Cellulose Synthesis in Agrobacterium tumefaciens Page: 4 of 13
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communication) and its structure is shown in Figure 5. We believe that the lipid is undecaprenol
(C55H900). We are currently determining the most appropriate method to visualize and identify the
isolated lipids from the wild type and CelC mutant.
Cloning, expression, and purification of the CeiC protein
The celC gene was cloned using PCR into the expression vector pPROTet.E133 (Clontech). This
vector has an Ltet0-1 promoter whose activity can be regulated by anhydrotetracycline (this compound
does not have antibiotic activity against E. coli). The multiple cloning site in the vector is preceded by a
six histidines (in frame) and a site for cleavage by enterokinase. The protein was purified using a nickel
resin to which it binds due to the his-tag at its beginning. The activity of the purified protein is currently
under investigation. Unfortunately this clone is very poorly tolerated by E. coli and it was difficult to
obtain much protein. In addition the imiazole required to elute the histidine-linked cloned protein from
the resin inhibits cellulose synthesis. We attempted to overcome these problems by cloning the E. coli celC
equivalent, yhjM, in a vector (pCalN) which results in the production of a calmodulin-linked protein. This
cloned protein can be purified on a calcium-containing resin and eluted with EGTA. Low concentrations
of EGTA do not interfere with in vitro cellulose synthesis. Using this system we have purified a protein
which is able to complement extracts of the celC mutant resulting in cellulose synthesis in vitro. We are
currently determining how to stabilize the protein for shipping to North Dakota for analysis of its
interactions with oligosaccharides.
Complementation of CelC mutants
A clone carrying the celC gene behind a constitutive promoter was constructed in a shuttle vector
(pBBRmcs-5) and introduced from E. coli into A. tumefaciens. This vector is low copy number plasmid
present in 5-10 copies per bacterium. The clone was able to complement the CelC mutation in vivo and
allowed the mutant to grow at a normal rate. The complemented mutant no longer accumulated the
oligosaccharides found in the uncomplemented CelC mutant. It made some cellulose in vivo but extracts
of the complemented mutant synthesized very little cellulose in vitro. One possible interpretation of this
result is that the ratio of the various Cel proteins to each other may be important for normal cellulose
synthesis.
In order to compare the functions of CelC homologues from various cellulose-synthesizing
bacteria, the homologous gene, yhjM, was cloned from E. coli and introduced into A. tumefaciens using the
same shuttle vector and promoter as was used for the A. tumefaciens celC gene. The results were the same
as those observed with the A. tumefaciens gene suggesting that the E. coli yhjM gene can substitute for
celC.
Regulation of cellulose synthesis in A. tumefaciens
Previous work had identified a Tn5 insertion that resulted in overproduction of cellulose by A.
tumefaciens. The insertion was cloned and the region surrounding it was sequenced. A gene (ceiR) was
identified which has homology to DNA binding proteins. Insertions were made in celR in wild type and
CelA and CelB mutant bacteria. In the wild type background the insertion led to overproduction of
cellulose. The CelR mutation had no apparent effect in the CelA or CelB mutant background. We
attempted to construct a CelR mutant in the CelC mutant background but the resulting bacteria grew very
poorly and died after a few transfers. The toxicity of this mutation in the CelC background may be due to
over-accumulation of the lipid-linked oligosaccharides that we have observed in the CelC mutant.
As mentioned above we found that an insertion in the celG gene resulted in bacteria that
overproduced cellulose. The mechanism underlying this effect is not understood.
The role of cellulose in bacterial adherence and biofilm formation
Cellulose-minus and cellulose-overproducing mutants were used to explore the role of cellulose in
the adherence of A. tumefaciens to plant (particularly root) surfaces and in biofilm formation on root and
plastic surfaces. The wild type bacteria bind well to roots and form a biofilm on both roots and plastic.
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White, Alan R. & Matthysse, Ann G. Cellulose Synthesis in Agrobacterium tumefaciens, report, July 31, 2004; United States. (https://digital.library.unt.edu/ark:/67531/metadc781133/m1/4/: accessed April 26, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.