Upload
corey-hazekamp
View
44
Download
0
Embed Size (px)
Citation preview
Using the Master Regulator, CtrA, to Understand Cell Cycle Regula7on in Sinorhizobium melilo.
Corey Hazekamp and Katherine Gibson University of Massachuse<s Boston
Introduction CtrA is a response regulator and transcription factor that is shared throughout many Alpha-proteobacteria. Within the water dwelling prokaryote C. crescentus, CtrA has been shown to regulate at least 95 genes, and either directly or indirectly regulates 25% of cell cycle-related genes.1 However, there have not been any in depth investigations of this master regulator in S. meliloti. S. meliloti is a symbiotic species that divides assymetrically during free-living growth and adopts a novel cell cycle as it transitions into a bacteriod during infection of its’ host, Medicago sativa. Previous research shows that CtrA is essential in S. meliloti and that there are certain genes which are likely regulated by CtrA.2,3,4 In S. meliloti, CtrA is influenced by a two-component signal transduction system consisting of histidine kinasese CbrA, PleC, DivJ, and the response regulator DivK (Figure 1).5,6 Providing insight into how the cell cycle of this bacterium is regulated will require an understanding of how CtrA is regulated, and how it affects cell cycle gene expression. Therefore, my goal is to identify cell cycle genes that are regulated by CtrA. To begin, I have constructed clones with the promoter region of ccrM, ftsE, minC, cpdR1, rcdA, pleC, cbrA, and ctrA on a plasmid upstream of the β-glucuronidase (GUS) reporter gene. This has allowed me to quantify target gene expression as indicted through levels of GUS activity. Additionally, I was able to detect bacterial GUS expression in nodules of M. truncatula. This reporter system will allow me to identify CtrA-regulated genes and provide insight into those cell cycle genes that might be involved in the development of nodules during symbiosis.
References 1) Genes directly controlled by CtrA, a master regulator of Caulibacter cell cycle. Laub MT, Chen SL, Shapiro L, McAdams HH. Proc Natl Acad Sci (2002) 99(7):4632-7. 2) A Homolog of the CtrA Cell Cycle Regulator Is Present and Essential in Sinorhizobium meliloti. Barnett M, Hung D, Reisenauer A, Shaprio L, and Long S. J Bacteriol (2001) 183(10): 3204-3210. 3) The diversity and evolution of cell cycle regulation in alpha-proteobacteria: a comparative genomic analysis. Brilli M, Fondi M, Fani R, Mengoni A, Ferri L, Bazzicalupo M, Biondi EG. BMC Syst Biol (2010) 28;4:52. 4) Global mapping of transcription start sites and promoter motifs in the symbiotic α-proteobacterium Sinorhizobium meliloti 1021. Shluter JP, Reinkensmeier J, Barnett MJ, Lang C, Krol E, Giegerich R, Long SR, Becker A. BMC Genomics (2013) 14:156. 5) The Sinorhizobium meliloti sensor histidine kinase CbrA contributes to free-living cell cycle regulation. Sadowski C, Wilson D, Shallies K, Walker G, Gibson KE. Microbiology. (2013) 159:1552-63. 6) The DivJ, CbrA, and PleC system controld DivK phosphoyrlation and symbiosis in Sinorhizobium meliloti. Pini F, Frage B, Ferri L, De Nisco NJ, Mohapatra SS, Taddei L, Fioravanti A, Dewitte F, Galardini M, Brilli M, Villeret V, Bazzicalupo M, Mengoni A, Walker GC, Becker A, Biondi EG. Mol Microbiol (2013) 90(1):54-71. 7) Molecular determinants of a symbiotic chronic infection. Gibson KE, Kobayashi H, Walker GC. Annu Rev Genet (2008) 42:413-41.
Construction and Assay of GUS Transcription Fusions So far, I have engineered 8 clones that will track expression of cell cycle genes that I hypothesize to be regulated by CtrA. CcrM is a DNA methyltransferase shown to be essential in both C. crescentus and S. meliloti, and its overexpression results in defects of cell division, cell morphology and DNA replication initiation. In many bacteria, FtsE is involved in regulating cell wall hydrolysis at the division site during cell division, and MinC inhibits FtsZ ring formation at the midpole during cell division in order to assure a proper division site. In C. crescentus, CpdR and RcdA are both required for the degradation of CtrA, while PleC functions to dephosphorylate DivK and competes with the kinases DivJ and, in S. meliloti, CbrA6. DivJ and CbrA are histidine kinases which act through DivK to repress CtrA levels, and are required for an effective symbiosis5,6. CtrA has been shown to regulate itself in C. crescentus and is predicted to have self-regulating binding sites in S. meliloti.2,4 pVO155 is a suicide plasmid with a promoter-less GUS reporter gene (uidA). Therefore, cloning the promoter region of a gene upstream of GUS will provide a method to quantify transcription at the native gene locus within the chromosome. Production of GUS can be measured several ways. 4-methylumbelliferyl-beta-D-glucuronide (MUG) is converted to 4-methylumbelliferone (4MU) in the presence of GUS enzyme. Therefore, I have optimized a protocol in which MUG substrate is added to cell lysates and enzyme activity is followed through fluorescence. 4MU is fluorescent when excited at 355nm and read at an absorption of 460nm. Below are figures showing data from MUG assays with several GUS transcription fusions. I have plotted the average “relative GUS activity” for each strain based on three independent biological replicates.
Results 1) Results so far suggest that several cell cycle genes are misexpressed in the ΔcbrA mutant when compared to WT. This
suggests that these target genes are regulated by one or more factors which are disrupted in the ΔcbrA mutant. For example ftsE, which is related to cellular division, shows a substantial increase in expression in ΔcbrA versus WT, whereas pleC is down-regulated in ΔcbrA versus WT.
2) The nodule data provides insight into which target genes contribute to development of root nodules during the symbiotic relationship between S. meliloti and M. sativa. Some of my targets, MinC and CcrM seem to only be expressed during the early stages of nodule development, primarily in the IT, but not in the nitrogen-fixing or senescent zones. Other target genes, such as RcdA, PleC, FtsE, and CtrA, are expressed in both early and late stages of nodulation and symbiosis. CbrA and CpdR1 shows little to no expression at all. This is surprising considering CbrA is required for an effective symbiosis4, however it is possible that these genes are expressed at such low levels that this method of detection is not sufficient to capture their expression pattern during symbiosis.
Future Direction The MUG assay provides accurate data that will be useful for future experiments and comparisons with additional gene fusions. I have already created a list of additional targets for consideration in attempt to create an even more thorough understanding of the CbrA signaling pathway and genes that it regulates. The plasmids that I engineered for these GUS fusions can be moved into other mutants created in our lab to gain a better understanding of how this pathway regulates cell cycle gene expression. Most importantly, plasmids engineered to overexpress certain genes in this pathway, such as CtrA, can be easily moved into my transcription fusion strains to test a direct relationship between aberrations in target gene expression and CtrA levels. The future direction for symbiosis will be to test GUS expression in the ΔcbrA mutant. This will require a marker that identifies all bacteria as a positive control since this mutant has colonization defects. This can be achieved through the use of constitutively expressed fluorescent proteins. Ideally, I will optimize a protocol to quantify the expression of target genes within nodules. Additionally, we will treat free-living bacteria with Nodule-specific Cysteine Rich Peptides, which have been shown to induce bacteroid formation, and assay GUS activity. This will provide insight into which target genes are regulated during bacteroid formation, and whether this regulation is CbrA-dependent.
Abstract Sinorhizobium meliloti is a Gram-negative alphaproteobacterium and nitrogen-fixing symbiont, which undergoes a novel cell cycle modification during its’ host-microbe interaction. I intend to identify and decipher the regulation of target genes under the control of the highly conserved transcription factor, CtrA in addition to monitoring their expression during symbiosis. Using genes known to be regulated by CtrA in C. crescentus or predicted to be regulated by CtrA in S. meliloti, I aim to show how certain cell cycle genes are regulated in S. meliloti.1,4 In C. crescentus, CtrA acts as a transcription factor that is active when phosphorylated and inactive when not phosphorylated.3 In S. meliloti, CbrA is a histidine kinase that ultimately inhibits CtrA phosphorylation.5,6 Using a ΔcbrA null mutant, which leads to increased levels of CtrA in S. meliloti5, and the β-glucuronidase (GUS) reporter gene, I can monitor the expression levels of target genes that are potentially regulated by CtrA. Promoter regions, transcription start sites, and translation start sites of target genes have been cloned into the plasmid pVO155 upstream of the GUS gene. Using a GUS activity assay, I measured target gene expression in the wild type and ΔcbrA mutant. Additionally, after infecting Medicago sativa seedlings with these fusions strains, I have used a GUS substrate to test for the presence of target gene expression in root nodules. Results thus far have shown some target genes with large differences in expression coinciding with varied CtrA levels and some target genes with only slight differences, if any at all. Tracking expression location and patterns of target genes in root nodules has shown some genes being expressed ubiquitously throughout the nodule while other genes are expressed in specific locations such as the infection zone, nitrogen fixing zone or the senescent zone. These results are significant because no one has looked yet at genes regulated by CtrA in S. meliloti, which is more applicable to host-microbe interactions than C. crescentus, especially since Agrobacterium tumefaciens and Brucella abortus both have a CbrA homologue. Additionally, I will provide critical insight into the molecular biology of the host-microbe interaction that S. meliloti participates in. .
Figure 3. GUS activity expressed from transcription fusions in WT and ΔcbrA strains. A Gain of 600 was used with excitation set at 355nm and absorption read at 460nm. Readings were taken at 30 minutes. A dilution series was created using 2uM to 10uM concentrations of 4MU to calculate the amount of 4MU produced per minute.
Acknowledgments Funding for this project was provided by funding from the University of Massachusetts Boston, NIH, and NSF awards to Katherine Gibson. I would like to extend a special thanks to Katherine Gibson, Karla Schallies, Craig Sadowski, Jamie Dombach, and the University of Massachusetts Boston for providing assistance in making this project possible.
Figure 5. The nodules shown below demonstrate different bacterial GUS expression patterns seen within root nodules of M. sativa. 1 is a WT nodule showing no GUS expression (negative control) but is used to illustrate approximate zones within the nodule: I is the apical meristem, II is the infection thread zone (IT), II-III is a mid-zone between the IT and nitrogen fixing zone, III is the nitrogen fixing zone and IV is the senescent zone. 2 is showing blue GUS expression ubiquitously, 3 is showing blue GUS expression restricted to the IT, and 4 is showing a novel GUS expression pattern in the IT, the periphery of the nodule, and in the senescent zone but not in the nitrogen fixing zone.
Figure. 2 Previous work from our lab shows an increase in CtrA levels in the ΔcbrA mutant.5 Lane 1 and 5 are purified CtrA, lane 2 is S. meliloti strain Rm1021, our wild type (WT), lane three and four are ΔcbrA mutants from a transposon mutagenes i s and a gene replacement, respectively. The ΔcbrA::cat strain (CS6000) is used to detect the difference in GUS expression between CtrA overexpression compared to WT.
CbrA DivK CtrA CcrM
FtsE
MinC
PleC
DivJ RcdA /CpdR1
Figure 4. Two single time points are used to show that cpdR1 is expressed in cpdR1::GUS strains. Fluorescent settings were identical to Figure 3. The first reading was taken at 30 minutes and the second at 24 hours. The enzymatic activity of WT cpdR1::GUS and ΔcbrA cpdR1::GUS is such that there is no significant difference versus WT or ΔcbrA. However, after a substantial amount of time, it becomes clear that cpdR1 is expressed, just at a slower rate.
Figure 1. Below is a simplified version of the DivK signaling pathway that is found in S. meliloti, B. abortus, and A. tumefaciens. This condensed model focuses on the genes discussed within this poster.
I
III
IV
II II-‐III
1 2 3 4
Figure 6. Below are pie charts showing the location of target gene expression during nodule formation within M. sativa. Assays were done at 3 and 5 weeks post-inoculation. Mature and immature nodules were grouped together for the purpose of space restriction on this poster. Examples for each category of expression are shown in Figure 5.
0
500
1000
1500
2000
2500
3000
3500
4000
4500
Wild Type ΔcbrA WT cpdR1::GUS ΔcbrA cpdR1::GUS
Arbitrary Fluo
rescen
t Unit
GUS Transcrip7on Fusion
Enzyma7c Ac7vity of GUS at 30 Minutes Versus 24 Hours
30 Min
24 Hours
pleC Expression AKer 3 Weeks ctrA Extression AKer 3 Weeks ctrA Expression AKer 5 Weeks pleC Expression AKer 5 Weeks
minC Expression AKer 3 Weeks
rcdA Expression AKer 3 Weeks
cbrA Expression AKer 3 Weeks cbrA Expression AKer 5 Weeks
5sE Expression AKer 5 Weeks 5sE Expression AKer 3 Weeks
ccrM Expression AKer 5 Weeks ccrM Expression AKer 3 Weeks
minC Expression AKer 5 Weeks
cpdR1 Expression AKer 3 Weeks cpdR1 Expression AKer 5 Weeks
rcdA Expression AKer 5 Weeks
LEGEND: n = Blue in IT and periphery of nodule n = Blue only in IT ! = Blue everywhere != No Blue
0
25
50
75
100
125
150
175
200
225
250
275
300
325
350
375
400
425
450
WT cbrA::GUS ΔcbrA cbrA::GUS WT ccrM::GUS ΔcbrA ccrM::GUS WT cpdR1::GUS ΔcbrA cpdR1::GUS WT ctrA::GUS ΔcbrA ctrA::GUS WT PsE::GUS ΔcbrA PsE::GUS WT minC::GUS ΔcbrA minC::GUS WT pleC::GUS ΔcbrA pleC::GUS
uM of 4
MU / m
inute
GUS Transcrip7on Fusion
Enzyma7c Ac7vity of GUS