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[Biofilms] PM Newsletter

Biolog, Inc. via biofilms%40net.bio.net (by Biolog_Inc from mail.vresp.com)
Thu Sep 20 12:02:44 EST 2007

Bacillus subtilis Metabolism and Gene Regulation Studied in Two Recent
Applications of Phenotype MicroArray Technology

Genome-scale reconstruction of metabolic network in bacillus subtilis
based on high-throughput phenotyping and gene essentiality data. Oh
YK, Palsson BO, Park SM, Schilling CH, Mahadevan R. Chemical
Engineering and Applied Chemistry, University of Toronto, Toronto, ON


This landmark paper is the first example of a careful and thorough use
of Phenotype MicroArray data to challenge and improve a whole-cell
bioinformatics model of Bacilllus subtilis. Network gap analysis was
used to identify 48 essential reactions missing from the genome
annotation. Detailed growth rate analysis revealed incorrect modeling
outcomes which were subsequently improved by adding 75 reactions.
Simulation of growth phenotypes of knock-out strains were verified in
720 of 766 cases. Overall, the integrated analysis of substrate
utilization and gene essentiality data led to identification of 80
required enzymes and improved the genome annotation for Bacillus

Regulatory Overlap and Functional Redundancy Among Bacillus subtilis
Extracytoplasmic Function (ECF) (sigma)-Factors. Mascher T, Hachmann
AB, Helmann JD. Department of Microbiology, Cornell University,
Ithaca, NY 14853-8101, USA.


The biological role of extracytoplasmic function sigma factors in
Bacillus subtilis was examined in this paper. Seven genes encode these
sigma factors, so the authors constructed strains with multiple gene
knockouts and then used Phenotype MicroArray technology to test the
phenotypes of their strains. A triple mutant in sigM, sigW, and sigX
exhibited greatly increased sensitivity to detergents, polymyxin B,
B-lactams, and D-cycloserine. 

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Brief description of PM technology

Phenotype MicroArray™ technology tests cellular phenotypes. Included
in the tests are assays of basic cellular nutritional pathways for C,
N, P, and S metabolism, osmotic and pH and sensitivity, and
sensitivity to chemical agents.

The most common application is to assess the phenotypic effects of
mutations. A change in genotype of a cell should lead to one or more
changes in phenotype. PMs allow testing of knock-out or knock-in
mutants to help discern the biological changes that occur consequent
to genetic changes.

Another common application is phenotypic characterization of a
collection of related strains. For example, it is possible to
determine the phenotypic relatedness of a collection of isolates of a
given species. PM analysis has been successfully implemented for a
wide variety of model microbial cells including Escherichia coli, 
Salmonella enterica, Pseudomonas aeruginosa, Pseudomonas putida,
Sinorhizobium meliloti, Saccharomyces cerevisiae, Bacillus subtilis,
Bacillus cereus, Shewanella oneidensis, Proteus mirabilis, and many
other species. In the last year, Biolog has successfully developed
universal protocols that allow us to test most species of interest.
Contact us for details.

Phenotype MicroArray™ technology uses Biolog's OmniLog® instrument,
which is also used for microbial identification. The OmniLog®
automatically incubates, reads and interprets the Biolog Phenotype
MicroArray MicroPlates™. It continuously processes samples but allows
the user complete access at any time during a sample run. Samples can
be loaded when ready and removed when complete without disturbing
other samples still in-process. Inside the Reader there are 25 trays.
Each tray holds 2 MicroPlates, giving the Reader a total capacity to
incubate and read 50 MicroPlates. Before the user inoculates the
appropriate MicroPlates, they log the MicroPlate information into the
OmniLog® software.

By simply following the software's instructions, the user then opens
the door of the OmniLog® Reader and places each MicroPlate in the
appropriate tray slot indicated by the software. Once all the
MicroPlates are loaded and the door of the Reader is closed, the
OmniLog® software takes over the responsibility for incubating,
reading, saving, and printing the results. The OmniLog® PM System
utilizes Windows-based software with an elegantly simple user
interface. The status of all the samples can be observed by simply
looking at the Read menu screen of the OmniLog® PM System software.
All of the information for a specific MicroPlate is contained on a
single line of this screen. Once the system has completed a specific
MicroPlate it indicates this status with a check mark icon. A clock
icon is used for those samples that are still incubating because a
result has not yet been determined. The OmniLog® PM System's software
is extremely flexible and can be integrated into virtually any
laboratory's workflow. New MicroPlates can be entered into the Reader
and MicroPlates that have already completed can be removed from the
Reader at almost any time. This allows the user to either perform
experiments one at a time or batch them.

PM Technology and the OmniLog® instrument are ideal for a core
facility or for purchase under a shared equipment grant.

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