09/27/12              Progress Report for Award #SCW1165                  Page 1
Title of Project:  Systems level investigation of uranium response and regulation by Caulobacter
crescentus
Institution:      Lawrence Livermore National Laboratory
Date Received:    August 2011
Principal Investigator: Dr. Yongqin Jiao
Project Results:
In the past year, we have fully started this project both in terms of laboratory setup and staffing.
We have outfitted a new laboratory that hosts most of the equipment needed for the project, and we hired
two postdoctoral fellows (Drs. Cho and Ma) who work on the project full time. Dr. Mimi Cho received her
Ph.D. in the laboratory of Dr. JoAnne Stubbe at MIT in early 2012. Her graduate work focused on the
biochemistry of polyhydroxybutyrate granule formation in Ralstonia eutropha. She has expertise in protein
purification and enzyme characterization, which will be useful in our biochemical studies on the putative
phytase. Additionally, she has experience in both fluorescence microscopy and TEM of bacterial cells,
which will help us to characterize the physiology of C. crescentus under uranium stress. Dr. Jincai Ma has
a Ph.D. in environmental sciences from Rutgers University. During his graduate studies, he employed a
combination of molecular genetics, chemical kinetics, and spectroscopy to elucidate the mechanisms of
selenium reduction by Enterobacter cloacae. After receiving his Ph.D., he studied the persistence and
survival of the food pathogen, E. coli 01 57:H7, in soils of leafy greens at the US Salinity Laboratory (U.C.
Riverside) for his postdoctoral training. Dr. Ma's role in the current project is to investigate the proteomics
of C. crescentus in response to various stresses and to examine microbe-uranium interaction. Dr. Cho
started at LLNL in March 2012 and Dr. Ma in June 2012. It should be noted that the PI, Dr. Jiao, took a 3-
month maternity leave (Nov 2011- Jan 2012). As a result, the pace of the progress in the last year was
not as fast as we had hoped. Nevertheless, we have begun several lines of experimental approaches
towards the overall goal of our research: deciphering the mechanism of uranium (U) resistance in
Caulobacter crescentus.
In the past year, our research has focused on one question: what are the genes and proteins that
are involved in U (and other heavy metal) resistance in C. crescentus and how do these genetic elements
function to confer resistance? We have started to test several of our hypotheses through a targeted
approach, in which we have focused on the roles of a putative phytase and an S-layer protein. In addition,
we employed three unbiased approaches towards an understanding of the fundamental processes that
underpin U resistance, including Tn-seq, proteomic profiling, and Fosmid library screening. For these
studies, we have integrated bacterial culturing, genetics, biochemistry, spectroscopy, electron
microscopy, and next-generation sequencing. Our major accomplishments for the first year of the ECRP
are listed below, categorized by specific aims.
Aim 1. Characterization of U resistance by C. crescentus
C. crescentus is noteworthy for its ability to survive high concentrations of U (7), but the
physiology with respect to U resistance and microbe-U interaction is not well understood. We have
established methods and techniques for characterizing U resistance and mineral transformation that will
be useful for characterization of mutants in the future. Through growth and cell-suspension experiments,
we found U resistance by C. crescentus is greatly dependent on medium composition, in particular,
calcium and phosphate concentrations. In both rich (PYE) and minimal (M2G and M5G) media, chemical

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