Transformation-deficient mutants of piliated Neisseria gonorrhoeae (original) (raw)
Related papers
Journal of Bacteriology, 2001
We created plasmids for use in insertion-duplication mutagenesis (IDM) of Neisseria gonorrhoeae. This mutagenesis method has the advantage that it requires only a single cloning step prior to transformation into gonococci. Chromosomal DNA cloned into the plasmid directs insertion into the chromosome at the site of homology by a single-crossover (Campbell-type) recombination event. Two of the vectors contain an erythromycin resistance gene, ermC, with a strong promoter and in an orientation such that transcription will proceed into the cloned insert. Thus, these plasmids can be used to create insertions that are effectively nonpolar on the transcription of downstream genes. In addition to the improved ermC, the vector contains two copies of the neisserial DNA uptake sequence to facilitate high-frequency DNA uptake during transformation. Using various chromosomal DNA insert sizes, we have determined that even small inserts can target insertion mutation by this method and that the insertions are stably maintained in the gonococcal chromosome. We have used IDM to create knockouts in two genes in the gonococcal genetic island (GGI) and to clone additional regions of the GGI by a chromosome-walking procedure. Phenotypic characterization of traG and traH mutants suggests a role for the encoded proteins in DNA secretion by a novel type IV secretion system.
Microbiology, 1988
~~ We have previously shown that some strains of Neisseria cinereu can serve as recipients in conjugation (Con+) with Neisseria gonorrhoeae while others cannot (Con-). To determine if a replication defect contributes to the inability of certain strains of N. cinerea to serve as recipients in conjugation, we attempted to introduce a naturally occurring gonococcal #l-lactamase plasmid into N. cinerea by transformation. Various methods were employed, and all proved unsuccessful. Since specific sequences are required for DNA uptake in transformation of N. gonomhoeae, we constructed a number of hybrid plasmids containing N. cinerea chromosomal DNA inserted into the N. gonotrkae/Esckerichia coli Plactamase shuttle vector, pLES2. When nine randomly selected plasmids with inserts were used to transform an N. cinerea strain which did not accept the gonococcal fl-lactamase plasmid by conjugation, transformants were observed with four of the hybrid plasmids. The presence of one of the hybrid plasmids, pCAG9, in transformants was confitmed by agarose gel electrophoresis, Southern hybridization, and /I-lactamase production. When an N. gonorrhue donor strain containing pCAG9 was used in conjugation with several N. cmerea strains, only those strains that were previously shown to act as recipients could accept and maintain pCAG9. The ability of pCAG9 and the other three hybrid plasmids to transform Constrains demonstrates that the Plactamase plasmid can replicate in Constrains , and, therefore, the Con-phenotype is due to a block in some other stage of the conjugation process.
Fate of donor DNA in pneumococcal transformation
Journal of Molecular Biology, 1967
The molecular fate of homologous (pneumococcal) and heterologous (Eacherichia coli) [32P]DNA was examined after introduction into pneumococcal cells. In both cases the composition of 3aP-containing products at the earliest time of observation was the same. About 60% of the incorporated donor material was in the form of single strands, identified by their density in alkaline CsCl gradients. Of the incorporated 32P, 20 to 30% was in the form of native DNA of pneumococcal composition. Since this was true for donor DNA from E. cobi as well as for donor DNA from pneumococcus, it appears that this label entered native DNA by way of de novo synthesis of DNA, presumably from nucleotides. The remaining 32P in the cell was in the form of dialyzable fragments composed of large amounts of inorganic phosphate and L-cc-glycerophosphate and smaller amounts of the four 5'-deoxynucleotides.
DNA Transformation and Type IV Pili in Neisseria gonorrhoeae
2024
This thesis braved the COVID-19 pandemic. It is also a time in which the world is united, for once (in a long time), to ensure the survival of humanity through these difficult times. This instance also reminds us that global health is of high importance. Scientists, healthcare workers, and hopefully, the rest of the population are now more aware of potential global health threats. One of them is antibiotic resistance. The World Health Organization has listed antibiotic resistance as one of humankind's top global health threats. As of 2013, antibiotic-resistant infections record more than 2.6 million cases and nearly 44000 deaths 1. While common perceptions towards antibiotic resistance have medical implications, antibiotic resistance impacts our lives in many ways, like food safety and environmental sustainability. 1.1.1 What are Antibiotics? How do they work? Antibiotics are molecular weapons against bacteria. They are low molecular weight molecules, usually less than one kiloDalton 2. In nature, antibiotics can be produced by microorganisms like fungi or bacteria. While most of the earlier laboratory work in microbiology relied heavily on culturing microorganisms in test tubes, researchers have slowly expanded the scope of understanding microorganisms through complex settings such as co-culture, biofilm, bacterial communities, and microbiome 3-7. This is primarily due to our realization that microorganisms exist in different forms in nature, and the context of their environment is equally important. In many cases, microorganisms cohabit an environment and are involved in several interactions (some will be discussed further in Chapter 6). It is also due to these interactions, that microorganisms evolved to equip themselves with means for survival, either through metabolic or survival fitness, colonization strategy or hijacking competitors 8. Some of the tools microorganisms evolved to equip include toxin-antitoxin system in bacteria and antibiotics. There are in general two classes of antibiotics based on their working mechanism: bactericidal and bacteriostatic 2. Bactericidal antibiotics work by killing target microorganisms 2. Whereas bacteriostatic antibiotics can inhibit the proliferation of bacteria, rendering the slowdown of its viability and population growth 2. In the context of medical treatment, while bactericidal antibiotics will be more efficient in eliminating infections, bacteriostatic antibiotics can be administered to stall infectious bacterial growth, giving time for the host immune system to ramp up and clear the infection out of the system. Generally, antibiotics, target on five key mechanisms in bacteria to achieve either bactericidal or bacteriostatic effect 2 : (a) inhibition of cell wall synthesis, (b) inhibition of protein synthesis, (c) inhibition of DNA or RNA synthesis, (d) inhibition of folate synthesis, and (e) membrane disruption (Figure 1.1). Susceptibility of antibiotics, especially those targeting cell wall and membrane disruption, are also highly dependent on the bacteria cell surface structure 2,9,10. Gram-negative bacteria are less susceptible to antibiotics. The extra outer membrane in Gram-negative bacteria make it difficult
Plasmid-mediated chromosomal gene transfer in Neisseria gonorrhoeae
Journal of Bacteriology, 1978
An indigenous Neisseria gonorrhoeae conjugative plasmid, pLE2450, was tested for its ability to mediate chromosomal gene transfer between gonococcal strains. Plasmid-mediated chromosomal transfer was detected at a low frequency and can be used to establish certain linkage relationships between amino acid and antibiotic resistance markers.
The Ecology of Gonococcal Plasmids
Journal of General Microbiology, 1979
Of 261 strains of Neisseria gonorrhoeae examined for plasmids, 6 were plasmid-free, 217 contained only a small multicopy 2.6 x lo6 dalton plasmid and 38 carried a large 24.5 x los dalton plasmid. Restriction enzyme digests and DNA-DNA hybridization studies revealed that the large plasmids isolated between 1940 and 1978 share a common core of DNA sequences (70 to 100%) and represent a group of closely related molecules.