SCSU Students Speak at Bennett College
Space Week

SCSU Biology majors Bradford Gillens and Daniel Howard presented the results of their summer research at a recent NASA-funded meeting at Bennett College. Gillens and Howard conducted their work under the direction of Dr. Waltena Simpson, Department of Bilological and Physical Sciences at SCSU. Their presentations at Bennett College were a part of a collaboration between SCSU and Bennett College under the NASA MUCERPI-2003 award to SCSU.

Identification of Plasmids from Two Novel Sphingomonas Strains

Student Presenter: Mr. Bradford Gillens, South Carolina State University
Faculty Mentor: Dr. Waltena Simpson, Department of Biological and Physical Sciences,
South Carolina State University

Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds made up of two or more benzene rings fused in either a linear, angular or cluster arrangement. Basically, they contain only carbon and hydrogen atoms, although sulfur, oxygen and nitrogen may sometimes substitute carbon atoms in the aromatic rings. PAHs are formed whenever organic matter is burnt but, as environmental pollutants, mainly issue from the processing, disposal and combustion of fossil fuels. Due to their potential trophic biomagnification and acute toxicity, and in some cases carcinogenic effects, they are considered environmentally unsafe.

A large number of bacteria with PAH-degrading capabilities have been reported. The microorganisms were able to either completely assimilate a defined range of compounds or to exhibit partial metabolism. Many Sphingomonas spp. have been recently recognized as being catabolically versatile in the degradation of aromatic compounds because they are capable of utilizing both low and high molecular weight aromatic compounds. Two novel Sphingomonas strains, BPF and BPH, were recently isolated from a waste lagoon in Poland. Like other Sphingomonas strains, BPF and BPH are capable of degrading various PAHs. Specifically, Sphingomonas strain BPH is capable of converting indole to indigo, and degrading naphthalene, phenanthrene, fluorene, acenaphthene, fluoranthene, pyrene, and benzo [b] fluoranthene. Examination of Sphingomonas strain BPH revealed that it had many of the same capabilities as strain BPH. However, in contrast to strain BPF, strain BPH was incapable of degrading naphthalene but capable of degrading anthracene.

Typically, Sphingomonas strains possess relatively large plasmids which often harbor genes involved in PAH degradation. To determine if strains BPF and BPH contained plasmids, both strains were taken through a Sphingomonas-specific plasmid isolation procedure. Plasmid preparations were electrophoresed on 0.7% agarose gels. Our studies revealed the presence of 2 plasmids in strain BPF and 1 plasmid in strain BPH. The 2 plasmids in strain BPF were designated pBPF1 and pBPF2. pbpf1 is 95 kb while pBPF2 is ~65 kb. The single plasmid found in strain BPH was designated pBPH1 and in >165 kb in size. Because these novel plasmids may harbor PAH-degrading genes, further studies will be performed to cure the plasmids from these strains. This will allow us to determine specifically which PAH-degrading genes are localized to them.

Employing a System of Random Transpositional Mutagenesis to Create Polycyclic Aromatic Hydrocarbons Utilization Mutants of Sphingomonas Strain BPH

Student Presenter: Mr. Daniel Howard, South Carolina State University
Faculty Mentor: Dr. Waltena Simpson, Department of Biological and Physical Sciences,
South Carolina State University

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that are a worldwide problem. They are organic compounds that may be carcinogenic, mutagenic, or genotoxic. PAHs. can be removed from the environment through bioremediation, which uses living organisms to degrade organic contaminants into less toxic forms. Many microorganisms, such as Sphingomonas spp, can degrade PAHs. Many Sphingomonas strains are capable of degrading organic contaminants including PAHs. A novel strain of Sphingomonas, designated Sphingomonas strain BPH, was isolated at SRS from an oil refinery in Katowice, Poland. BPH can convert indole to indigo and is capable of degrading the PAHs anthracene, phenanthrene, and fluoranthene, and fluorene. However, nothing is known regarding the genes responsible for the phenotype of Sphingomonas strain BPH. The overall purpose of this study was to create PAH-degrading mutants which would allow us to begin to identify the specific genes in Sphingomonas strain BPH that confer upon it the ability to degrade these PAHs. To produce PAH-degrading mutants, a system of random transpositional mutagenesis was employed. The Tn5 mini-transposon contained on the suicide vector pUT:miniTn5KmluxAB was transferred to Sphingomonas strain BPH using Escherichia coli S17 ?pir as the donor strain.

Transpositional mutagenesis with miniTn5Km resulted in the production of 2,150 kanamycin-resistant transconjugants of BPH. R2A agar plates containing the transconjugants were then oversprayed with phenanthrene, or streaked onto plates containing 1mM indole to select transconjugants which were unable to degrade/convert these compounds. Three phenanthrene degradation mutants (transconjugants 257, 1778 and 1782) were identified. Biodegradation of phenanthrene was determined by clear zones around the periphery of the Sphingomones colony. Four mutants deficient in their ability to convert indole to indigo, a dioxygenase-mediated activity, were also identified. Specifically, the indole conversion mutants were transconjugants 1778, 2106, 2124, and 2135. Interestingly, the phenanthrene degradation mutants 257 and 1778 were also unable to convert indole to indigo, indicating that a dioxygenase gene has been disrupted in these transconjugants.

Sphingomones strain BPH was also found to possess a large plasmid, as is typical of Sphingomonas strains. The plasmid in strain BPH, designated pBPH, is >165 kb. Our studies indicate that in our mutants, Tn5 has inserted into the genome and not into pBPH. Analysis of the genetic lesion produced by Tn5 in these mutants will allow us to identify novel genes involved in PAH degradation in Sphingomonas strain BPH.

 

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