Plasmids of the Gonococcus

Sparling, P. Frederick; Biswas, G D; Graves, James F.; Blackman, E

doi:10.1007/978-1-4684-3983-0_23

Cited by 24 publications

(32 citation statements)

References 22 publications

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“…TF+ or LF+ transformants were selected on CDM agar plates supplemented with Desferal and 25% saturated 2.5 ,uM TF or LF, respectively. Drugresistant transformants were selected on GCB agar plates overlaid with the appropriate antibiotic after allowing 5 h for phenotypic expression of the introduced marker (30 (14).…”

Section: Methodsmentioning

confidence: 99%

“…Unfortunately, there are few data by which to relate genetic distances to physical map distances in the gonococcus. In an earlier report, recombination data were related to physical sizes of transforming DNA for certain linked gonococcal ribosomal genes; loci that exhibited 10 to 20% cotransformation were estimated to be separated by 10 to 15 kilobases (30). This suggests that mutations that exhibit an RI of 0.5 may be separated by several kilobases and conceivably could affect separate proteins.…”

Section: Methodsmentioning

confidence: 99%

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Genetic evidence that Neisseria gonorrhoeae produces specific receptors for transferrin and lactoferrin

et al. 1990

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Transferrin (TF) and lactoferrin (LF) are probably the major sources of iron (Fe) for Neisseria gonorrhoeae in vivo. We isolated mutants of N. gonorrhoeae FA19 that were unable to grow with Fe bound to either TF (TF-) or LF (LF-) or to both TF and LF ([TF LF]-). The amount of Fe internalized by each of the mutants was reduced to background levels from the relevant iron source(s). The wild-type parent strain exhibited saturable specific binding of TF and LF; receptor activity was induced by Fe starvation. The TF(-)-specific or LF(-)-specific mutants were almost completely lacking in receptor activity for TF or LF, respectively, whereas the [TF LF]- mutants bound both TF and LF as well as the wild-type strain. All mutants utilized citrate and heme normally as Fe sources. These results demonstrate that ability to bind TF or LF is essential for gonococci to scavenge appreciable amounts of Fe from these sources in vitro. In addition, the TF and LF Fe acquisition pathways are linked by the mutual use of a nonreceptor gene product that is essential to Fe scavenging from both of these sources; this gene product is not required for Fe acquisition from other sources.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Genetic evidence that Neisseria gonorrhoeae produces specific receptors for transferrin and lactoferrin

et al. 1990

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“…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).…”

Section: 65-kb Hinfl Fragment Of the 42-kb Plasmidmentioning

confidence: 99%

Construction of isogenic gonococcal strains varying in the presence of a 4.2-kilobase cryptic plasmid

Biswas¹,

Graves²,

Schwalbe³

et al. 1986

J Bacteriol

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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...

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“…A second class was morphologically normal and took up DNA into a DNase-resistant state normally, but was deficient in both chromosomal and plasmid transformation; mutations in these mutants may affect entry of DNA into the cell proper. A third class was similar to the second but was fully competent for plasmid transformation, suggesting that there was a defect in a late stage of chromosomal transformation.Genetic transformation is known to be the only mode of chromosomal gene transfer in Neisseria gonorrhoeae (11,34), and transformation has been postulated to be involved in genetic exchange among gonococci in vivo (18, 32). Gonococci are known to be competent (i.e., to be able to take up DNA irreversibly) when they are in the piliated (P+) state (7,17,33,34); virtually all gonococci are P+ during growth on mucosal surfaces (19,21,35,36).…”

mentioning

confidence: 99%

Transformation-deficient mutants of piliated Neisseria gonorrhoeae

1989

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Seven transformation-deficient mutants of piliated, competent Neisseria gonorrhoeae were isolated by screening them for their inability to be transformed by chromosomal DNA after chemical mutagenesis. Three distinct classes of mutants were obtained, each of which was piliated, as determined by electron microscopy. One class exhibited abnormal colony morphology and was unable to take up DNA into a DNase-resistant state. A second class was morphologically normal and took up DNA into a DNase-resistant state normally, but was deficient in both chromosomal and plasmid transformation; mutations in these mutants may affect entry of DNA into the cell proper. A third class was similar to the second but was fully competent for plasmid transformation, suggesting that there was a defect in a late stage of chromosomal transformation.Genetic transformation is known to be the only mode of chromosomal gene transfer in Neisseria gonorrhoeae (11,34), and transformation has been postulated to be involved in genetic exchange among gonococci in vivo (18, 32). Gonococci are known to be competent (i.e., to be able to take up DNA irreversibly) when they are in the piliated (P+) state (7,17,33,34); virtually all gonococci are P+ during growth on mucosal surfaces (19,21,35,36). Competence for transformation is expressed constitutively in P+ gonococci under in vitro growth conditions; there is no evidence for competence-enhancing factors (7, 33). Loss of pili (P-) in vitro results in a drastic (2104) decrease in competence for transformation and in loss of the ability to take up DNA (7,17,33).Although competence is strongly correlated with expression of pili, there is no evidence that pili bind DNA (29) or otherwise directly promote entry of DNA. Klimpel and Clark (22) recently showed that the P+ to P-phase transition (3, 31) is associated with changes in multiple proteins, in addition to pilin. It has been suggested that gonococci contain a surface receptor for DNA, since gonococci preferentially take up homologous DNA (18), and some fragments of a gonococcal 4.2-kilobase (kb) plasmid are taken up in preference to others (10, 18). The nature of the putative receptor and the DNA sequence(s) required for uptake are unknown. DNA is taken up by competent gonococci in the double-stranded form (8) and may be processed into smaller double-stranded fragments during or shortly after uptake (4). Transforming DNA remains double stranded throughout most of the transformation process, since there is no evident eclipse in biological activity of donor DNA after uptake (34).A recA mutant decreases the transformation efficiency of piliated gonococci (23); but otherwise, there are no known mutants that are useful in helping us to understand the mechanisms of gonococcal DNA uptake, processing, and recombination. In this report, we describe the isolation and characterization of several types of transformation-defective mutants of P+ gonococci that should be useful in helping us further dissect the biochemistry of gonococcal transformation. * Corresponding...

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Plasmids of the Gonococcus

Cited by 24 publications

References 22 publications

Genetic evidence that Neisseria gonorrhoeae produces specific receptors for transferrin and lactoferrin

Genetic evidence that Neisseria gonorrhoeae produces specific receptors for transferrin and lactoferrin

Construction of isogenic gonococcal strains varying in the presence of a 4.2-kilobase cryptic plasmid

Transformation-deficient mutants of piliated Neisseria gonorrhoeae

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