Mark S. Wilson, Ph.D.
Genetic and Biochemical Characterization of PAH-degrading Microorganisms
from Marine and Estuarine Environments
Studies of naphthalene degradation
are significant because naphthalene is a common pollutant that serves as a
chemical model for the degradation of polycyclic aromatic hydrocarbons (PAHs),
which are often carcinogenic. Genetic and biochemical studies of PAH
degradation expand our abilities to effectively manage and treat polluted
environments and to engineer novel technologies for pollution abatement.
Additionally, these studies provide insights into the behavior and evolution of
conjugative plasmids, the lateral transfer of genetic information among
bacteria, and the diversity and function of oxygenase genes and enzymes.
In all pure cultures of
naphthalene-mineralizing bacteria that have been examined, the aerobic metabolism
of naphthalene is initiated by a multicomponent enzyme system called
naphthalene dioxygenase (NDO). The ability to degrade naphthalene has been
demonstrated for a wide range of bacterial genera, and these organisms have
been shown to possess a number of distinct NDOs that have widely varying
degrees of nucleotide and amino acid sequence similarities. Frequently, the genes for PAH
degradation are located on large conjugative plasmids that range from 60-500 kbp
in size. These plasmids are transmitted between a variety of bacteria in
polluted environments, thus disseminating the ability to derive carbon and
energy from the chemical pollutants.
The vast majority of studies
characterizing PAH degradation have examined bacteria residing in soils and
freshwater habitats. The few studies focusing on marine and estuarine
environments have indicated that distinct bacterial genera, using novel NDO and
other gene sequences, are responsible for attenuation of PAHs in these sites.
This study examines the genetic basis and molecular diversity of PAH
degradation in Humboldt Bay in northern CA, with a focus on the diversity of
genes, plasmids and organisms responsible for naphthalene catabolism.
Objectives and Approaches
Objective 1: Isolate and characterize a range of microorganisms
from Humboldt Bay that are capable of naphthalene catabolism.
Naphthalene-degrading microorganisms will be isolated by plating dilutions of
PAH-impacted sediments onto an artificial seawater-based medium supplemented
with nitrogen and phosphorous and solidified with purified noble agar. Naphthalene will be provided in vapor
phase. In preliminary studies done last year, 2 undergraduates working with me
isolated 50 PAH-degrading bacteria from sediments at the base of a
creosote-soaked piling in Humboldt Bay.
The students characterized the morphology, Gram Stain reactions, and
biochemical characteristics of the isolates. They cloned and sequenced 16S rRNA
genes from 12 of the isolates and demonstrated that they belonged to 4
different taxonomic groups, including Gram positive Rhodococci and Gram
negative alpha and gamma proteobacteria. These 50 isolates are stored as frozen
stocks. Starting in fall of 2004, two other undergraduates have begun to
isolate naphthalene-degrading bacteria from an area of Humboldt Bay that was
heavily impacted by a 5000-gallon fuel oil spill in 1997 and again by a
2000-gallon oil spill in 1999.
Objective 2: Characterize the diversity of NDO genes that are
present in the Humboldt Bay isolates. DNA will be extracted from
naphthalene-degrading isolates and the presence of characterized NDO genes will
be examined by attempting to PCR-amplify previously described NDO gene
sequences using a suite of primer sets. Some of these primers will be designed
by students as part of this project.
PCR products will be cloned, sequenced and compared. DNA from strains
not producing a PCR product will be examined in Southern hybridizations using
labeled probes produced from strains that are positive for amplification. We will attempt to clone novel NDO
genes from strains that don’t produce a PCR product or hybridize with NDO
probes. These cloning attempts will use an NDO-indicative indole-to-indigo
color change reaction. This
reaction allows us to directly screen colonies resulting from ligations of
size-selected genomic DNA samples partially digested with restriction enzymes.
Starting in fall of 2004, two additional undergraduates (not those involved in
culturing efforts) have begun these experiments with a subset of the isolates
from the creosote site.
Objective 3: Characterize the large conjugative plasmids that are
present in the Humboldt Bay isolates. We will identify the presence and size of
large plasmids in site isolates using Pulsed-Field Gel Electrophoresis (PFGE). Transfer of PFGE-separated DNA to
nitrocellulose membranes will allow us to assess the plasmid or chromosomal
location of NDO genes, and potentially whether multiple copies of NDO genes
exist in site organisms. Restriction Fragment Length Polymorphisms of plasmid
DNA will be used to assess the relatedness of different plasmids. Future
studies will use labeled plasmid DNA as probes to examine plasmid diversity via
Southern hybridization.
Future studies will examine the transmission of
conjugative plasmids among site isolates and in situ in Humboldt Bay. We will
use long-term non-selective culturing to produce plasmid-cured derivatives of a
diverse set of naphthalene degraders. Rifampicin-resistant, plasmid-cured
derivative strains will be obtained using rifampicin gradient plates. The host range and transmission
characteristics of the plasmids will be studied by using these derivative
strains. These derivative strains can also be used to trap and recover
conjugative plasmids being transferred in situ in Humboldt Bay. I intend to use
these results to determine the relative significance and distribution of the
NDO analogs in situ. Additionally, I intend to seek funding for the sequencing
and comparison of several of the distinct conjugative plasmids involved in PAH
degradation that are discovered during the course of the projects outlined
above.
Greater understanding of these processes can assist with
the development of new technologies and management strategies for use in
contaminated field sites. For example, we expect to identify rapidly-transmissible
and widely-transmissible plasmids involved in natural attenuation of PAHs in
Humboldt Bay. Comparison of
baseline and post-treatment levels of the genes and plasmids identified in
these studies can be used to monitor the efficacy of remediation efforts.
Additionally, engineering efforts may be brought to bear to increase the rate
at which naturally-occurring plasmids are disseminated, and particular plasmids
may be engineered for specific applications or enhanced activities. The results
will also help us to understand how these plasmids evolve, and the diversity of
plasmids and genes present in these environments.
A
major goal of this project is to engage several undergraduates (4-6 per
semester) in independent research projects that train them in techniques
important in diverse biotechnological fields. These techniques include microbiological isolation and
culturing techniques, DNA extractions, PCR, PCR primer design, pulsed field gel
electrophoresis, molecular cloning, DNA sequencing, RFLP analysis, and Southern
hybridizations. Additionally,
these students will be introduced to the fields of biodegradation and
bioremediation.
The following
abstract is for a poster presentation on this work at the 106th General Meeting
of the American Society for Microbiology, Orlando FL May 21-25.
Isolation
and Genetic Characterization of Naphthalene-Degrading Bacteria from Humboldt
Bay, CA
Donnie L. Carter*, Amber L. Orloff*, Erika
L. Kraft* and Mark S. Wilson
The ability of microorganisms
to catabolize PAH is frequently the consequence of acquiring large,
low-copy-number conjugative plasmids that carry genes for degradative
pathways. This study seeks to
understand the diversity of microorganisms, genes and conjugative plasmids
responsible for the biodegradation of naphthalene and other PAHs in marine and
estuarine systems, with a focus on Humboldt Bay, CA. PAH-degrading bacteria were isolated from creosote-contaminated
salt marsh sediments by liquid enrichment with naphthalene followed by plating
and single colony isolation on a synthetic seawater medium exposed to
naphthalene vapors. Of >50 isolates obtained, six isolates were identified
for further study because 16S rRNA sequences, microscopy and biochemical tests
indicated that they represented taxonomically diverse groups. Pulsed field gel electrophoresis (PFGE)
was used to identify the size and presence of large plasmids in the isolates.
PCR and Southern hybridization were used to identify the presence and location
(chromosome or plasmid) of previously characterized dioxygenase genes. Isolates were identified as Rhodococcus
opacus, Thalassolituus
oleivorans, Chromohalobacter
salinarum and
Sphingomonas sp. PFGE indicated that the two Sphingomonas isolates each had 2 circular plasmids (~
220 and 270 kbp), and T. oleivorans had 3 (possibly linear) plasmids (~ 170, 220 and 250 kbp).
No plasmids were detected in the Chromohalobacter isolate, and problems
with lysing the Gram positive R.
opacus isolates have so
far led to inconclusive results with those strains. PCR and Southern
hybridization indicated that diverse alleles of dioxygenases were present in
these strains, that some strains had multiple dioxygenase alleles, and that
some of these genes were located chromosomally. Taxonomically
diverse strains of PAH-degrading bacteria have been isolated from
creosote-contaminated sediments of Humboldt Bay. Characterization of these
organisms indicates that diverse large plasmids and diverse genetic systems for
PAH degradation are present in microorganisms living in the estuary.
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