Biosc 1280 - 11 April 2001 - 1
Enteric bacteria as model systems
Three closely related species
 Escherichia coli is a commensal of mammalian gut environments, is adapted for periods of rapid
growth at 37 C, and is rarely pathogenic.
 Salmonella enterica is a commensal of mammalian, avian and reptilian guts, and frequently adopts a
pathogenic lifestyle. The Salmonella lineage separated from the E. coli lineage about 120 MYrs ago.
 Klebsiella aerogenes is a common soil bacterium that can also exploit insect gut habitats. It is
metabolically more versatile than either E. coli or Salmonella. Klebsiella diverged from the
E. coli/Salmonella ancestor about 150 Myr ago.
The power of comparative genetics
 All three organisms grow well in laboratory environments.
 All three organisms perform respirations and fermentation, are prototrophic, can utilize numerous
sources of carbon, nitrogen and sulfur, can grow either aerobically or anaerobically and exhibit large
numbers of inducible functions. Therefore, an enormous breadth of biology can be learned from
studies of their biology.
 Cross-organism comparisons can test which features are conserved over evolutionary time, and
detect recent alterations of genetic or physiology.
 Differences between the organisms can help identify which events contribute to organismal
diversification.
 Having close relatives sometimes assist in solving problems that may arise in the investigation of a
single organism.
A multitude of tools
 Conjugation can employ several factors, including F and R plasmids, which can be mobilized within
and between species.
 Conjugative elements can be integrated into the genome for facile mapping.
 Transducing phages are available for all three organisms.
 Electroporation and chemical transformation allow direct DNA introduction.
 Bacteriophage lambda allows for integration of foreign DNA into the genome as well as transient
gene expression.
 Bacteriophage Mu allows for facile insertion mutagenesis and in vivo cloning
 Numerous antibiotic-resistant transposons, including derivative of Tn5, Tn10, Mu and , allow for
insertion mutagenesis.
 Numerous compatible plasmids allow for ectopic gene expression
 Linear transformation allows for directed gene knock-out
Powerful transducing phages
 Bacteriophage P22 infects Salmonella enterica. Mutants with defective packaging allow for cell
lysates bearing 40% transducing particles, vastly facilitating genetic manipulation. P22 can package
~42kb of DNA.
 Bacteriophage P1 can infect all three organisms and packages ~105 kb of DNA, although lysates
usually bear ~0.1% transducing particles.
 Other transducing phages, like P52 in Klebsiella or ES18 in Salmonella, allow for “emergency”
transductions when the normally-employed phage is unusable.
Biosc 1280 - 11 April 2001 - 2
A test case - Raffinose degradation
Variation in raffinose degradation abilities among enteric bacteria
 Mammalian ingestion of raffinose - a sugar found in many legumes - leads to excess production of
CO2 ands H2S in the digestive system
 This is because most E. coli - the primary sugar consumer of the digestive tract - cannot consume
raffinose. It performs a fermentation of sugars and secretes harmless organic acids.
 Instead, sulfate reducers obtain the raffinose and respire it, producing large amounts of CO2 from the
sugar and large amounts of H2S from reduced sulfate (the electron acceptor)
 You discover that natural isolates of Salmonella can degrade raffinose just fine
 Your company, Chili-Friendly Inc., wants you to characterize the mechanism by which Salmonella
degrades raffinose
Step 1 : Identify mutant loci
 Genetically, we can examine raffinose metabolism by mutating the genes responsible for this
activity, and isolating mutants which cannot degrade raffinose.
 We can use transposon mutagenesis to couple the desired mutation (loss of raffinose degradation)
with a positive, selectable phenotype (like antibiotic resistance)
 We can deliver the transposon in a number of ways
 A self-contained bacteriophage vector can introduce a defective transposon along with a cisacting transposase. Since the phage cannot integrate or replicate, antibiotic resistance can be
inherited only by transposition. The phage vector then degrades.
 Bacteriophage P22 transduction can move a transposon from heterologous DNA, copackaged
with a transposase-producing gene, into the target strain. Since inheritance of the antibiotic
resistance gene cannot occur by homologous recombination, antibiotic-resistant cells must arise
by transposition.
 Similarly, a suicide plasmid can be delivered by conjugation; if the plasmid cannot replicate, the
transposition must occur to allow inheritance of the antibiotic-resistance gene.
 We then screen for mutants by replica printing from rich media bearing antibiotics to defined media
containing either raffinose or glucose. Colonies that fail to grow on raffinose but do grow on
glucose are defective for raffinose degradation.
 Several rounds of mutagenesis are performed to generate mutants bearing insertions with several
different antibiotic-resistance genes
Step 2 : How many loci are involved?
 To characterize all of the genes involved in raffinose degradation, we must sort them into linkage
groups. Operons may allow all of the genes to be located in one location, but we don’t know that
this is true.
 To determine the number of groups, we can use transduction. This approach has the added benefit
of verifying that the antibiotic resistance gene we are following has indeed inserted in a gene
contributing to raffinose degradation (rather than inserting somewhere at random in a strain that
picked up a random point mutation in the raffinose degradation gene).
 We can use transduction to determine is raf insertions with different antibiotic resistances are located
at the same location; this is a two-factor transduction. If inheritance of one drug-resistance gene
removes a resident drug-resistance gene, then those genes were located very close to one another on
the chromosome. The more often this occurs, the more closely the genes are linked.
Biosc 1280 - 11 April 2001 - 3
 Similarly, we can transduce the drug-resistance gene into a wild-type background. If this mutant
now fails to degrade raffinose, then the antibiotic-resistance gene is linked to the mutant phenotype
of interest, and there wasn’t just a spurious point mutation.
Step 3 : Physical characterization
 The drug resistance genes could be cloned and the regions flanking the insertion sequenced. This
could provide the DNA sequence of the raf genes.
 These days, complete genome sequences are available, so we need only determine a small region of
sequence adjacent to our insertion, and gather the remaining information from the published genome
sequence.
 This can be accomplished by cloning, or by PCR-based methods. Alternatively, we can sometimes
determined small amounts of DNA sequence directly from chromosomal DNA without cloning.
Step 4 : Rough Regulation
 How are the raf genes turned on? There are two major ways of determining expression patterns.
 First, we can assay enzyme activity directly. This is certainly accurate, but requires a specific
enzyme assay for every system we examine.
 Alternatively, we can use a reporter gene to tell us how genes are expressed. Reporter genes have
well characterized products that are easy to assay. The task is to place reporter gene expression
under control of the raf promoters.
 This can be accomplished in many ways
 First, we can use transposons that create reporter gene fusions during mutagenesis. This is the
easiest way, and most often employed.
 Second, we can clone the promoter onto a plasmid bearing the reporter gene. While this can be
accomplished after genes are isolated, the disadvantage is that the promoter is now on a multicopy plasmid
 Third, we could engineer an integrating bacteriophage that could carry our constructed fusion.
The integrated phage would be in single copy. These constructs are more difficult to construct.
Step 5 : Fine Regulation
 Reporter fusions are mutants. What if a regulatory gene is encoded downstream of your fusions and
polarity affects regulation? What if raffinose itself is not controlling expression but some metabolic
intermediate which is not produced in your fusion mutant?
 To circumvent these complexities, we must use a single-copy fusion that is not a mutant. We can
construct this from our fusion in several ways.
 First, transduction can be used to move the fusion into a preëxisting duplication. This leaves
regulation intact.
 Alternatively, a directed duplication can be formed with the fusion at the join-point. This construct
has the advantage of being a stable genetic marker that can be moved from strain to strain.
Step 6 : Fine-scale analysis
 Insertion mutants are null mutations, and most are polar. To examine mutations in individual genes,
we would like to create point mutations at each raf locus. These mutants can also dissect the
regulatory region in a way that insertion mutants cannot.
 But doing a big mutant hunt with a chemical mutagen does not employ all of the information we
have gathered. We would be back to square 1 in terms of mapping and screening.
 Instead, we can do a local mutagenesis to isolate large numbers of point mutants in our region of
interest.
Biosc 1280 - 11 April 2001 - 4
 We start by isolating an insertion linked to, but not actually in, our raf locus. This mutant is raf+, but
carries a closely-linked antibiotic resistance gene. This is easily created by transductional repair of a
raf mutant with an insertional library, selecting for drug resistance and raf+.
 Next, we make a bacteriophage lysate on this strain and treat the phage with a mutagen.
 We than transduce a wild-type strain to drug resistance with this lysate. We control the mutagenesis
so that the DNA surrounding the antibiotic resistance gene has been heavily mutagenized. Most of
our transductants are point mutants at the raf locus.