The Isolation and Characterization of Multiply Antibiotic Resistant Strains of Fish Pathogenic Flavobacterium Species

Since the development of the first antibiotics in the 1940’s, there has been widespread overuse in both clinical and agricultural applications. Antibiotic resistance has become a significant problem as a result of subsequent dissemination of antibiotics into the environment, and multiply-resistant strains of bacteria are now a major pathogenic threat. In this study eight separate strains of Flavobacterium responsible for recent disease outbreaks in fish hatcheries throughout Maine were collected and analyzed. All eight strains were found to be resistant to high levels of a number of different antibiotics, including those used for aquaculture as well as human chemotherapeutic applications. Flavobacterium isolates were also shown phenotypically to transfer antibiotic resistance determinants using a conjugation mating system in which Flavobacterium was the donor and Escherichia coli DH5- alpha was the recipient. This experiment suggests that it may be possible for Flavobacterium strains to transfer their multiple antibiotic resistance determinants to human pathogenic bacterial strains. Importantly, none of the hatcheries from which the Flavobacterium isolates were obtained had ever used antibiotics to treat their fish stock. It is possible that there is another selective agent responsible for the development of antibiotic resistance in the absence of antibiotic pressure. Mercury is one possible candidate, as all of the strains tested were resistant to mercuric chloride and it is known that genes encoding antibiotic resistance can be carried on the same mobile genetic elements that encode for mercury resistance. Preliminary data also suggest that the majority of the Flavobacterium isolates contain genes for mercuric ion reduction, which would confirm the mercury resistance genotype.

Characterization of AFN1, a Gene Associated with Cereal Grain Germination

The AFN1 gene is transiently expressed in germinating oat grains. As AFN1 is not expressed in dormant oat grains during imbibition, we hypothesize that AFN1 may be involved in stimulating the germination process. Sequence analysis of an AFN1 cDNA clone indicates that the AFN1 polypeptide is similar to a previously identified abscisic acid (ABA) glucosyl transferase. This suggests that AFN1 may be acting to glucosylate ABA, thereby inactivating it. As the hormone ABA is known to inhibit germination, ABA glucosylation/inactivation could lead to germination in grains expressing AFN1. To test this hypothesis, we have constructed an expression plasmid that encodes an MBP::AFN1 (maltose binding protein) fusion protein. E. coli cells carrying the expression plasmid were found to produce the MBP::AFN1 fusion protein as a substantial fraction of total protein. We are currently in the process of purifying the MBP::AFN1 fusion protein by affinity chromatography, so that it can be assayed for ABA glucosyl transferase activity. We also wish to test the effect of AFN1 gene expression during grain imbibition on the germination behavior of the grains. To this end, we have constructed plasmids for the overexpression and RNAi-based suppression of AFN1 in transgenic plants. These plasmids have been introduced into oat cells by particle bombardment and we are in the process of regenerating transgenic plants for study.

Characterization of the 4.5S RNA molecule in Escherichia coli

The 4.5S RNA molecule of Escherichia coli is essential to cell viability. It has been shown that depletion of this molecule inhibits protein synthesis, induces the heat shock response, and generally slows cell growth. The molecule has also been implicated in protein secretion, as in cells depleted of 4.5S RNA, an unsecreted precursor to ?-lactamase accumulates (pre-?-lactamase). A role in protein secretion is further supported by structural similarities with the 7S RNA molecule of eukaryotic SRP, specific binding to SRP54, and its homolog in E. coli, P48, and the ability of 7S RNA from certain archaebacteria to suppress 4.5S RNA depletion. In this study I have utilized strains with mutant forms of the 4.5S RNA genes in order to study the effect of altered 4.5S RNA on cell physiology. These strains have their mutant 4.55 RNA under the control of the tryptophan synthetic operon. Decreased growth rates, inhibited cell division, and altered protein synthesis all result from these mutations.

Intent and Method: A Study of Female Characters in Aeschylean and Euripidean Drama

Aeschylus and Euripides used tragic female characters to help fulfill the purpose of religious celebration and to achieve the motivation of public reaction. The playwrights, revising myths about tragic woman and redefining the Greek definition of appropriate femininity, supported or questioned the very customs which they changed. Originally composed as part of a religious festival for Dionysus, the god of wine, revelry and fertility, the tragedies of Aeschylus and Euripides were evaluated by Aristotle. He favored Aeschylus over Euripides, but it appears as if his stipulations for tragic characterization do not apply to Aeschylean and Euripidean women. Modem critics question both Aristotle's analysis in the Poetics as well as the tragedies which he evaluated. As part of the assessment of Aeschylus, the character of the Persian Queen, Atossa, appears as a conradiction the images that Greeks maintain of non-Greeks. The Persians is discussed in relation to modem criticisms and as on its function as a warning against radical changes in Athenian domestic life. The Oresteia, a trilogy, also charts the importance of an atypical woman in Aeschylean tragedy, and how this role, Clytaemnestra, represents an extreme example of the natural and necessary evolution of families, households and kingdoms. In contrast to Aeschylus' plea to retain nomoi (traditional custom and law), EUripides' tragedy, the Medea, demonstrates the importance of a family and a country to provide security, especially for women. Medea's abandonment by Jason and subsequent desperation drives her to commit murder in the hope of revenge. Ultimately, Euripides advocates changes in social convention away from the alienation of non-Greek, non-citizens, and females. Euripides is, unfortunately, tagged a misogynist by some in this tragedy and another example-the Hippolytus. Euripides' Phaedra becomes entangled in a scheme of divine vengeance and ultimately commits suicide in an attempt to avoid societal shame. Far from treatises of hate, Euripidean women take advantage of the little power they possess within a constrictive social system. While both Aeschylus and Euripides revise customary images and expectations of women in the context of religiously-motivated drama, one playwright intends to maintain civic order and the other intends to challenge the secular norm.