A Functional Genomics Clearinghouse as an Outcome of Undergraduate& High School Education – An Update of Efforts at Hiram College

Brad Goodner1, Cathy Wheeler1, Prudy Hall1, Stuart Gordon1, Kathryn Reynolds1, Stephanie Lammlein2, Lindsey Wilson1,

Garrett Temkiewicz1, 2002-2007 Molecular & Cellular Biology courses1, 2002-2007 Genetics courses1, 2006-2007 Introductory Biology courses2, & 2006-2007 Hiram Genomics Academy sessions3

1 Hiram College, Hiram, OH (goodner@hiram.edu, 330-569-5260), 2 Rootstown High School, Rootstown, OH,& 3 81 high school students from all over Ohio, Michigan, Pennsylvania, & Indiana

Abstract

Obtaining the complete genome sequence of any organism is really just a new beginning – you have a lot of tools now available but are not yet sure how best to use them. Functional genomics is really just an extension of how molecular biology and microbiology have made progress over the past 25 years or more through characterization of mutants through forward or reverse genetics. The extension is one of breadth to include as many genes as possible. Over the past 6 years, Hiram College faculty and students have accumulated a large set of mutations in the A. tumefaciens C58 genome (and to a lesser extent in the A. rhizogenes A4 genome). Students in the Molecular & Cellular Biology course use reverse genetics to test functional predictions based on bioinformatics analyses of gene function. We will highlight the work done by the 2006 and 2007 iterations of the course. Students in the Genetics course, along with high school students during the past two years, have used forward genetics to link genes to functions through a large scale mutant hunt. We will highlight some of the more interesting findings in several functional categories. Finally, we will look forward to future efforts and we are certainly open to new suggestions from others.

2007 Molecular & Cellular Biology Course – Penicillin Binding Proteins

The bacterial cell wall is crucial for withstanding turgor pressure and also as a cell shape determinant. The wall is composed of a heteropolymer called peptidoglycan that has both polysaccharide and peptide characteristics. The major enzymes involved in wall synthesis, maintenance, and turnover are nicknamed Penicillin-Binding Proteins (PBP’s) because the antibiotic penicillin and its derivatives act as irreversible enzyme poisons on these proteins. Most rod-shaped bacteria have 6 or more PBPs that fall into 4 categories. Class A PBP’s construct new peptidoglycan chains and crosslink them. Class B PBP’s are only involved in cros-linking peptidoglycan chains, but do so in either a elongation-growth or septation-growth mode depending on the enzyme. The low molecular weight PBP’s modify peptidoglycan chains before or after crosslinking. Finally, cell wall-localized b-lactamases, besides their impact on antibiotic resistance, can be involved in peptidoglycan turnover.

E. coli and Bacillus subtilis have been the models for studying PBP activities. Data from these two organisms have shown that the Class B PBP’s influence cell shape in terms of the lengths of rods or rod-to-coccus transitions because of their differential activities during the cell cycle, and the low MW PBP’s influence the uniformity of cell shape by altering the cross-linking level and substrate availability.

The Fall 2007 Molecular & Cellular Biology course at Hiram College (50 students) set out to look at the function of the 11 PBP’s encoded by the A. tumefaciens C58 genome. During the course of a 12-week semester, the students were able to make mutations in 9 of the 11 genes and to obtain phenotypic data on 8 of the mutants. Here are their initial observations (also see Table 1):

* While some of the mutants might have a slight change in cell shape, the Atu1341- mutant had a dramatic cell shape change and showed a large decrease in growth rate. The Atu1341- cells had a variety of amorphous shapes with few regular rods.

* Most of the mutants had a definite impact on cell motility. The Atu1499- mutant is very hypermotile, as compared to wildtype, on both 0.3% and 0.9% agar plates. The Atu1341- mutant is hypermotile only on 0.3% agar. The Atu1067-, Atu1505-, Atu2513-, and Atu3694- mutants were hypomotile on all agar concentrations tested.

Interesting Stories from Forward Genetics

Screen for Biofilm Defects (change in colony morphology on LB agar minus NaCl plus Coomassie Blue and Congo Red; Figure 2):

Atu2121 on ChrIencodes Lyc glycosyl hydrolasemay be involved in exopolysaccharide synthesis or maintenance

Atu2660 on ChrI (HCA61)encodes a conserved hypothetical protein

Atu3327 on ChrII (HCA41)encodes ExoY succinoglycan exopolysaccharide synthesis protein

Atu3437 on ChrIIencodes a ABC transporter ATP-binding protein

Atu3740 on ChrII (HCA82)encodes fructose bisphosphate aldolasemay influence substrate availability for making exopolysaccharide

Atu4000 on ChrII (HCA40)encodes BioA adenosylmethionine-8-amino-7-oxononanoate aminotransferase

role of biotin in biofilm? – in E. coli, bioF- mutant has altered biofilmAtu4053 on ChrII

encodes ExoA succinoglycan biosynthesis proteinAtu4668 on ChrII

encodes a ABC transporter permease

Screen for pH Tolerance (lack of growth on LB agar pH 5.5 or LB 10):15 acid sensitive mutants

all of them only show defect on solid medium! Why?4 base sensitive mutants (Figure 3)

most interesting is Atu0512 encoding PhaA pH adaptation K+ efflux system component

mutation in Atu0512 homolog of Sinorhizobium is base-sensitive & is nodulation-negative

experiments underway to see if Atu0152- mutant is virulent

Screen for Auxotrophy:identified steps in pathways for aspartate, branched chain amino acid, histidine,

methioinine, purine, pyrimidine, serine & glycine, & tryptophan synthesismutations in glucose dehydrogenase & transaldolase point out crucial role for Entner-

Doudoroff & pentose phosphate pathwaysmutation in glycogen synthase impacts use of glucose but not of glycerolmutation in Atu3885 encodes a inositol monophosphatase family member that is

neededfor growth on both glucose and glycerol; role?

mutation in Atu1457 leads to auxotrophy for sulfur; gene encodes a conserved hypothetical protein ; in many organisms, this gene is right next to Atu1456 encoding CysI sulfite reductase hemoprotein beta subunit (Figure 4); our

working hypothesis is that Atu1457 encodes a previously unknown protein of the

cysteine synthesis pathway; students at Rootstown High School are currently working

to see if the auxotrophic phenotype in Atu1457- mutant is due to that mutation or just a polar effect within the operon

Table 1. Data on PBP- mutants obtained by 2007 Molecular & Cellular Biology course.

Gene Current Best Hit Domains identified Possibly Growth Impact onNumber Annotation to E. coli by InterProScan* Essential Defect MotilityClass A PBP’s (have both glycosyltransferase & transpeptidase activities):Atu0103 PBP1A PBP1B 1,2,3,4,14 N N HypomotileAtu0931 PBP PBP1B 1,2,3,4 N N NAtu1341 PBP1A PBP1A 1,2,3,4 N Very slow HypermotileAtu3694 PBP PBP1C 1,2,4,12,13 N N Hypomotile

Class B PBP’s (have only transpeptidase activity):Atu1067 PBP PBP2/3 2,8,9 N N HypomotileAtu2100 PBP2 PBP3 2,4,8,9 N no data no data

Low MW PBP’s (have either DD-carboxypeptidase or endopeptidase activity):Atu1499 Dac PBP5/6 4,5,6,7 N Slow Very hypermotileAtu1505 Dac PBP5/6 2,6,10 N N HypomotileAtu2321 Dac PBP5/6 2,6,10 Y?Atu3634 DacF PBP5/6 2,6 Y?

-Lactamases:Atu2513 PBP -lactamase 11 N N N

* InterProScan Domains:1 = Family 51 glycosyl transferase domain2 = PBP transpeptidase domain3 = PBP1a domain4 = Beta-lactamase transpeptidase domain5 = Peptidase S13 D-Ala-D-Ala carboxypeptidase C domains6 = Peptidase S11 D-Ala-D-Ala carboxypeptidase A domains7 = PBP-associated domain8 = Class A Beta-lactamase9 = PBP dimerization domain10 = Cell division protein domain11 = Beta-lactamase family12 = PBP1c domain13 = PBP C-terminal domain14 = WW/Rs5/WWP domain

Summary of Accomplishments Up to Date

Reverse Genetics75 specific genes disrupted so far & mutant phenotypes characterized

Forward Genetics~10,000 Agrobacterium transposon insertion mutants generated ~10,000 mutants screened for 10 different phenotypes 160 mutants with interesting phenotypes saved for further analysis genomic DNA isolated from 119 of these mutants the site of transposon insertion

cloned out & sequenced

Figure 1. Cell shape of the Atu 1341- mutant is highly altered. A, B: Atu1341- mutant. C, D: Atu1499- mutant. E, F: Atu2513- mutant. A, C, E: Light microscopy of crystal violet-stained cells. B, D, F: Fluorescent confocal microscopy of Bocillin FL-labeled cells (fluorescent penicillin derivative).

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2006 Molecular & Cellular Biology Course – 24 Two-ComponentSystem Mutants, 12 Environmental Variables, 54 Treatments

Two-component systems are a common regulatory mechanism in bacteria and the A. tumefaciens genome is loaded with 34 paired teams, 5 hybrid proteins, 17 orphan histidine sensor kinases, and 16 orphan response regulators. Previous work by others had characterized 7 proteins that fall into this family. The 2005 and 2006 iterations of the MolCell course knocked out 11 and 16, respectively, of the response regulator genes. The 2006 course used a new microtiter plate reader to study the phenotype of 20 of the response regulator mutants. A summary of their findings is given in Table 2.

Table 2. Summary results for a phenotypic microarray of 20 response regulator mutants.

Mutant Strain Condition*Atu0050- (actR) glucose-low, glucose, sucrose, xylose, succinate, ammonium-low,

nitrate, glutamine, arginine, 1%NaCl, 2%NaCl, 1.5%KCl, 2%NaCl, 3%KCl, Mg-both5.5, Mg-low7, temp-35C, pH10, Ca-all

Atu0114- (dctD) succinate, 2%NaCl, Mg-both5.5, pH5.5, Ca-0.1mM, Ca-none,0.1uM,10mM

Atu0343- (barA) succinate, Atu0527- glucose-low, succinate, 2%lactate Atu0629- pH5.5Atu0970- (phoP) succinate, 3%KCl, Mg-both5.5, pH5.5, temp-15CAtu0978- (ragA) glucose-low, Mg-both5.5, pH5.5, Fe-DIPAtu1116- (nwsB) glucose-low, succinate, Mg-high5.5, pH5.5Atu1297- (pleD) proline, 2%NaCl Atu1446- (ntrC) glucose-low, succinate, serine(C), 2%NaCl, 3%KCl, Fe-M9 Atu1448- (ntrX) succinate, 2%NaCl, pH5.5 Atu1900- (agp1) glucose, glucose-low, sucrose, xylose, glycerol, succinate,

histidine(C), serine(C), ammonium-low, nitrate, proline,1%NaCl, 1.5%KCl, 2%NaCl, 3%KCl, Mg-both7, 1%lactate, 2%lactate,1%urea, 2%urea, temp-all, pH5.5, phosphate-all, Ca-all, Fe-all, H2O2,Cu

Atu2165- (agp2) glucose, glucose-low, sucrose, xylose, glycerol, succinate, histidine(C), serine(C), ammonium-low, nitrate, serine, proline,1%NaCl, 1.5%KCl, 2%NaCl, 3%KCl, Mg-both7, 1%lactate, 2%lactate,1%urea, 2%urea, temp-all, pHall, pH10, phosphate-all, Fe-all, H2O2, Cu, Oxyrase, anaerobic

Atu2206- (petR) 1.5%KCl, 3%KCl Atu2332- succinate Atu2434- (ctrA) succinate, 2%NaCl, Mg-high5.5 Atu3907- (nasT) 2%NaCl, temp-15C Atu4047- (cvgS) succinate, proline, Mg-both5.5, pH5.5Atu4300- (nwsB) glucose, sucrose, xylose, histidine(C), serine(C), nitrate, 1%NaCl,

2%NaCl, 1.5%KCl, 2%NaCl, 3%KCl, Mg-both5.5, Mg-low7, 1%lactate, 2%lactate, 1%urea, 2%urea, pH10

Atu4805- xylose, 2%lactate, 2%urea, Ca-10mM

* Interpretation of impact results:blue means the mutant showed growth >50% greater than that seen for wtred means the mutant showed >50% less growth than that seen for wtbold means in a ratio to wt under that condition (e.g., mutant at pH5.5/wt at pH5.5)underlined means as a ratio to itself at baseline condition (mutant pH5.5/mutant pH7 compared to wt 5.5/wt 7)

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Figure 4. Atu1457 orthologs are in a consistent genomic context. A: Position of the Atu1457 ortholog (pale green arrow) just behind Atu1456 CysI ortholog (bright red arrow) in a wide variety of genera within the alpha-Proteobacteria. B, C: Similar alignment for members of the gamma-Proteobacteria and beta-Proteobacteria, respectively. Atu1456 CysI ortholog shown in pale green while Atu1457 ortholog shown in bright red.

This work was funded by Hiram College and by

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Figure 3. Atu0512- mutant is base-sensitive. Growth curve of Atu0512- mutant in LB broth at different pH levels.

Figure 2. Biofilm defective mutants. A: Colony morphology of selected mutants on LB-NaCl+dyes. Wildtype is shown at the top. B: Pellicle morphology of selected mutants. Wildtype is shown at the top. C: Assay for binding to polystyrene microtiter wells under different [Pi].

Hiram genomics academy

Atu1456 Atu1457 Atu1456 Atu1457