TnWO insertions were selected on the basis of resistance to the lipopolysaccharide (LPS)-specific bacteriophage U3. The majority of these were located in a 2-kilobase region within the rfa locus, a gene cluster of about 18 kb that contains genes for LPS core biosynthesis. The rfa::TnlO insertions all exhibited a deep rough phenotype that included hypersensitivity to hydrophobic antibiotics, a reduction in major outer membrane proteins, and production of truncated LPS. These mutations were complemented by a Clarke-Carbon plasmid known to complement rfa mutations of Salmonella typhimurium, and analysis of the insert from this plasmid showed that it contained genes for at least six polypeptides which appear to be arranged in the form of a complex operon. Defects in two of these genes were specifically implicated as the cause of the deep rough phenotype. One of these appeared to be rfiaG, which encodes a function required for attachment of the first glucose residue to the heptose region of the core. The other gene did not appear to be directly involved in determination of the sugar composition of the core. We speculate that the product of this gene is involved in the attachment of phosphate or phosphorylethanolamine to the core and that it is the lack of one of these substituents which results in the deep rough phenotype.

No abstract

Pseudomonas aeruginosa, an opportunistic pathogen that often initiates infec

A 4.2-kilobase (kb) cryptic plasinid is present in 96% of isolates of Neisseria gonorrhoeae. An inability to construct isogenic derivatives which vary in the presence of the 4.2-kb plasmid has prevented the study of its function. We report a method to deliver an intact 4.2-kb plasmid into plasmidless gonococcal strains. The method involved transformation with novel 15.7-kb hybrid penicillinase-producing (Pcr) plasmids, which were cointegrates containing two copies of the 4.2-kb plasmid arranged in tandem direct repeat plus one copy of the 7.2-kb Pcr plasmid pFA3. When the 15.7-kb hybrid Pcr plasmids were introduced into a gonococcal recipient lacking evident plasmids, they dissociated at a relatively high frequency into plasmids identical to their parents: the 4.2-kb cryptic plasmid and pFA10 (a stable 11.5-kb plasmid containing one copy of each of the 7.2-kb Pce plasmid pFA3 and the 4.2-kb cryptic plasmid pFAl). Curing strains of their Pcr plasmids resulted in isogenic strains which varied only in the presence of the 4.2-kb plasmid. A phenotype can be assigned to a plasmid either by curing the host strain of the plasmid or by transferring the plasmid of interest into a plasmid-free strain. Previous attempts failed to eliminate the plasmid from strains that carry it (24), and transformants or trahsconjugants that acquired a 7.2-kb Pcr plasmid failed to acquire the donor cryptic plasmid (39).In this paper, we describe a method to deliver an intact 4.2-kb plasmid into a plasmid-free recipient by using a novel, unstable 15.7-kb penicillin-resistant hybrid plasmid that contains two copies of the 4.2-kb plasmid in direct tandem repeat orientation. We also studied putative functions ascribed to the 4.2-kb plasmid, using the constructed isogenic * Corresponding author.strains, and searched for evidence for chromosomal sequences homologous with the 4.2-kb plasmid. MATERIALS AND METHODSBacterial strains and growth. The bacteria and plasmids used in this study are described in Tables 1 and 2. Media and growth conditions were as described previously (4). GC Medium base broth or agar from Difco Laboratories, Detroit, Mich., was used throughout. The presence of Ilactamase production was confirmed by a chromogenic cephalosporin indicator, compound 87/312 (Glaxo Research Ltd., Greenford, England) (32).Preparation of DNAs and restriction endonuclease digestion. Chromosomal DNA of nonpiliated cells (Pil-) was isolated by the method described by Marmur (26). Plasmid DNA was isolated by alkali denaturation and phenol extraction of whole cell (Pil-) DNA followed by one cycle of ethidium bromide-cesium chloride density gradient centrifugation (4). Restriction endonuclease digestions were performed as recommended in instructions supplied by the manufacturers (Bethesda Research Laboratories, Inc., Gaithersburg, Md., and New England Biolabs Inc., Beverly, Mass.).Transformation. The transformation protocol for gonococci was similar to that described previously (4,38). Escherichia coli strains were transformed by the method of Dagert and...

Piliated, competent gonococci are known to preferentially take up homologous transforming DNA into the cell. We examined the mechanism for DNA uptake with pFA10, a hybrid 11.5-kilobase (kb) penicillin-resistant (Pcr) plasmid composed of heterologous DNA from a 7.2-kb Pcr plasmid and homologous DNA from a 4.2-kb gonococcal cryptic plasmid. The presence of the gonococcal cryptic plasmid DNA in the hybrid resulted in markedly increased transformation efficiencies in isogenic crosses as compared with the parent 7.2-kb Pcr plasmid. Uptake of 32P-end-labeled MspI or TaqI restriction fragments of the hybrid was limited to fragments entirely derived from the 4.2-kb gonococcal cryptic plasmid, indicating that DNA uptake was probably dependent on the presence of a specific DNA sequence. Since Haemophilus DNA did not inhibit transformation by the hybrid Pcr plasmid, the gonococcal DNA uptake sequence is different from the known sequence involved in homologous DNA uptake by Haemophilus spp.