pfl mutant lacking pyruvate formate lyase cannot convert pyruvate to acetyl coenzyme A, the required precursor for acetate and ethanol production, and could not produce this additional ATP. E. coli pfl mutants failed to grow under anaerobic conditions in xylose minimal medium without any negative effect on their survival or aerobic growth. An ackA mutant, lacking the ability to generate ATP from acetyl phosphate, also failed to grow in xylose minimal medium under anaerobic conditions, confirming the need for the ATP produced by acetate kinase for anaerobic growth on xylose. Since arabinose transport by AraE, the low-affinity, high-capacity, arabinose/H+ symport, conserves the ATP expended in pentose transport by the ABC transporter, both pfl and ackA mutants grew anaerobically with arabinose. AraE-based xylose transport, achieved after constitutively expressing araE, also supported the growth of the pfl mutant in xylose minimal medium. These results suggest that a net ATP yield of 0.67 per pentose is only enough to provide for maintenance energy but not enough to support growth of E. coli in minimal medium. Thus, pyruvate formate lyase and acetate kinase are essential for anaerobic growth of E. coli on xylose due to energetic constraints.     

The Maltose Permease Encoded by the Mal61 Gene of Saccharomyces Cerevisiae Exhibits Both Sequence and Structural Homology to Other Sugar Transporters

The MAL61 gene of Saccharomyces cerevisiae encodes maltose permease, a protein required for the transport of maltose across the plasma membrane. Here we report the nucleotide sequence of the cloned MAL61 gene. A single 1842 bp open reading frame is present within this region encoding the 614 residue putative MAL61 protein. Hydropathy analysis suggests that the secondary structure consists of two blocks of six transmembrane domains separated by an approximately 71 residue intracellular region. The N-terminal and C-terminal domains of 100 and 67 residues in length, respectively, also appear to be intracellular. Significant sequence and structural homology is seen between the MAL61 protein and the Saccharomyces high-affinity glucose transporter encoded by the SNF3 gene, the Kluyveromyces lactis lactose permease encoded by the LAC12 gene, the human HepG2 glucose transporter and the Escherichia coli xylose and arabinose transporters encoded by the xylE and araE genes, indicating that all are members of a family of sugar transporters and are related either functionally or evolutionarily. A mechanism for glucose-induced inactivation of maltose transport activity is discussed.     

Physical and genetic characterization of cloned enterobactin genomic sequences from Escherichia coli K-12

We have cloned genes responsible for enterobactin synthesis (entD) and transport (fepA,fes) from Escherichia coli K-12. Relevant recombinant plasmids enabled EntD- and transport-defective mutants to grow on iron-limiting medium. Subcloning and deletion analysis demonstrated that the gene order is entD-fepA-fes. Protein synthesis studies in minicells suggest that FepA is first translated as an Mr 84 000 precursor, which is subsequently cleaved to the active Mr 81 000 receptor; the fes gene product is an Mr 44 000 protein; no polypeptide has been identified as the entD gene product.     

Location of a structural gene for xylose-H+ symport at 91 min on the linkage map of Escherichia coli K12

Mutations in the xylose-H+ transport activity of Escherichia coli K12 were isolated using Mud(ApRlac). The initial selection was for simultaneous acquisition of ampicillin and xylose resistance in an fda background. Colonies were then screened for xylose-inducible beta-galactosidase and for growth on xylose of their fda+ derivatives. Two of the xylose-positive derivatives were shown to be impaired in xylose-H+ symport in whole cells and in xylose transport into subcellular vesicles. Their xylose transport in whole cells showed increased sensitivity to arsenate. The site of prophage insertion was mapped to 91.4 min on the E. coli genome between pgi and malB. It is proposed that the gene for the xylose-H+ symport system be called xylE.     

The narK gene product participates in nitrate transport induced in Escherichia coli nitrate-respiring cells

The nucleotide sequence of the Escherichia coli narK gene, which is located in the upstream region of the narCHJI operon, was determined. The narK gene encodes a very hydrophobic protein with 463 amino acid residues (Mr 49,693). A narK deletion mutant, under conditions for the induction of nitrate respiration, was unable to perform nitrate transport. Loss of transport activity was recovered by transforming the mutant with a narK+ plasmid. Thus, we conclude that the narK gene encodes a transmembrane protein participating in nitrate transport. In the narK promoter region, we defined a unique sequence that we designate as a 'nitrate box', functioning as a putative NarL-binding site, in addition to the consensus sequence of the 'anaero-box'.     

cys154 Is important for lac permease activity in Escherichia coli

The lac Y gene of Escherichia coli which encodes the lac permease has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys154 is replaced with either gly or ser. Permease with gly in place of cys154 exhibits essentially no transport activity, while substitution of cys154 with ser also causes marked, though less complete loss of activity. The findings suggest that cys154 plays an important role in lactose:H+ symport.     

Mathematical modeling of the low and high affinity arabinose transport systems in Escherichia coli

A mathematical model was developed for the low and high affinity arabinose transport systems in E. coli. The model is a system of three ordinary differential equations and takes the dynamics of mRNAs for the araE and araFGH proteins and the internal arabinose into account. Special attention was paid to estimate the model parameters from the literature. Our analysis and simulations suggest that the high affinity transport system helps the low affinity transport system to respond to high concentration of extracellular arabinose faster, whereas the high affinity transport system responds to a small amount of extracellular arabinose. Steady state analysis of the model also predicts that there is a regime for the extracellular concentration of arabinose where the arabinose system can show bistable behavior.     

Mapping, sequence, and apparent lack of function of araJ, a gene of the Escherichia coli arabinose regulon.

We report the mapping, sequencing, and study of the physiological role of the fourth arabinose-inducible operon from Escherichia coli, araJ. It is located at 9 min on the chromosome and codes for a single 42-kDa protein that shows no significant homology to other known proteins. Destruction of the chromosomal araJ gene does not detectably affect either of the two arabinose transport systems, the ability of cells to grow on arabinose, or the induction kinetics of the araBAD operon, and thus the physiological role of AraJ, if any, remains unknown. We have also found a long open reading frame upstream of araJ. The sequence of this upstream open reading frame was found to be identical to the previously reported sequence of the sbcC gene (I. S. Naom, S. J. Morton, D. R. F. Leach, and R. G. Lloyd, Nucleic Acids Res. 17:8033-8044, 1989). The carboxyl region of SbcC has an amino acid sequence consistent with this region of SbcC forming an extended alpha-helical coiled-coil.     

Molecular cloning of gltS and gltP, which encode glutamate carriers of Escherichia coli B.

Two genes encoding distinct glutamate carrier proteins of Escherichia coli B were cloned into an E. coli K-12 strain by using a cosmid vector, pHC79. One of them was the gltS gene coding for a glutamate carrier of an Na+-dependent, binding protein-independent, and glutamate-specific transport system. The content of the glutamate carrier was amplified about 25-fold in the cytoplasmic membranes from a gltS-amplified strain. The gltS gene was located in a 3.2-kilobase EcoRI-MluI fragment, and the gene product was identified as a membrane protein with an apparent Mr of 35,000 in a minicell system. A gene designated gltP was also cloned. The transport activity of the gltP system in cytoplasmic membrane vesicles from a gltP-amplified strain was driven by respiratory substrates and was independent of the concentrations of Na+, K+, and Li+. An uncoupler, carbonylcyanide m-chlorophenylhydrazone, completely inhibited the transport activities of both systems, whereas an ionophore, monensin, inhibited only that of the gltS system. The Kt value for glutamate was 11 microM in the gltP system and 3.5 microM in the gltS system. L-Aspartate inhibited the glutamate transport of the gltP system but not that of the gltS system. Aspartate was taken up actively by membrane vesicles from the gltP-amplified strain, although no aspartate uptake activity was detected in membrane vesicles from a wild-type E. coli strain. These results suggest that gltP is a structural gene for a carrier protein of an Na+-independent, binding protein-independent glutamate-aspartate transport system.     

L-arabinose transport and the L-arabinose binding protein of escherichia coli

The active accumulation of L-arabinose by arabinose induced cultures of Escherichia coli is mediated by 2 independent transport mechanisms. One, specified by the gene locus araE, is membrane bound and possesses a relatively “low affinity.” The other, specified in part by the genetic locus araF, contains as a functional component the L-arabinose binding protein and functions with a “high affinity” for the substrate. The L-arabinose binding protein has been purified, partially characterized, crystallized, and sequenced.     

A unique arabinose 5-phosphate isomerase found within a genomic island associated with the uropathogenicity of Escherichia coli CFT073

Previous studies showed that deletion of genes c3405 to c3410 from PAI-metV, a genomic island from Escherichia coli CFT073, results in a strain that fails to compete with wild-type CFT073 after a transurethral cochallenge in mice and is deficient in the ability to independently colonize the mouse kidney. Our analysis of c3405 to c3410 suggests that these genes constitute an operon with a role in the internalization and utilization of an unknown carbohydrate. This operon is not found in E. coli K-12 but is present in a small number of pathogenic E. coli and Shigella boydii strains. One of the genes, c3406, encodes a protein with significant homology to the sugar isomerase domain of arabinose 5-phosphate isomerases but lacking the tandem cystathionine beta-synthase domains found in the other arabinose 5-phosphate isomerases of E. coli. We prepared recombinant c3406 protein, found it to possess arabinose 5-phosphate isomerase activity, and characterized this activity in detail. We also constructed a c3406 deletion mutant of E. coli CFT073 and demonstrated that this deletion mutant was still able to compete with wild-type CFT073 in a transurethral cochallenge in mice and could colonize the mouse kidney. These results demonstrate that the presence of c3406 is not essential for a pathogenic phenotype.     

The cloning and DNA sequence of the gene xylE for xylose-proton symport in Escherichia coli K12

The gene xylE, coding for xylose-proton symport in Escherichia coli, was cloned and its DNA sequence determined. The cloning strategy utilized lambda placMu insertions and exploited the proximity of xylE to malB. A 2.8-kilobase HincII fragment of cloned DNA restored [14C]xylose transport and xylose-proton symport activities to a xylose transport-negative strain. The xylE gene was identified as a 1473-base pair open reading frame, located 373 base pairs downstream of malG, encoding a hydrophobic protein of Mr 53,607. The amino acid sequence of XylE bore little resemblance to the lactose-proton LacY symporter or melibiose-sodium MelB symporter, but a high degree of homology was found with the arabinose-proton AraE symporter of E. coli and glucose transport proteins of mammals. Structural analyses and comparisons suggest that 12 membrane-spanning segments may occur in the XylE protein.     

Characterization and functional expression in Escherichia coli of the sodium/proton/glutamate symport proteins of Bacillus stearothermophilus and Bacillus caldotenax

The genes encoding the Na+/H+/L-glutamate symport proteins of the thermophilic organisms Bacillus stearothermophilus (gltTBs) and Bacillus caldotenax (gltTBc) were cloned by complementation of Escherichia coli JC5412 for growth on glutamate as sole source of carbon, energy and nitrogen. The nucleotide sequences of the gltTBs and gltTBc genes were determined. In both cases the translated sequences corresponded with proteins of 421 amino acid residues (96.7% amino acid identity between GltTBs and GltTBc). Putative promoter, terminator and ribosome-binding-site sequences were found in the flanking regions. These expression signals were functional in E. coli. The hydropathy profiles indicate that the proteins are hydrophobic and could form 12 membrane-spanning regions. The Na+/H+ coupled L-glutamate symport proteins GltTBs and GltTBc are homologous to the strictly H+ coupled L-glutamate transport protein of E. coli K-12 (overall 57.2% identity). Functional expression of glutamate transport activity was demonstrated by uptake of glutamate in whole cells and membrane vesicles. In accordance with previous observations (de Vrij et al., 1989; Heyne et al., 1991), glutamate uptake was driven by the electrochemical gradients of sodium ions and protons.