Advanced Bacterial Genetics:

Genetics and Physiology of Bacterial Membrane Proteins

January 21-26, 2008.

Over 70 years ago microbial genetics led to the revolution in molecular biology, providing powerful new
methods for exploring the fundamental mechanisms of life. In the mid-1970s the development of
molecular cloning coupled with the characterization of transposons led to a second revolution in
molecular genetics, providing a set of indispensible tools that facilitated the expansion of basic genetic
selections and screens to a wide variety of bacteria. Recently another revolution in molecular genetics
has been stimulated by the sequence determination of hundreds of complete genomes in combination
with methods to determine RNA and protein expression levels as well as a complete library of sets of
interacting proteins and genome-scale 3-dimensional protein structure determinations. These new
approaches provide insights into how each of the multiple different cell components changes in response
to a particular stimulus – a holistic perspective called systems biology. Despite all of these genomic and
post-genomic advances, genetic analysis is needed to verify the roles of particular gene products in vivo.

These genetic methods provide tools for testing hypotheses raised from genomics and systems biology
approaches, elucidating the structure, function, regulation, and physiology of bacteria. In addition to
providing novel insights into basic biological processes, combining systems biology and genetic
approaches will provide practical insights into the role of bacteria in the environment, health and
disease, and the application of bacteria in biotechnology.

To illustrate how genetic methods can be used to answer complex biological questions, this course will
use directed readings to focus on bacterial membrane proteins. Membrane proteins lay at the interface
between the inside of the cell and the outside environment, and catalyze many critical cellular processes,
from transport of nutrients into the cell to metabolism to sensing environmental signals and regulating
gene expression. However, because membrane proteins are more difficult to study than soluble proteins,
we still know surprisingly little about questions such as how do proteins associate with the membrane,
how is the function of membrane proteins modulated by membrane physiology, and how do membrane
proteins transmit information from outside the cell to inside the cell. The directed readings will focus on
the genetic and biochemical analysis of membrane proteins involved in genetic regulation.

The course will integrate three components:

• lectures on bacterial genetics, genomics, and post-genomic methods
• practical experience solving experimental problems in bacterial genetics and physiology
• directed readings of research papers that emphasize the use of these approaches to characterize
bacterial membrane proteins

Daily schedule: 9:30 - 12:00 PM Lectures (coffee break about 10:30 AM)
12:00 - 12:30 PM Problem solving / present assigned papers
12:30 - 1:30 PM Lunch
1:30 - 4:30 PM Read assigned papers
4:30 - 6:00 PM Discuss papers

11/22/07
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General References:
Hughes K. and Maloy S. (Eds). 2007. Advanced Bacterial Genetics. Methods Enzymol. Volume
421. Academic Press, San Diego, CA.
Maloy S, Stewart V, and Freifelder D. 2008. Microbial Genetics. Jones and Bartlett Publishers, MA.

Supplemental Website:
Microbial Genetics <http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/>

Day 1 (Mon, January 21): Bacterial genomes, genes, and mutations

Introductions and course overview

Bacterial genomes
Genome organization
Gene expression
Operons and polarity
Genetic regulatory mechanisms
Mutations
Types of mutations (Loss of function vs Gain of function)
The zen of mutagenesis (Selection, Screen, Enrichment; Frequency vs Rate)
Compensating second-site mutations – suppressors
Complementation analysis

Background Reference:
Microbial Genetics <http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/>

Research Papers:
Figueroa-Bossi N, Lemire S, Maloriol D, Balbontín R, Casadesús J, Bossi L. 2006. Loss of Hfq
activates the σE-dependent envelope stress response in Salmonella enterica. Mol Microbiol. 62:
838-852. [This paper describes the identification of small regulatory RNAs that mediate the
adaptive response to incorrectly folded membrane proteins.]
Button JE, Silhavy TJ, Ruiz N. 2007. A suppressor of cell death caused by the loss of sigmaE
downregulates extracytoplasmic stress responses and outer membrane vesicle production in
Escherichia coli. J Bacteriol. 189: 1523-1530.
or
Nehme D, Poole K. 2007. Assembly of the MexAB-OprM multidrug pump of Pseudomonas
aeruginosa: component interactions defined by the study of pump mutant suppressors. J
Bacteriol. 189: 6118-6127. [This paper describes the use of suppressor mutations to dissect the
structure and function of an antibiotic resistance efflux pump.]

Day 2 (Tues, January 22): Transposons

Use of transposons as mutagens

In vivo transposition
In vitro transposition
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Transposons as portable regions of homology

Directed deletions and duplications
Identification of essential genes
Localized mutagenesis
Special transposons
Operon and gene fusions
Portable promoters
DNA sequence tags
Protein tags

Background References:
Hughes K, Maloy S. (Eds). 2007. Advanced Bacterial Genetics. Methods Enzymol. Volume 421.
Academic Press, San Diego, CA.
Mazurkiewicz P, Tang C, Boone C, Holden D. 2006. Signature-tagged mutagenesis: barcoding
mutants for genome-wide screens. Nat Rev Genet. 7: 929-939.
Rediers H, Rainey P, Vanderleyden J, De Mot R. 2005. Unraveling the secret lives of bacteria: use
of in vivo expression technology and differential fluorescence induction promoter traps as tools
for exploring niche-specific gene expression. Microbiol Mol Biol Rev. 69: 217-261.

Research Papers:
Bucarey S, Villagra N, Martinic M, Trombert A, Santiviago C, Maulén N, Youderian P, Mora G.
2005. The Salmonella enterica serovar Typhi tsx gene, encoding a nucleoside-specific porin, is
essential for prototrophic growth in the absence of nucleosides. Infect Immun. 73:6210-6219.
[This paper describes the role of genetics in uncovering novel functions for membrane proteins,
including the use of T-POP fusions.]
Groh J, Luo Q, Ballard J, Krumholz L. 2007. Genes that enhance the ecological fitness of
Shewanella oneidensis MR-1 in sediments reveal the value of antibiotic resistance. Appl Environ
Microbiol. 73: 492-498. [This paper describes the importance of membrane export functions
commonly associated with antibiotic resistance in survival in the environmental.]

Day 3 (Wed, January 23): Genetic Regulation

Dissecting regulatory networks

Environmental cues

Background Reference:
Ptashne M. 1986. A Genetic Switch: Gene Control and Phage λ. Blackwell Scientific, NY.

Research Papers:
Walthers D, Carroll R, Navarre W, Libby S, Fang F, Kenney L. 2007. The response regulator SsrB
activates expression of diverse Salmonella pathogenicity island 2 promoters and counters
silencing by the nucleoid-associated protein H-NS. Mol Microbiol. 65: 477-493.
Batchelor E, Walthers D, Kenney L, Goulian M. 2005. The Escherichia coli CpxA-CpxR envelope
stress response system regulates expression of the porins ompF and ompC. J Bacteriol. 187:
5723-5731.
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Day 4 (Thurs, January 24): From genomes to evolution

Comparative genomics
Genome directed genetics
Mapping using microarrays and whole genome amplification
Construction of defined genomic mutations via Red-swaps
Evolution of niche specific functions
Horizontal gene exchange – environmental gene pools
Mutation – Indels, SNPs, and pseudogenes
Metagenomics

Background References:
Sawitzke J, Thomason L, Costantino N, Bubunenko M, Datta S, Court D. (2007) Recombineering: in
vivo genetic engineering in E. coli, S. enterica, and beyond. Methods Enzymol. 421: 171-199.
Brown E, Maloy S. 2006. Facile approach for constructing TEV insertions to probe protein structure
in vivo. Biotechniques 41: 721-724.
Miller R, Day M. 2004. Microbial Evolution: Gene Establishment, Survival, and Exchange. ASM
Press, Washington DC.
Casas V, Rohwer F, Maloy S. 2008. Evolution of bacterial virulence: an environmental pool of
Staphylococcal enterotoxin genes in a psychrophilic pseudomonad. Submitted.
Weissman S, Moseley S, Dykhuizen D, Sokurenko E. 2003. Enterobacterial adhesins and the case
for studying SNPs in bacteria. Trends Microbiol. 11: 115-117.

Research Papers:
Hermann J, Marti-Arbona R, Fedorov A, Fedorov E, Almo S, Shoichet B, Raushel F. 2007.
Structure-based activity prediction for an enzyme of unknown function. Nature 448: 775-779.
[This paper describes an approach to determine the substrate of proteins with a known 3D
structure determined but no previously known function.]
Winfield M, Groisman E. 2004. Phenotypic differences between Salmonella and Escherichia coli
resulting from the disparate regulation of homologous genes. Proc Natl Acad Sci USA. 101:
17162-17167. [This paper describes distinctive phenotypes conferred by differential regulation
of membrane components.]

Day 5 (Fri, January 25): Synthetic biology

Synthetic biology
Genome design and assembly
Optimizing gene function
Complex metabolic pathways (surrogate hosts, shuffling)
New regulatory networks
Single cell biology

Background References:
Tucker J, Zilinskas R. 2006. The promise and perils of synthetic biology. New Atlantis Spring 25-45.
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Research Papers:
Krishnamoorthy G, Tikhonova E, Zgurskaya H. 2007. Fitting periplasmic membrane fusion proteins
to inner membrane transporters: mutations that enable Escherichia coli AcrA to function with
Pseudomonas aeruginosa MexB. J Bacteriol. Nov 16; [Epub ahead of print] [This paper takes
advantage of the structure of related efflux systems with different functions to design mutants
that allow assembly of heterologous efflux complexes.]

Course overview and general discussion

Exam

Day 6 (Sat, January 26): Mini-symposium

We will finish the course with a mini-symposium bringing together a series of scientific talks on the
genetics and physiology of bacterial membrane proteins. Each talk will be 25 min plus 5 min for
questions.

9:30 AM Guido Mora – Welcome and overview

9:45 AM Edmundo Calva – A Salmonella regulatory system for sensing environmental cues
10:15 AM TBA –
10:45 AM Coffee break / Discussion
11:15 AM TBA –
11:45 AM TBA –
12:15 PM Linda Kenney – ***
12:45 PM Lunch / Discussion
2:00 PM TBA –
2:30 PM TBA –
3:00 PM Coffee break / Discussion
3:30 PM Claudia Saavedra – Sensitivity of Salmonella to external toxins: role of OmpW in export
4:00 PM TBA –
4:30 PM Stanley Maloy – Bacterial minicells as vaccine delivery systems
5:00 PM Summary and Discussion