Transcript Experimental Ecology

The following slides are provided by
Vincent O’Flaherty.
Dr.
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Experimental Ecology
• What is present, where is it and what is it
doing?
• Numbers, Biomass and Metabolic Activity are
the fundamental basic biotic parameters of
microbial ecosystems
• Much needs to be done to improve our
accuracy and sensitivity in measuring key
parameters - especially re: scale.
• All approaches that are currently used
have advantages and disadvantages - if
these are appreciated the best use can be
made of data
What do we want to know??
• What microbes are present?
Detection/identification
• Where are they? Detection/localisation
• How many of each population are present
(No.’s of cells or mass of cells)
Numbers/biomass
• What are they doing and how is activity
influenced by changes in the environment?
Activity/metabolism
Methods - Summary
1. Detection and Numbers:
•
Culture-based methods for detection and
enumeration
•
Non-culture and non-DNA based methods for
detection, enumeration - immunology and lipids
•
DNA(molecular)-based methods for detection,
localisation and enumeration
1. 2. Methods for determination of Biomass
2. 3. Activity and metabolism determinations
Precautions
• Need to know limitations of each measurement
procedure e.g. knowing that a “total viable
count” typically enumerates around 1% of a
microbial community
• A combination of methods usually gives the
best results
• In some cases numbers, biomass and activity
show proportional correlations - mostly they do
not - how to interpret this??
Sampling
1. Destructive sampling - removing a sample
from the environment for analysis in the lab. very important to be as non-invasive as
possible e.g. soil cores, tissue biopsies, sea
water, sediment, rumen fluid etc etc.
2. Micro and mesocosms -model systems
3. Field studies - at present very difficult and
expensive to undertake
1. Detection and Enumeration of
Microbes in the Environment
• Culture-based
• Immunology-based
• Membrane lipid
• Genotypic Methods
Culture-based detection
methods
• Organisms must be recovered from
environmental samples - recognisable and
specific phenotypes must be expressed during
in vitro culture
• Classical approach - selective plating and
enrichment procedures - useful because they
may provide physiological information useful
in analysing microbes ecological function
Culture vs Counts
Habitat
Typical
microscopic
counts
Typical
cultivability
(%)
0.01-0.1
Soil
109-1010 cells/g
Lakes/rivers
Ground water
106-107 cells/ml 0.01-0.1
104-105 cells/ml <1
Marine
104-106 cells/ml 0.001-0.1
(surface)
Marine (depth) 104-105 cells/ml ND
Sediments
106-109 cells/g
<1
Why can’t we grow
environmental bacteria?
• Little is known about the specific growth
requirements of most microbes - e.g. O2 levels,
nutrients, co-factors, cross-feeding with other
populations
• Many microorganisms in the environment will
have a very low metabolic activity or are
quiescent
• Most aggregates contain a zone of proliferation
and a zone of quiescence e.g. biofilms -these
microbes are not growing but are not dead waiting for favourable conditions
BioLOG System
• Method of rapid testing of environmental
samples - can simultaneously assay for a range
of metabolic characteristics
• Results are based on a colour-change and thus
can be read automatically - rapid
• Based on the pattern of substrate utilisations a statistical analysis can be carried out - gives
a “physiological profile” of the sample
• Available for G(-) , G(+) specific or general use
• Most frequently used culture-based method in
ecological studies - labour, time and money
• Advantages: Cultivation media are formulated
to take advantage of specific traits of organism
i.e. nutritional capabilities and/or resistance to
specific antibiotics - target microorganisms are
favoured over others
• Can detect growth automatically in broth
more sensitive than plates
• Only viable bacteria will grow
• Can work further with isolated bacteria
-
• Problems - “unculturability”, totally artificial
environment
• Can’t examine interactions of mixed group of
microbes
• Lab. and in vivo phenotype may well be
different
Immunological detection
methods
• Based on the fact that
bacterial cell wall polymers
such as proteins and
lipopolysaccharides have
strong antigenic properties
• Can be used to raise
antibodies, usually in rabbits
• After repeated
exposure to antigen
counts of antibody
become very high
• Can be harvested
from serum for use
to detect antigens in
samples
• Labelled with either fluorochrome, biotin or
gold and analysed using fluorescence or
electron microscopy - can be very specific if
monoclonal antibodies are used - specific for
one bindng site, polyclonal antibodoes are more
common
• Usually cells immobilised on slides and
antibodies added - observe using a microscope
or detect electronically. Also Direct
immunofluorescence used to detect organisms
in a variety of environments - water, soils, root
surfaces etc.
• Advantages: Can be used to detect viable but
non-culturable microorganisms; can be used to
count microbes; can be automated; can be used
in situ in samples
• Problems: cross-reactivity, can’t raise
antibodies if you don’t have a pure culture and
so can’t predict if any other microbe will also
react; change of antigenic properties is
response to environment; sometimes not very
sensitive; can be very time-consuming
Membrane lipid analysis
• Bacteria can be characterised on the basis of
different lipids that are found in their
membranes - Number of carbons, saturation,
branching all characteristic of different
organisms
• The fatty acids that are important for bacterial
identification are the branched chain fatty
acids containing from 9 to 20 carbons
• Lipids are extracted from the sample and
treated by attaching an ester group- so they can
be dissolved
• Methylated phospholipid ester-linked fatty
acids - (PL)FAME or PLFA profiles
• Consists of esterification of the lipids and
injection, separation, identification and
quantitation (using known standards) of the
fatty acid methyl esters by gas chromatography
(GC)
• Can read the outputs as peaks - profile of
community structure - individual microbes will
have individual profiles ( again can do stats)
• Using this approach a signature profile can be obtained
for samples
• Community members are identified also some info on
their physiological state e.g. - a ratio of > 1 of trans to
cis- isomers of monosaturated PLFAs is indicative of
starvation or other environmental stress
• Advantages: Important chemotaxonomic
approach, culture independent; Statistically
valid; straightforward and rapid, many
samples can be processed, and change can be
observed over time
• Problems: Organisms which lack signature
profiles will not be distinguished, not very
sensitive and environmental conditions
(substrate, temperature etc.) can cause major
changes in the patterns
Genotypic Detection Methods
• Based on the ability to detect specific signature
gene sequences of organisms in the
environment - detect sequence unique to a
microbe => detect microbe
• Extremely valuable in detection of the
microbial communities present in the
environment increasingly being used to infer
function - main method of community analysis
currently employed
• Also used in phylogeny - determination of the
evolutionary relationship between microbes
Principals of genotypic
detection methods
• Methods are based on the fact that
nucleic acids are made up of 4 bases
arranged in a specific order
• Base sequences are conserved from one
generation to the next
• DNA molecules are double-stranded
• A nucleic acid sequence will only stick or
hybridise to a complimentary sequence
• DNA and RNA can be made single
stranded or denatured by raising the
temperature
• Two detection approaches used: Nucleic
acid Probes and DNA Amplification
• Probing and Amplification are linked as you
need to know the target sequence before you
design a probe
• Must recover sequence information, analyse it
and use it to produce probes
• Sequences got from the environment through:
1. Extraction of nucleic acids and 2.
Amplification via PCR
Extraction of Nucleic Acids
Two approaches to isolating DNA from the
environmental samples:
1. Isolation of microbial cells followed by cell lysis
and purification of nucleic acid (Cell
extraction)
2. Direct lysis of microbial cells in the
environmental matrix followed by nucleic acid
purification (Direct extraction)
• For water samples cells can be collected by
filtration and then lysed to obtain nucleic acids
- cells subjected to enzymatic lysis and/or
phenol-chloroform extraction
• Cell extraction methods also developed for soils
- normally combine vortexing, centrifugation
steps
• Direct DNA extraction increasingly favoured
for environmental studies - more representative
of populations present - crude extracts purified
to remove interfering substances
PCR
• Mimics the natural DNA replication in
microbes
• Uses polymerase to synthesise a complimentary
strand of DNA/RNA from a single strand
• Small sequences (primers) added to create
double- stranded template
• A series of amplification cycles used to increase
initial target
• Target sequence amplified – can detect very low
initial numbers
• Amplified DNA can be used for probing or can
be cloned and/or sequenced
• Sequencing and comparison with known
sequences provides information on diversity
and types of microbe present and also can be
used to design probes
• Advantages of PCR: no culture, allows
detection of very low starting numbers,
applicable to a wide range of samples, allows
the sequencing of amplified target
• Problems: sampling is destructive, need to
know some sequence information on target,
does not distinguish between viable and nonviable, can be inhibited easily, absolutely
dependent on success of nucleic acid extraction
Nucleic acid Probes
• Probes and nucleic hybridisation techniques
used to detect target sequences diagnostic of
specific groups of organisms in environmental
samples
• Probe is a relatively short nucleotide sequence
that can hybridise with a homologous sequence
in the target micro-organism
• Can be designed to target either DNA
(chromosome) or RNA (usually the rRNA)
How probes work
• Sequence of events is that nucleic acids
are extracted from the sample, denatured
and immobilised - e.g. on a nitrocellulose
filter
• Labeled probe is then added and allowed
to hybridise
• Unbound probe is then washed off and
finally hybrids are detected
• Normally carry out hybridisation on an
immobilised target or probe on a solid phase
e.g. - nitrocellulose or nylon filter surface
• Normally probe is labelled (32P) and after
hybridisation and washing can detect target
binding by autoradiography
• Relative amounts of nucleic acid can be
quantified by comparison with signal obtained
with universal probe - variations include use of
Dot blot manifold etc.
• Advantages: Do not require culture,
applicable to a wide range of samples,
can be quantitative
• Problems: destructive, requires some
sequence information, may not detect
low-numbers very well (combination with
PCR overcomes this), no distinction
between viable and non-viable
In-situ hybridisation
• Alternative approach is to carry out specific
hybridisation between labelled probe and
specific target sequence inside intact cell with
minimum sample disturbance
• Most direct method - morphology of the cell
fixed, membrane made permeable to allow
penetration of probe (usually with
paraformaldehyde)
• Fixed cells bound to glass
slide and hybridised with
oligonucleotide probe in a
moist chamber - probes
can be labelled with
radioactivity, biotin
combined with antibodies
etc
• Most commonly labelled
with a flourescent dye like
fluorescein (green) or
rhodamine (red)
FISH
• Fluorescent signals detected by epifluorescence
or confocal laser scanning microscope (much
more detail)
• Excellent technique for detection of
unculturables e.g. symbionts of protozoa etc.
• Very useful for identifying bacteria in complex
environments - soil, biofilms, activated sludge
etc.
• Advantages: no culture, can detect both
culturable and unculturable organisms,
localise specific cells within a community,
estimate numbers
• Problems: difficulties in getting “clean”
hybridisation with some samples, cells
have to be fixed to get probe in, need
sequence information on target microbes
Reporter Genes
• Genetic markers used to track specific
genetically modified microbial populations in
the environment - genetic element that permits
detection of an unrelated biological function
e.g. lacZ gene useful and commonly employed can cleave X-gal to create a blue pigment
readily visable on plates - versatile biomarker
• Also
green
fluorescent
protein
and
bioluminescence genes used for this purpose
1 (b): Determination of numbers
• Direct counts - either stains or nucleic
acid probes
• Viable Counts
• Numbers obtained by direct counts typically 2
orders of magnitude higher than counts
obtained by cultural techniques and applicable
to a variety of habitats without culture-based
biases
• Numbers of specific microbes can be estimated
using fluorescent antibody or gene probes
• Multiple populations in the same sample can be
counted by using several probes with different
colours
Stains used for direct counts
• Acridine Orange (water) - nucleic acid
• DAPI (water/solids) - DNA stain
• Fluorescein isothiocyanate (FITC) protein stain
Dead or Alive?
• Very important to determine if cells that you
are counting are viable - are they alive or dead number of procedures attempt to do this
• i.e. use of 2-[p-indophenyl]-3[p-nitrophenyl]-5phenyl tetrazolium chloride (INT) which
deposits red dye in cells that have active
dehydrogenases
• Similar respiration assay involves the use of 5cyano-2,3-ditolyl tetrazolium chloride (CTC)
• Also membrane potential-sensitive
fluorochromes can distinguish between
active, injured (dying) and dead cells
• rRNA targeted probes - bind to
ribosomes - these are present in live cells
only
• Using such methods it appears most of the cells
observed by direct microscopy are alive - viable
but non-culturable, concept first introduced by
Rita Colwell in 1987
• Demonstrated organisms carry out active
metabolism and retain virulence
• Use of gene probes/PCR etc. can classify
unculturables - can infer properties based on
cultured homologues - need to be careful!
• Otherwise, Plate count and MPN the two basic
approaches used to cultivate viable organismsboth rely on separation of microorganisms into
individual reproductive units
• All viable count procedures are selective - the
degree of selectivity varies with the particular
viable count procedure - impossible to get a
“Total Viable Count”