Zechun Yuan and Rahat Zaheer
This projected has progressed from a
characterization of the phoCDET gene cluster that in S. meliloti
Rm1021 is required for the formation of N2-fixing
nodules (Fix-). The phoCDET transport system was demonstrated
to encode a high
affinity
Pi and phosphonate transport system and expression of this system was
positively regulated by the phoB regularory protein.
Two classes of second site mutations
that suppressed the Fix- phenotype of phoCDET matants were identified.
One class mapped to the promoter region of an alternate
Pi transport system encoded
by the orfA-piti genes. Expression of orfA-piti was negatively regulated
by phoB. The second suppressor class mapped to
the phoB gene – and
phoCDET phoB knock-out mutants were shown to transport
Pi via the orfA-piti transport system.
More recently the Rm1021 strain used
in the above studies has been found to carry a
single nucleotide C-deletion mutation – in the pstC
gene. The characterization of this mutation and regulation
of expression of
the pstSCAB
gene cluster has been the subject of a detailed study in our laboratory.
The pstSCAB gene cluster in S. meliloti has been characterized to encode
a high
affinity, high velocity Pi-specific transport system. S. meliloti cells
growing in excess Pi media transport phosphate
through the low affinity, low velocity
orfA-piti transporte system. In Pi-limiting conditions, the orfA-piti genes
are negatively regulated and therefore repressed where as the pstSCAB
and phoCDET
genes are induced. The Fix- phenotype of Rm1021 phoCDET mutants has been
shown to be dependent on the single nucleotide-C deletion mutation in
the pstC gene.
This mutation has been repaired in S. meliloti strain Rm1021 and the
resulting strain, RmP110, is now being used as
a wild type.
Members of the Pho regulon – We have
applied a whole genome in silico analysis using a frequency
matrix based on known Pho-box sequences from E. coli and S.
meliloti, to identify over 30 genes as putative members
of the
S. meliloti Pho regulon, followed by reporter fusion studies
to examine their regulation by PhoB protein. Some of these
regulons falling in the category of “unknown genes” are
being characterized.
1. Construction of the reporter plasmid
pTH1522 and its derivatives……
Jiujun Cheng, Alison Cowie, Cheryl Patten and Rahat Zaheer
We have constructed a reporter vector
for the purpose of generating transcriptional fusions of
cloned DNA
fragments to readily assayable genes. The vector
contains a single XhoI cloning site flanked on one
side by the gusA (ß-glucuronidase)
gene tdimer2 (RFP) gene and on the other side by the gfp+ (GFP) gene and
lacZ (B-galactosidase) gene. Thus there can be expression of a fluorescent
protein
and an enzyme activity for either orientation of insert DNA. The plasmid
carries gentamicin resistance, an attP site for phage FC31 and an FRT site
for directed
recombination. It replicates in E. coli but not in S. meliloti. Ttransfer
of the plasmid into S. meliloti by conjugal mating will only produce antibiotic
resistant colonies if homologous recombination occurs between the genome
and
the inserted DNA fragment. Derivatives of this vector, containing multiple
cloning sites have also been constructed: pTH1703, pTH1704, pTH1705; and
more recently pTH1945, pTH1946 and pTH1947 in which there has been in vivo
replacement
of the Gm gene in pTH1522, pTH1703 and pTH1705 with Neo/Km gene.
An E. coli strain that expresses the
phage FC31 integrase and thus catalyzes direct
recombination of pTH1522 with pTH1508 has been made. In addition,
for high-throughput microtiter plate screenings, we have optimized fast
two step
reporter enzyme assays for ß-glucuronidase and ß-galactosidase
assays.
2. A transcriptional gene fusion library for the Sinorhizobium meliloti genome
Jiujun Cheng, Alison Cowie, Chris Sibley, Bridget Kelly,
Ying Fong, Richard Morton
We have constructed a library of random
fragments of the S. meliloti genome in the reporter plasmid
vector pTH1522. These clones have been introduced back
into the S. meliloti genome by homologous recombination to generate gene
fusions to the reporter genes gfp+ and lacZ or gusA and tdimer2(12).
From 5869 S. meliloti
clones we have 4375 gene fusions. Of these 1827 unique genes are gusA/tdimer2(12)
fusions and 1810 unique genes are gfp/lacZ fusions. In addition, we have
identified approximately 100 essential genes and isolated about
10 auxotrophic mutants.
All of the S. meliloti fusion strains
have been assayed for reporter gene activity in complex
and minimal media, by highthroughput screening.
By selecting fusion
strains that are not expressed in these conditions we can ask what compounds
will induce expression and thereby begin to elucidate the function of
some of the unknown genes in the genome.
The reporter gene fusion library contains fusions to many of the genes
involved in amino acid and purine and pyrimidine biosynthesis. We are
assaying the expression
of these genes in the root nodules of alfalfa plants inoculated with
the various strains. This will allow us to further understand the metabolic
state of S. meliloti when it is the symbiotic state. The library also contains fusions
to many of the genes involved in nodulation and nitrogen fixation, these
are also being assayed for activity in root nodules. By in situ staining
of sectioned
root nodules we will be able to locate where within this structure particular
genes are expressed and we can compare how expression of these genes
differs in the symbiotic and free-living state.
3. The minimal expression library
Chris Sibley, Bridget
Kelly and Ying Fong …
Following the high throughput
reporter enzyme assay screen of the S. meliloti gene
fusion
library in
complex and M9 minimal
media two minimal libraries were constructed. 1563
gene fusions were assigned to either the Minimal
gusA
Library (646) or the
Minimal lacZ Library (917). Fusions assigned to these
minimal libraries have to satisfy three conditions:
1) recombination of
the reporter plasmid into the S. meliloti genome generates
a transcriptional fusion 2) the reporter genes
are transcriptionally fused to a
gene of “unknown” function and 3) the correctly
fused reporter enzymes do not have greater than two fold wildtype
enzyme activities in M9 glucose and M9 succinate. Thus
all strains in
the minimal library represent gene fusions to genes
of
unknown function that are not expressed in M9 minimal
media. The minimal
libraries were then grown in the many different carbon
and nitrogen source cocktail mixtures and reporter
enzymes assayed. Activation
of gene expression (at least 3 fold) was detected in
~13% of the fusions in both minimal libraries. All
the fusions that showed
induction of reporter enzyme activity in a particular
cocktail were then assayed in the individual constituents
of the cocktail.
To date we have identified individual compounds that
induce the expression of 91 of the fusions from the
S. meliloti fusion library
and this number is ever growing. This information will
provide a focused direction in defining the metabolic
role of unknown
gene clusters.
4. The transportome project
Jane Fowler and Andrea Sartor
Almost all compounds metabolized
by bacteria have first to be transported across
the cytoplasmic membrane. In many cases, for free-living bacteria with large
genomes, the actual transport systems responsible for the transport of indivual
substrates are not synthesized until the substrate is present in the media.
Thus, in general, expression of individual transport systems is induced in
the presence of the transported ligand.
In this project, we wish to identify
conditions under which the transport systems of Sinorhizobium meliloti are expressed. To monitor expression of the transport
systems, we are employing readily assayed reporter gene fusions. The genes
encoding the transport systems in S. meliloti are known from annotation
and
informatics and reporter gene fusions in the pTH1522 fusion library to
transport genes were identified. These fusions are being
screened under diverse nutritional
conditions for expression. Reporter fusions to transport systems not present
in the library are being constructed and tested for expression.
Knock-out mutants of various transporters and their respective metabolism
genes have been tested on the inducing compounds in order to elucidate
no growth
phenotypes to indicate transporter function.
5. Malic enzyme project
Laura Smallbone
In the past, we focused on
elucidating the pathways through which succinate
and malate (C4-dicarboxylic acids) were metabolized
by Sinorhizobium meliloti. This involved characterizing
various
mutants that fail to grow on succinate as a sole carbon
source. That work led to the characterization
of the C4-dicarboxylate
transport genes (dctABD), genes encoding phosphoenolpyruvate
carboxykinase and two malic enzymes – the
NAD+-malic enzyme DME, and the NADP-dependent
malic enzyme TME. We are now continuing this
study
with a focus on identifying the functions of both DME
and TME by conducting a metabolic analysis of
mutants and identifying
possible binding partners.
6. Biodegradation by Sinorhizobium meliloti – Characterization
of Protocatechuate metabolism
Allyson MacLean
Aromatic compounds represent an important energy and carbon source for soil-dwelling
bacteria. The beta-ketoadipate pathway is involved in the conversion of the
aromatic compound protocatechuate into succinate and acetyl-CoA. My work has
focused upon a basic characterization of the pca genes in S. meliloti. I have
demonstrated that ORFs Y20587 and Y20588 encode subunits of the beta-ketoadipate
succinyl-CoA transferase, and have identified a transcriptional regulator involved
in the regulation of expression of these genes. We are also interested in expanding
our current work to include the study of genes involved in the metabolism of
diverse compounds present in the soil through the use of root or soil exudates.
7. The FRT deletion analysis of the Sinorhizobium meliloti genome
Branislava Poduska
The 6.7 megabase S. meliloti
genome contains many non-essential genes that presumably
play roles in allowing the bacteria
to compete and survive in soil environment and in
the rhiozosphere. Deleting large non-essential regions
of the genome will help us identify phenotypes associated
with these genes. Hence mutants with 4-500 kb dekletions
can be screened for phenotypes.
While this project
has a separate designation, it integrates
with almost all other projects in the laboratory.
This is so, as the deletions generated in this
project are
used in other studies to: a) confirm, and examine
predicted mutant phenotypes, b) generate strains
lacking particular
pathways, c) identify essential genes, d) identify
novel gene functions.
8. Microarray analysis of Sinorhizobium meliloti – collaboration with Dr. Richard Morton and Dr. Brian Golding, Biology, McMaster University.
Jiujun
Cheng
Using Nimblegen microarrays for the S. meliloti genome, we have
examined gene expression in Sinorhizobium meliloti following growth in
complex media (LBmc), and minimal medium containing glucose and succinate as sole sources of carbon.
That serves as baseline gene expression data S. meliloti.
