Replica Plating

This chapter describes how Esther Lederberg, in daring to re-purpose her compact makeup pad as a kind of ink stamp, developed the new Replica Plating Technique but later her husband Joshua seemed to claim sole credit for this discovery. The Lederbergs demonstrated the effectiveness of the new plating technique in their study of spontaneous bacterial mutations, using the technique to resolve a longstanding question of bacterial mutations: Did mutations occur spontaneously or was some other process of adaptation involved? While at first, Joshua defended Esther’s co-equal contribution to the invention of replica plating, in later publications, he seemed to claim sole credit, by ignoring her contributions. This is a classic example of the Matilda Effect, when a male colleague is given sole credit for an invention by unfairly ignoring the female colleague’s contribution. Some of the Lederbergs’ colleagues recognize Esther as the primary inventor of replica plating, since she had the reputation of an experimental genius; others assumed that the creative insight was Joshua’s.

Increased Quinolone-Resistant Mutations of gyrA and parC Genes after Pouchitis Treatment with Ciprofloxacin

Background: Oral antibiotics, such as ciprofloxacin (CFX), are widely used for the treatment of acute and chronic pouchitis. Most bacterial mutations that confer quinolone resistance are at Ser-83 and Asp-87 in the gyrA gene and Ser-80 and Glu-84 in the parC gene. Methods: We obtained 51 stool samples from 43 patients who were diagnosed with ulcerative colitis and underwent ileal pouch-anal anastomosis. Patients were divided into 2 groups: 13 patients with CFX treatment of pouchitis and 30 patients without pouchitis. After extraction of fecal DNA, the amount of Escherichia coli 16S rRNA, gyrA, and parC gene DNA were measured using real-time polymerase chain reaction (PCR). Possible mutations at gyrA 83 and 87 and at parC 80 and 84 were investigated by PCR cloning and sequencing, and mutation rates were quantified by rapid PCR-restriction fragment length polymorphism. Results: Samples from both CFX-treated and -untreated patients had comparable levels of gyrA and parC gene DNA. Nucleic acid and amino acid mutations were identified at gyrA 83 and 87, and at parC 80 and 84. We successfully quantified mutation rates at gyrA 83 and 87, and at parC 84, all of which were significantly higher in samples from CFX-treated patients (70, 84, and 38%) than from CFX-untreated patients (13, 11, and 5%). Conclusion: E. coli in patient pouches may have mutations in their gyrA and parC genes that produce CFX resistance. Mutation rates of these genes were significantly higher in samples from CFX-treated patients. This study contributes to understanding the decrease and loss of CFX effectiveness against pouchitis.

ROS-based lethality of Caenorhabditis elegans mitochondrial electron transport mutants grown on Escherichia coli siderophore iron release mutants

Caenorhabditis elegans consumes bacteria, which can supply essential vitamins and cofactors, especially for mitochondrial functions that have a bacterial ancestry. Therefore, we screened the Keio Escherichia coli knockout library for mutations that induce the C. elegans hsp-6 mitochondrial damage response gene, and identified 45 E. coli mutations that induce hsp-6::gfp. We tested whether any of these E. coli mutations that stress the C. elegans mitochondrion genetically interact with C. elegans mutations in mitochondrial functions. Surprisingly, 4 E. coli mutations that disrupt the import or removal of iron from the bacterial siderophore enterobactin were lethal in combination with a collection of C. elegans mutations that disrupt particular iron–sulfur proteins of the electron transport chain. Bacterial mutations that fail to synthesize enterobactin are not synthetic lethal with these C. elegans mitochondrial mutants; it is the enterobactin–iron complex that is lethal in combination with the C. elegans mitochondrial mutations. Antioxidants suppress this inviability, suggesting that reactive oxygen species (ROS) are produced by the mutant mitochondria in combination with the bacterial enterobactin–iron complex.

Sinorhizobium meliloti ExoR and ExoS Proteins Regulate both Succinoglycan and Flagellum Production

ABSTRACT The production of the Sinorhizobium meliloti exopolysaccharide, succinoglycan, is required for the formation of infection threads inside root hairs, a critical step during the nodulation of alfalfa (Medicago sativa) by S. meliloti. Two bacterial mutations, exoR95::Tn5 and exoS96::Tn5, resulted in the overproduction of succinoglycan and a reduction in symbiosis. Systematic analyses of the symbiotic phenotypes of the two mutants demonstrated their reduced efficiency of root hair colonization. In addition, both the exoR95 and exoS96 mutations caused a marked reduction in the biosynthesis of flagella and consequent loss of ability of the cells to swarm and swim. Succinoglycan overproduction did not appear to be the cause of the suppression of flagellum biosynthesis. Further analysis indicated that both the exoR95 and exoS96 mutations affected the expression of the flagellum biosynthesis genes. These findings suggest that both the ExoR protein and the ExoS/ChvI two-component regulatory system are involved in the regulation of both succinoglycan and flagellum biosynthesis. These findings provide new avenues of understanding of the physiological changes S. meliloti cells go through during the early stages of symbiosis and of the signal transduction pathways that mediate such changes.

p-Chlorophenyl Methyl Sulfide, p-Chlorophenyl Methyl Sulfoxide, and p-Chlorophenyl Methyl Sulfone. I. Acute Toxicity and Bacterial Mutagenicity Studies

An acute toxicity battery was performed on a series of three chemicals (p-chlorophenyl methyl sulfide, p-chloro-phenyl methyl sulfoxide, and p-chlorophenyl methyl sulfone) which have been identified as ground water contaminants at the U.S. Army's Rocky Mountain Arsenal near Denver. The results indicate that these materials exhibit oral LD50 values in the ranges of 400–620 mg/kg in rats and 330–880 mg/kg in mice. Following dermal exposures, only the sulfide induced death in rabbits. This agent and the sulfoxide induced central nervous system depression for a period of up to 7 days postapplication. Skin irritation potencies in rabbit tests were in the order of sulfoxide > sulfone >> sulfide, whereas ocular test results revealed irritation potencies to be sulfoxide > sulfide > sulfone. Results of guinea pig testing indicated a lack of sensitization potential for all compounds. None of the test materials induced bacterial mutations in Salmonella (five strains) assays that employed Arochlor 1254- and phenobarbital-induced S-9 rat liver activation systems. The most overt short-term effects following exposure to one or more of these agents are the ocular effects and the neurologic/lethal potentials following dermal or oral contact.