Nucleotide sequence of the uhp region of Escherichia coli.

The Escherichia coli uhp region encodes the transport system that mediates the uptake of a number of sugar phosphates as well as the regulatory components that are responsible for induction of this transport system by external glucose 6-phosphate. Four uhp genes have been identified by analysis of the complementation behavior and polypeptide coding capacity of plasmids carrying subcloned regions or transposon insertions. The nucleotide sequence of a 6.5-kilobase segment that contains the 3' end of the ilvBN operon and the entire uhp region was determined. Four open reading frames were identified in the locations expected for the various uhp genes; all were oriented in the same direction, counterclockwise relative to the genetic map. The properties of the polypeptides predicted from the nucleotide sequence were consistent with their observed features. The 196-amino-acid UhpA polypeptide has the composition characteristic of a soluble protein and bears homology to the DNA-binding regions of many regulatory activators and repressors. The 518-amino-acid UhpB and the 199-amino-acid UhpC regulatory proteins contain substantial segments of hydrophobic character. Similarly, the 463-amino-acid UhpT transporter is a hydrophobic protein with numerous potential transmembrane segments. The UhpC regulatory protein has substantial sequence homology to part of UhpT, suggesting that this regulatory protein might have evolved by duplication of the gene for the transporter and that its role in transmembrane signaling may involve sugar-phosphate-binding sites and transmembrane orientations similar to those of the transport protein.     

Genetic control of the hexose phosphate transport system of Escherichia coli: mapping of deletion and insertion mutations in the uhp region.

The Escherichia coli transport system responsible for the accumulation of a number of sugar phosphates is encoded by the uhp region and is induced by external, but not intracellular, glucose 6-phosphate. To delineate the genetic organization of the uhp region, a total of 225 independent point, deletion, and transposon Tn10 insertion mutations were collected. Mutations conferring the Uhp-phenotype were obtained on the basis of their resistance to fosfomycin and their inability to use sugar phosphates as carbon source. Deletions of uhp sequences were obtained as a consequence of imprecise excision of Tn10 insertions located on either side of uhp. Conjugal crosses between these deletions and the point of insertion mutations allowed determination of the relative order of the uhp alleles and of the deletion endpoints. Specialized lambda transducing phages carrying a uhpT-lac operon fusion and various amounts of adjacent uhp material were isolated and used as genetic donors. Results from these crosses corroborated those obtained in the conjugal crosses. The locations of the mutant alleles were compared with the regulatory properties of Uhp+ revertants of these alleles. This comparison suggested the existence of at least three genes in which mutation yields the Uhp-phenotype. Mapping experiments were consistent with the gene order pyrE-gltS-uhpTRA-ilvB, where uhpT encodes the transport system and uhpR and uhpA are regulatory genes whose products are necessary for proper uhp regulation.     

Location of the gene specifying hexose phosphate transport (uhp) on the chromosome of Escherichia coli.

The uhp gene, which specifies the uptake of hexose phosphates, and several other genes in the vicinity of minute 81 on the E. coli linkage map have been located by phage-mediated transductions. The order found is mtl-gpsA-pyre-gltc-uhp-tna-dnaa. Alleles specifying the Uhp- and Uhp+ characters were separated from that specifying constitutivity of hexose phosphate uptake (Uhpc). Although cotransduction frequencies between gltC and uhp as high as 90%, and between uhp and tna as high as 80%, were observed, these frequencies were unusually strongly dependent on which marker was selected. This may be due to the proximity of the uhp region to the point of origin of chromosome replication.     

Engineering EGFP reporter constructs into a 200 kb human beta-globin BAC clone using GET Recombination

GET Recombination, a simple inducible homologous recombination system for Escherichia coli, was used to target insertion of an EGFP cassette between the start and termination codons of the beta-globin gene in a 200 kb BAC clone. The high degree of homology between the promoter regions of the beta- and delta-globin genes also allowed the simultaneous generation of a delta-globin reporter construct with the deletion of 8.8 kb of intervening sequences. Both constructs expressed EGFP after transient transfection of MEL cells. Similarly, targeting of the EGFP cassette between the promoter regions of the gamma-globin genes and the termination codon of the beta-globin gene enabled the generation of reporter constructs for both (A)gamma- and (G)gamma-globin genes, involving specific deletions of 24 and 29 kb of genomic sequence, respectively. Finally the EGFP cassette was also inserted between the epsilon- and beta-globin genes, with the simultaneous deletion of 44 kb of intervening sequence. The modified constructs were generated at high efficiency, illustrating the usefulness of GET Recombination to generate large deletions of specific sequences in BACs for functional studies. The establishment of stable erythropoietic cell lines with these globin constructs will facilitate the search for therapeutic agents that modify the expression of the individual globin genes in a physiologically relevant manner.     

Use of lambda Red-mediated recombineering and Cre/lox for generation of markerless chromosomal deletions in avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) are bacteria associated with extraintestinal diseases in poultry. A method to generate markerless deletions of APEC genome is described. Lambda Red recombination is used to introduce a LoxP cassette (loxP-rpsL-neo-loxP) containing the rpsL gene for streptomycin sensitivity and the neo gene for kanamycin/neomycin resistance into the APEC genome, with attendant deletion of a desired chromosomal gene. The loxP sites are incorporated into primers used to amplify the rpsL-neo marker during the construction of the LoxP cassette, making the method rapid and efficient. The cassette is specifically integrated into the fiu gene or intergenic region 2051-52, and the Cre/lox system is used to remove the marker, hence deletion of the drug-resistance genes. The results demonstrate that the Cre/lox system can successfully be used to generate markerless deletions in APEC, and rpsL counter-selection can be used to select the deletions so that one does not have to pick and test to find the desired product.     

Size Of Gene Specific Inverted Repeat - Dependent Gene Deletion In Saccharomyces cerevisiae

We describe here an approach for rapidly producing scar-free and precise gene deletions in S. cerevisiae with high efficiency. Preparation of the disruption gene cassette in this approach was simply performed by overlap extension-PCR of an invert repeat of a partial or complete sequence of the targeted gene with URA3. Integration of the prepared disruption gene cassette to the designated position of a target gene leads to the formation of a mutagenesis cassette within the yeast genome, which consists of a URA3 gene flanked by the targeted gene and its inverted repeat between two short identical direct repeats. The inherent instability of the inverted sequences in close proximity facilitates the self-excision of the entire mutagenesis cassette deposited in the genome and promotes homologous recombination resulting in a seamless deletion via a single transformation. This rapid assembly circumvents the difficulty during preparation of disruption gene cassettes composed of two inverted repeats of the URA3, which requires the engineering of unique restriction sites for subsequent digestion and T4 DNA ligation in vitro. We further identified that the excision of the entire mutagenesis cassette flanked by two DRs in the transformed S. cerevisiae is dependent on the length of the inverted repeat of which a minimum of 800 bp is required for effective gene deletion. The deletion efficiency improves with the increase of the inverted repeat till 1.2 kb. Finally, the use of gene-specific inverted repeats of target genes enables simultaneous gene deletions. The procedure has the potential for application on other yeast strains to achieve precise and efficient removal of gene sequences.     

A simple method for multiple modification of the Escherichia coli K-12 chromosome

We developed a simple method of generating markerless deletions in the Escherichia coli chromosome. The method consists of two recombination events stimulated by lambda Red recombinase. The first recombination replaced a target region with a marker cassette and the second then eliminated the marker cassette. The marker cassette included an antibiotic resistant gene and a negative selection marker (Bacillus subtilis sacB). Since sacB makes E. coli sensitive to sucrose, a markerless deletion strain was successfully selected using its sucrose-resistant phenotype. To stimulate these recombination events, 1-kbp homologous sequences adjacent to the target region were connected to both ends of the marker cassette or connected to each other by PCR. The average efficiency of the recombinations was 24% and 93% respectively. Eliminating the marker cassette with a fragment including an additional sequence, insertion was also possible. This markerless deletion method should be useful in creating a highly modified E. coli chromosome.     

Novel Topology of BfpE, a Cytoplasmic Membrane Protein Required for Type IV Fimbrial Biogenesis in Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) produces the bundle-forming pilus (BFP), a type IV fimbria that has been implicated in virulence, autoaggregation, and localized adherence to epithelial cells. The bfpE gene is one of a cluster of bfp genes previously shown to encode functions that direct BFP biosynthesis. Here, we show that an EPEC strain carrying a nonpolar mutation in bfpE fails to autoaggregate, adhere to HEp-2 cells, or form BFP, thereby demonstrating that BfpE is required for BFP biogenesis. BfpE is a cytoplasmic membrane protein of the GspF family. To determine the membrane topology of BfpE, we fused bfpE derivatives containing 3' truncations and/or internal deletions to alkaline phosphatase and/or beta-galactosidase reporter genes, whose products are active only when localized to the periplasm or cytoplasm, respectively. In addition, we constructed BfpE sandwich fusions using a dual alkaline phosphatase/beta-galactosidase reporter cassette and analyzed BfpE deletion derivatives by sucrose density flotation gradient fractionation. The data from these analyses support a topology in which BfpE contains four hydrophobic transmembrane (TM) segments, a large cytoplasmic segment at its N terminus, and a large periplasmic segment near its C terminus. This topology is dramatically different from that of OutF, another member of the GspF family, which has three TM segments and is predominantly cytoplasmic. These findings provide a structural basis for predicting protein-protein interactions required for assembly of the BFP biogenesis machinery.     

Identification, cloning, and sequencing of piv, a new gene involved in inverting the pilin genes of Moraxella lacunata.

Moraxella lacunata is a bacterium that is a causative agent of human conjunctivitis and keratitis. We have previously cloned the Q and I pilin (formerly called beta and alpha pilin) genes of Moraxella bovis and determined that an inversion of 2 kilobases (kb) of DNA determines which pilin gene is expressed. Using an M. bovis pilin gene as a hybridization probe to screen a lambda ZAP library of M. lacunata DNA, we have isolated a clone that not only contains the entire type 4 pilin gene inversion region of M. lacunata but inverts the 2-kb region on a plasmid subclone (pMxL1) in Escherichia coli. Deletion derivatives of pMxL1 yielded some plasmids that still had the entire inversion region but were phase locked into one or the other of the two potential orientations. Similarly, insertions of a 2-kb streptomycin-resistant element (omega) within some regions outside of the inversion also resulted in phase-locked plasmids. These deletions and insertions thus localize a probable invertase necessary for the inversion event. The region was sequenced, and an open reading frame with over 98% DNA sequence homology to an open reading frame that we previously found in M. bovis and called ORF2 appeared to be a strong candidate for the invertase. This conclusion was confirmed when a plasmid containing the M. bovis ORF2 supplied, in trans, the inversion function missing from one of the M. lacunata phase-locked inversion mutants. We have named these putative invertase genes piv(ml) (pilin inversion of M. lacunata) and piv(mb) (pilin inversion of M. bovis). Despite previously noted sequence similarities between the M. bovis sites of inversion and those of the Hin family of invertible segments and a 60-base-pair region within the inversion with 50% sequence similarity to the cin recombinational enhancer, there is no significant sequence similarity of the Piv invertases to the Hin family of invertases.     

The family of organo-phosphate transport proteins includes a transmembrane regulatory protein

This review article briefly summarizes aspects of our current understanding of the Uhp sugar phosphate transport system in enteric bacteria, particularly the mode of genetic regulation of its synthesis. This regulation occurs by a process that involves an example of the very widespread and ever-growing group of so-called two-component bacterial regulatory systems, a mechanism of response to environmental signals that employs phosphate transfer reactions between constituent proteins. Of emphasis here is the unusual involvement in transmembrane signaling of the UhpC protein which is related in sequence and structure to some transport proteins, including the very protein whose synthesis it helps regulate.     

Sequences of the Escherichia coli BtuB protein essential for its insertion and function in the outer membrane.

The Escherichia coli btuB gene encodes the outer membrane transporter for vitamin B12, the E colicins, colicin A, and bacteriophage BF23. Several series of mutant forms of BtuB resulting from the insertion of dipeptide sequences and from overlapping in-frame deletions and duplications were constructed. Strains expressing the variant genes in single and multiple copy numbers were analyzed for BtuB function, for the level of BtuB polypeptide in the outer membrane, and for changes in the outer membrane permeability barrier. Most dipeptide insertions had normal transport function and assembly in the membrane. Only 2 of the 27 deletions spanning residues 5 and 514 possessed transport function, and most of the remainder were not stably inserted in the membrane. Most duplications (19 of 21) retained transport function and were inserted in the outer membrane, although some were subject to proteolysis. Even long duplications containing as many as 340 repeated amino-terminal residues retained function, suggesting considerable plasticity in the sequence requirements for membrane insertion of BtuB. Expression of many deletion and duplication proteins conferred increased susceptibility to structurally unrelated inhibitors that are normally excluded by the outer membrane. These results could be consistent with the mutational disruption of extracellular loops or transmembrane segments of BtuB that constitute a gated channel, but the finding that alterations throughout the length of BtuB affect membrane permeability properties suggests that the altered proteins might perturb the outer membrane structure itself.