Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains

Summary A cosmid gene bank of the virulent Salmonella typhimurium C5 was constructed in Escherichia coli K12. The bank was repackaged into bacteriophage heads and transduced into the semi-rough S. typhimurium strain AS68 which expresses the LamB receptor protein. Approximately 6000 ampicillin-resistant transductants were pooled and used as host for the propagation of bacteriophage P22. The P22 lysate was able to transduce cosmid recombinants to smooth strains of S. typhimurium and individual transductants were selected which complemented various S. typhimurium auxotrophic mutations. A stable mutation was introduced into the aroD gene of S. typhimurium C5. The resulting aroD ^- mutant, named CU038, was highly attenuated compared with the wild-type parent strain and BALB/c mice immunised orally with CU038 were well protected against challenge with the virulent C5 parental strain. Using the cosmid bank repackaged into bacteriophage P22 heads it was possible to isolate cosmid recombinants that could complement the aroD mutation of CU038 either by in vitro selection using minimal medium or in vivo selection for restoration of virulence in BALB/c mice. Repackaged P22 cosmid banks could provide a simple system for selecting in vivo for Salmonella virulence determinants. A Salmonella typhi strain harbouring mutations in aroA and aroD was constructed for potential use as a live oral typhoid vaccine in humans.     

Oxygen concentration profiles and exchange in sediment cores with circulated overlying water

SUMMARY. 1. The overlying water of intact sediment cores was constantly stirred with an impeller at a rate sufficient to mix turbulently the water column and maintain the diffusive boundary layer at a determined thickness. The system allowed standardization of water circulation in laboratory sediment core experiments.
2. Both oxygen concentration and oxygen penetration depth in the sediments decreased, the former by 70% and the latter from 4.2 mm to 2.0 mm, when the overlying water was not stirred for 24 h, as measured with oxygen microelectrodes in a lake sediment core.
3. Oxygen profiles measured in sediment cores in the laboratory were similar to those measured in situ when the overlying water was stirred with an impeller at such a rate that a similar thickness of the diffusive boundary layer at the sediment-water interface developed in the laboratory as that in situ.
4. Sediment oxygen consumption was calculated from: (1) measured oxygen profiles in the diffusive boundary layer and the molecular diffusion coefficient for oxygen in water; (2) the measured oxygen decrease in the top of the sediments and the estimated diffusion coefficient in the sediment; and (3) by oxygen differences in the overlying water after incubation of sediment cores.     

Cloning and nucleotide sequence of the aroA gene of Bordetella pertussis.

The aroA locus of Bordetella pertussis, encoding 5-enolpyruvylshikimate 3-phosphate synthase, has been cloned into Escherichia coli by using a cosmid vector. The gene is expressed in E. coli and complemented an E. coli aroA mutant. The nucleotide sequence of the B. pertussis aroA gene was determined and contains an open reading frame encoding 442 amino acids, with a calculated molecular weight for 5-enolpyruvylshikimate 3-phosphate synthase of 46,688. The amino acid sequence derived from the nucleotide sequence shows homology with the published amino acid sequences of aroA gene products of other microorganisms.     

Cloning of chromosomal DNA encoding the F41 adhesin of enterotoxigenic Escherichia coli and genetic homology between adhesins F41 and K88.

The genetic determinant for production of the adhesive antigen F41 was isolated from a porcine enterotoxigenic Escherichia coli strain by cosmid cloning. The cloned DNA included sequences homologous to those of hybridization probes prepared from the K88 adhesive antigen operon. Transposon insertions which inactivated F41 production mapped to the same region of DNA showing homology with the K88 genes, demonstrating the genetic relatedness of F41 and K88. Hybridization of a K88 gene probe to plasmid and total DNA from the porcine E. coli isolate from which the F41 gene was cloned indicated that F41 is chromosomally encoded by this strain. This observation was extended to other F41-producing animal isolates. A large number of animal E. coli isolates were examined with K88, F41, and K99 gene probes and for mannose-resistant hemagglutination of human group O erythrocytes and K88 and F41 antigen production. All K88 and F41 antigen producers possessed genetic homology with the K88 and F41 gene probes. Most, but not all, F41-producing strains possessed homology to the K99 gene probe, reflecting the previously observed association of F41 and K99 antigen production. In the strains examined, homology with the K99 gene probe was plasmid associated, whereas homology with the F41 gene probe was chromosomal. The K88 antigen-producing strains showed no homology with the K99 probe. A number of strains possessed homology with the K88 and F41 gene probes and were mannose-resistant hemagglutination positive, but did not produce K88 or F41 antigens. This suggests that there are adhesins among animal isolates of E. coli which are genetically related to but antigenically distinct from K88 and F41.     

Living Vessel Elements in the Late Metaxylem of Sheathed Maize Roots

The two types of nodal roots of field-grown maize, sheathedand bare, were found to have such different water conductivitiesthat an investigation of the anatomy of their large metaxylemvessels was made. While the vessels of the bare roots were openfor scores of centimetres, those of the sheathed roots werefound to be not vessels but developing vessel elements, withcross walls at 1 mm intervals, and protoplasts. The cross wallsbetween the elements had several unique histochemical properties.Previous investigators have often failed to find the cross wallsbecause they are very easily dislodged during the usual methodsof tissue preparation. They are best identified by microdissectionof fresh xylem. The living elements persist in the late metaxylemup to 20 – 30 cm from the tip. As the roots become longerthan this both the cross walls and the soil sheaths disappearand there is a transition to a bare root with open vessels inthe proximal region. The soil sheath persists a little longerthan the cross walls. The two types are thus stages in a developmentalsequence through which all nodal roots pass. A fundamental differencebetween the two types is in their water status, since the estimatedconductive capacity of a bare root is about 100 times greaterthan that of a sheathed root. These observations point to theneed for a reassessment of the published work on transport ofions into the xylem of grass roots through a reinvestigationof the ‘maturity’ of their xylem vessels. Grass roots, dimorphic roots, ion secretion to xylem, soil sheaths, xylem vessels, xylem differentiation, water conduction, Zea mays L     

Reconstitution of a GTPase Activity a 50S Ribosomal Protein from <Emphasis Type="Italic">E. coli</Emphasis>

DURING each step of peptide chain elongation the ribosome shifts up one triplet along the messenger RNA with concomitant movement of the peptidyl-transfer RNA from the donor to the acceptor site. This process, commonly known as translocation, is triggered by a supernatant protein, factor G, which in association with the ribosome cleaves GTP into GDP and inorganic phosphate^1,2 and it has been argued that the energy liberated in this reaction is used “to carry the complex one triplet forward”³.     

Cloning and characterisation of the serC and aroA genes of Yersinia enterocolitica, and construction of an aroA mutant

A gene library of Yersinia enterocolitica 8081 was constructed in the cosmid vector pHC79. Recombinants containing the aroA gene, encoding 5-enolpyruvylshikimate 3-phosphate synthase, were identified by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829. All six recombinant plasmids which complemented aroA also complemented the serC mutation in E. coli K-12 strain KL282. Tn5 mutagenesis suggested serC encoding 3-phosphoserine aminotransferase was the proximal gene in an operon with aroA. The nucleotide sequence of a 3-kb HindII-EcoRV fragment encoding the two genes was determined. The serC and aroA open reading frames contain 362 and 428 codons, respectively, and the deduced amino acid sequences share 78% and 81% homology, respectively, with the corresponding E. coli genes. Sequence inspection revealed no obvious terminators or promoters in the intergenic region. The cloned Y. enterocolitica aroA gene was inactivated in vitro and reintroduced into the parental Y. enterocolitica 8081 strain using the suicide vector pJM703.1. Stable aroA insertion mutants of Y. enterocolitica were isolated.