Precise physical mapping of theEscherichia coli rnb gene, encoding ribonuclease II

Ribonuclease II (encoded byrnb) is one of the two main exonucleases involved in mRNA degradation inEscherichia coli. We report the precise physical mapping ofrnb to 29 min on the chromosomal map in the vicinity ofpyrF, and clarify the genetic and physical maps of thisE. coli chromosomal region. The results were confirmed by the construction of a strain partially deleted forrnb.     

DNA sequencing and expression of the gene rnb encoding Escherichia coli ribonuclease II

The Escherichia coli ribonuclease II (RNase II) is an exonuclease involved in mRNA degradation that hydrolyses single-stranded polyribonucleotides processively in the 3′ to 5′ direction. Sequencing of a 2.2 kb Msel–Rsal fragment containing the rnb gene revealed an open reading frame of 1794 nucleotides that encodes a protein of 598 amino acid residues, whose calculated molecular mass is 67 583 Da. This value is in good agreement with that obtained by sodium dodecyl sulphate/ polyacrylamide gel electrophoresis of polypeptides synthesized by expression with the T7 RNA polymerase/promoter system. This system was also used to confirm the correct orientation of rnb. Translation initiation was confirmed by rnb–lacZ fusions. The mRNA start site was determined by S1 nuclease mapping. Two E. coli mutants harbouring different rnb alleles deficient in RNase II activity were complemented with the expressed fragment carrying the rnb gene.     

Members of the pogo superfamily of DNA-mediated transposons in the human genome

Bacillus subtilis, likeEscherichia coli, possesses several sets of genes involved in the utilization ofβ-glucosides. InE. coli, all these genes are cryptic, including the genes forming thebgl operon, thus leading to a Bgl^? phenotype. We screened forB. subtilis chromosomal DNA fragments capable of reverting the Bgl⁺ phenotype associated with anE. coli hns mutant to the Bgl^? wild-type phenotype. OneB. subtilis chromosomal fragment having this property was selected. It contained a putative Ribonucleic AntiTerminator binding site (RAT sequence) upstream from thebglP gene. Deletion studies as well as subcloning experiments allowed us to prove that the putativeB. subtilis bglP RAT sequence was responsible for the repression of theE. coli bgl operon. We propose that this repression results from the titration of the BglG antiterminator protein ofE. coli bgl operon by our putativeB. subtilis bglP RAT sequence. Thus, we report evidence for a new cross interaction between heterologous RAT-antiterminator protein pairs.     

Identification of RNase-resistant RNAs in Saccharomyces cerevisiae extracts: Separation from chromosomal DNA by selective precipitation

High-quality chromosomal DNA is a requirement for many biochemical and molecular biological techniques. To isolate cellular DNA, standard protocols typically lyse cells and separate nucleic acids from other biological molecules using a combination of chemical and physical methods. After a standard chemical-based protocol to isolate chromosomal DNA from Saccharomyces cerevisiae and then treatment with RNase A to degrade RNA, two RNase-resistant bands persisted when analyzed using gel electrophoresis. Interestingly, such resistant bands did not appear in preparations of Escherichia coli bacterial DNA after RNase treatment. Several enzymatic, chemical, and physical methods were employed in an effort to remove the resistant RNAs, including use of multiple RNases and alcohol precipitation, base hydrolysis, and chromatographic methods. These experiments resulted in the development of a new method for isolation of S. cerevisiae chromosomal DNA. This method utilizes selective precipitation of DNA in the presence of a potassium acetate/isopropanol mixture and produces high yields of chromosomal DNA without detectable contaminating RNAs.     

Characterisation ofEscherichia coli isolated from raw milk cheeses

The aim of this work was: (i) to verify the level ofEscherichia coli in Pannerone and Valtrompia Formaggella, two artisanal Italian raw-milk cheeses ripened for less than 60 days; (ii) to phenotypically and genotipycally type theE. coli isolates; (iii) to detect the presence ofE. coli O157:H7 and of intestinal enteropathogenicE. coli by PCR. The levels ofE. coli in the cheeses ranged from 3.89 to 8.47 log CFU g^?1. NoE. coli O157:H7 was detected in 25 g of cheese. The 76E. coli strains (68 cheese isolates and 8 reference strains) were widely diverse, since a high number of both PCR fingerprinting profiles and PhenePlate® phenotypes were shown. Within the 68 cheese isolates, no toxin production and virulence-associated genes were shown by multiplex PCR. Non-pathogenicE. coli were isolated at high levels in raw-milk cheeses, where they may contribute to the development of desirable characteristics of some of these products, e.g. Pannerone.     

Identification and Validation of Novel Chromosomal Integration and Expression Loci in Escherichia coli Flagellar Region 1

Escherichia coli is used as a chassis for a number of Synthetic Biology applications. The lack of suitable chromosomal integration and expression loci is among the main hurdles of the E. coli engineering efforts. We identified and validated chromosomal integration and expression target sites within E. coli K12 MG1655 flagellar region 1. We analyzed five open reading frames of the flagellar region 1, flgA, flgF, flgG, flgI, and flgJ, that are well-conserved among commonly-used E. coli strains, such as MG1655, W3110, DH10B and BL21-DE3. The efficiency of the integration into the E. coli chromosome and the expression of the introduced genetic circuit at the investigated loci varied significantly. The integrations did not have a negative impact on growth; however, they completely abolished motility. From the investigated E. coli K12 MG1655 flagellar region 1, flgA and flgG are the most suitable chromosomal integration and expression loci.     

Chromosomal tet(O)-Harboring Regions in Campylobacter coli Isolates from Turkeys and Swine

In turkey-derived Campylobacter coli isolates of a unique lineage (cluster II), the tetracycline resistance determinant tet(O) was chromosomal and was part of a gene cassette (transposon) interrupting a Campylobacter jejuni-associated putative citrate transporter gene. In contrast, the swine-derived C. coli strain 6461 harbored a chromosomal tet(O) in a different genomic location.     

Analysis of chimeric UmuC proteins: identification of regions inSalmonella typhimurium UmuC important for mutagenic activity

UnlikeEscherichia coli, the closely related bacteriumSalmonella typhimurium is relatively unresponsive to the mutagenic effects of DNA-damaging agents. Previous experiments have suggested that these phenotypic differences might result from reduced activity of theS. typhimurium UmuC protein. To investigate this possibility, we have taken advantage of the high degree of homology between the UmuC proteins ofE. coli andS. typhimurium and have constructed a series of plasmid-encoded chimeric proteins. The possibility that the phenotypic differences might be due to differential expression of the respective UmuC proteins was eliminated by constructing chimeric proteins that retained the first 25 N-terminal amino acids of either of the UmuC proteins (and presumably the same translational signals), but substituting the remaining 397 C-terminal amino acids with the corresponding segments from the reciprocal operon. Constructs expressing mostlyE. coli UmuC were moderately proficient for mutagenesis whereas those expressing mostlyS. typhimurium UmuC exhibited much lower frequencies of mutation, indicating that the activity of the UmuC protein ofS. typhimurium is indeed curtailed. The regions responsible for this phenotype were more precisely localized by introducing smaller segments of theS. typhimurium UmuC protein into the UmuC protein ofE. coli. While some regions could be interchanged with few or no phenotypic effects, substitution of residues 212–395 and 396–422 ofE. coli UmuC with those fromS. typhimurium resulted in reduced mutability, while substitution of residues 26–59 caused a dramatic loss of activity. We suggest, therefore, that the primary cause for the poor mutability ofS. typhimurium can be attributed to mutations located within residues 26–59 of theS. typhimurium UmuC protein.     

Escherichia coli Clone Sonnei (Shigella sonnei) Had a Chromosomal O-Antigen Gene Cluster Prior to Gaining Its Current Plasmid-Borne O-Antigen Genes

O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria. The surface-exposed O antigen is subject to selection by the host immune system, which may account for the maintenance of many different O-antigen forms. Characteristically, all genes specific to O-antigen synthesis are clustered in a region close to the his and gnd genes on the chromosome of Escherichia coli and related species. Shigella sonnei, essentially a clone of E. coli (E. coli clone Sonnei), is an important human pathogen and is unusual in that its O-antigen gene cluster is located on a plasmid. Our results suggest that it once had a normal chromosomal O-antigen gene cluster which has been largely deleted. We suggest that the O antigen encoded by the plasmid-borne genes offered a selective advantage in adapting to a new environment and that the chromosomal O-antigen genes were eventually inactivated. We also identified, by PCR and sequencing, a potential ancestor of E. coli Sonnei among the 166 known E. coli serotype strains.     

A genetic study of tolerance and resistance to colicin A inCitrobacter freundii

Colicin A-insensitive mutants ofCitrobacter freundii were isolated and grouped into six phenotypic classes characterized by sensitivity, insensitivity or partial insensitivity to the bacteriocins S6, DF 13 and colicin A, and sensitivity or insensitivity to deoxycholate (DOC) and ampicillin. Mapping by the gradient-of-transmission method revealed the chromosomal regions in which the responsible genes are situated. Res-3 mapped nearpur betweenpur andthr; Tol-5 mapped betweenaro andilv and Tol-4 betweengal andpyr; Tol-1, Tol-2 and Tol-3 are situated close togal. All the mutations that mapped neargal rendered the bacteria more sensitive to DOC and ampicillin. Complementation analysis withE. coli plasmids showed that the three phenotypic groups that map neargal were complemented byE. coli plasmids and fall into three complementation groups. Two of these are equivalent with thetol A andtol B genes inE. coli.     

Recombination of Escherichia coli Chromosomal Segments in Salmonella typhosa

Approximately half of Salmonella typhosa hybrids resulting from mating with Escherichia coli Hfr donors inherit the selected donor marker by recombination, and the length of the E. coli chromosomal segment most frequently incorporated in these recombinants is between 1 and 2 min.     

Partial characterization ofPseudomonas fluorescens subsp.cellulosa endoglucanase activity produced inEscherichia coli

Summary The recombinant plasmid, pPFC4, which carriesPseudomonas fluorescens subsp.cellulosa chromosomal DNA was previously isolated on the basis of its ability to direct the expression of endoglucanase inEscherichia coli. In the present study, some physical and chemical properties of this activity were characterized. The major portion (78.4%) of the endoglucanase activity is found in the periplasmic space ofE. coli. This plasmid-encoded endoglucanase has a pH optimum of approximately 6.0 and a temperature optimum of approximately 50°C. With carboxymethylcellulose-zymograms, after polyacrylamide gel electrophoresis, periplasmic extracts fromE. coli carrying pPFC4 show six distinct bands with endoglucanase activity. The molecular mass of the major endoglucanase band is approximately 29 kDa while the remaining bands with endoglucanase activity range from 48 to 100 kDa. Although the basis of this heterogeneity is not known, the DNA insert of pPFC4 that encodes endoglucanase activity is not large enough to contain six separate genes; hence, the observed array of endoglucanases may result from post-translational modification of one or two primary gene products.     

A method for rapid mapping of mutations by plasmid rescue strategy inSaccharomyces cerevisiae

The products ofPRP17 andPRP18 genes are required for the second step of pre-mRNA splicing reactions inSaccharomyces cerevisiae. Temperature-sensitive mutants at either of these loci accumulate products of the first splicing reaction at nonpermissive temperature. To characterize functional regions in these proteins the mutations in three temperature-sensitive alleles ofPRP17 and two temperature-sensitive alleles ofPRP18 were mapped by the plasmid rescue strategy, One of the procedures adopted in the past is plasmid rescue of the mutant allele followed by sequencing of the entire gene. In this work we describe an adaptation of the above procedure that allows, first, rapid mapping of chromosomal segments bearing the mutations, followed by sequence characterization of the minimal segment. The strategy adopted was to integrate a wild-type copy of the gene at the homologous mutant chromosomal locus, followed by recovery of the chromosomal fragments from these integrants as plasmids inE. coli. The recovered plasmids were screened by a complementation assay for those that contained in them the chromosomal mutation. The mutations in all the three alleles ofPRP17 map to a small region in the N-terminal half of the protein, whereas the temperature-sensitive mutations in the two alleles ofPRP18 map to different regions of the PRP18 protein. The recovered mutant plasmids from all five alleles at the two loci were sequenced and the nucleotide changes were found to result in missense mutations in each case. Our strategy is therefore a rapid method to map chromosomal mutations and is of general use in structure-function analysis of cloned genes.     

Bdellovibrio possesses a prey-derived OmpF protein in its outer membrane

Bdellovibrio bacteriovorus 109D andBdellovibrio stolpii derive one of their major outer membrane proteins from the outer membrane of their prey. This prey-derived protein corresponds to the OmpF protein ofEscherichia coli. Bdellovibrios cultivated onSalmonella typhimurium prey acquire theSalmonella OmpF protein; this protein is distinguishable electrophoretically from the OmpF protein ofE. coli. Bdellovibrios containing the prey-derived OmpF protein are sensitive to killing by colicin A but not colicin E1, whereas bdellovibrios without this protein are completely resistant to colicin killing.