MICROBIAL ECOLOGY: A CHALLENGES IN

OMIC ERA
M. Saifur R., PhD
Lab. of Agricultural Microbiology
Department of Agricultural Microbiology

History of Microbial Ecology

Development staining
method (Micr. Obs)

Understanding on
the
microbial
physiology

Microbial Ecology

Microscope
become
principle tool.

Differ in number (Why?)

Microbial
community study
(1976)

Growing
invisible
organism and develop
isolation
technique
(1888) (Plate obsv.)

Winogradsky Create
media mimic the
nature conditions

Trend in microbial ecology

Classic Microbial
Ecology

Modern/Molecular
Microbial Ecology

Microbial Ecology in Omics era

Microbial Cell Culture: An old school technique

Consortium
Mixed culture

Pure culture

Isolation technique: An old school technique

Flow cytometry: An old school technique

Flow cytometer

Output data

Flow cytometry is a laser- or impedance-based, biophysical technology employed

in cell counting, cell sorting, biomarker detection and protein engineering, by
suspending cells in a stream of fluid and passing them by an electronic detection
apparatus.

FISH: An old school technique

2001 Nature Publishing Group Cheung, V. G. et al.

Integration of cytogenetic landmarks into the draft
sequence of the human genome. Nature 409, 954 (2001)

qPCR: An old school technique

Ct

Standard curve

Omic era

Flow of biological information

(bidirectional)

The numbers are the approximate quantity

at each functional level

Omic technologies: genomics, transcriptomics, proteomics and metabolomics

The Obstetrician & Gynaecologist

Volume 13, Issue 3, pages 189-195, 18 JUL 2011 DOI: 10.1576/toag.13.3.189.27672
http://onlinelibrary.wiley.com/doi/10.1576/toag.13.3.189.27672/full#f1

Genomics
The study of the structure,
function and expression of all the
genes in an organism
Genome
The total DNA of a cell or organism
Polymorphism
Variations in DNA at a specific site
Transcriptomics
The study of the mRNA within a
cell or organism
Transcriptome
The total mRNA in a cell or organism

Proteomics
The large-scale study of proteins, including
their structure and function, within a
cell/system/organism. A name coined as an
analogy with the genome

Omic era

The Obstetrician & Gynaecologist

Volume 13, Issue 3, pages 189-195, 18 JUL 2011 DOI:
10.1576/toag.13.3.189.2767http

Proteome
The set of all expressed proteins in
a cell, tissue or organism

Flow of biological information

(bidirectional)

The numbers are the approximate quantity

at each functional level

Omic technologies: genomics, transcriptomics, proteomics and metabolomics

Metabolomics
The study of global metabolite
profiles in a system (cell, tissue or
organism) under a given set of
conditions
Metabolome
The total quantitative collection of low
molecular weight compounds (metabolites)
present in a cell or organism that participate
in metabolic reactions. It also includes those
metabolites
taken
in
from
external
environments or symbiotic relationships

Metabonomics
A measure of the fingerprint of biochemical
perturbations caused by disease, drugs and
toxins; some would say that metabonomics
and metabolomics are the same and the
terms are occasionally used interchangeably

Metagenomic

Gitig, 2010. BioTechniques

48:361-365 doi 10.2144/000113416

one of the most remarkable events in the field

of microbial ecology in the past decade

Definition

the direct genetic analysis of genomes contained with an

environmental sample
(Thomas et al., 2012. Microbial Informatics and Experimentation 2:3)

Metagenomic
Potentially novel
biocatalyst/enzymes

tool
tool for
for generating
generating
novel
novel hypotheses
hypotheses of
microbial
microbial function
function

Metagenomic

Evolutionary
Evolutionary profiles
profiles of
of
community function
function and
and
structure
structure

genomic linkages
between function and
and
phylogeny
phylogeny for
for
uncultured
organisms
uncultured organisms

Metagenomic Project
Sampling and processing
Sampling
Sampling

Crucial
step
in
metagenomic,
High quality of nucleic
acid is required for library
production,
Sample: soil, biological
material, water.

Sample
Sample fractionation
fractionation
DNA
DNA extraction
extraction
DNA
DNA sequencing
sequencing

Sequencing
Assembly
Assembly

Binning

Annotation
Annotation

Statistical
Statistical analysis
analysis
Data
Data Storage
Storage
Data
Data sharing
sharing

Meta data

Library production for most

sequencing technologies require
high nanograms or micrograms
amounts of DNA
Over the past 10 years
metagenomic
shotgun
sequencing has gradually shifted
from
classical
Sanger
sequencing technology to nextgeneration sequencing (NGS)

Ribosomal RNA
(rRNA) for unculture bacteria and its community)

16SrRNA

16SrRNA gene

Ribosomal RNA (rRNA)

for unculture bacteria and its community
Do not have any ortholog-paralog issues or horizontal gene transfer

Occurs in all living

bacteria/archaea

Generally stable

16S rRNA

Preserved their evolutionary

history
(molecular
chronometer)

Long enough 1500 bp

Contains independently evolving domains

18SrRNA for eukaryote

SH
I
F

Nucleic
Nucleic
acid/Probes
acid/Probes

ra

Phylogenetic trees

Nu
c

Bulk
Bulk RNA/DNA
RNA/DNA

l.
hy
br

id
i za
tio
n

rDNA
rDNA
sequences
sequences and
and
database
database

Se
qu
en
cin
g

PCR

Probe design

Co

a
mp

a
it ve

sis
y
l
a

Environmental
Environmental
sample
sample

Ex
tra
ct i
on

Community
Community
rRNA/rDNA
rRNA/rDNA

rDNA
rDNA clones
clones

ng
i
on
l
C

'Full-cycle' rRNA approach to characterizing microorganisms in their

natural settings without the need for cultivation

Review
Exploring prokaryotic diversity in the genomic era
Philip Hugenholtz

rRNA for unculture bacteria and its community

Next Generation Sequencing (NGS)

Illumina
Illumina
Bridge
Bridge
amplification
amplification

Ion
Ion Torrent
Torrent

454
454

Emulsion
Emulsion
PCR
PCR

Emulsion
Emulsion
PCR
PCR

NGS
SOLiD
SOLiD
Emulsion
Emulsion
PCR
PCR

SMRT
SMRT

Next Generation Sequencing (NGS)

Technology

Amplifica
tion

Chemistry

Sequencing
method

Highest
Average
Read
Length

Error
Rate

Disadvant
age

Advantage

Yield
(Gb/run)

Bridge

Seq by
synthesis

Incorporation
fluorescent nt

300 bp

>0.1

Short read
and long
runtime

High
throughput
and low
cost

1-60

SOLiD (life
Technology)

Emulsion
PCR

Seq by
ligation

Fluorescent
short linker

75

>0.06

Long run
time

Low error
rate

454 (Roche)

Emulsion
PCR

Seq by
synthesis
(pyroseque
ncing)

iorporation of
normal
nucleotidesnc

700

High error
rate in
homopoly
mer

Long read

0.7

SMRT
(Pacific Bio)

N/A
(single
molecule)

Single
Molecule
Real Time
(SMRT)

incorporation
of
fluorescent
nucleotides

5000 bp

16

No PCR
longest
read

High error

0.3-0.5

Ion Torrent
(Life
Technology)

N/A

Seq bu
synthesis
(Proton
detection)

measuring pH
change

400

New

Short read

Illumina

Bridge PCR

Genomic DNA preparation

Bridge formation

Slide with anchored oligo

Amplification

Emulsion PCR

Sequencing by Synthesis

Pyrosequencing (Sequencing by synthesis)

Pyrophosphate

Sequencing by ligation

Oligos:

Size: Octamer (8) dan nonamer (9)

NGSequencing Data (example)

NGSequencing Data (example)

MICROBES AND ENVIRONMENT

M. Saifur R., PhD
Laboratory of Agricultural Microbiology
Department of Agricultural Microbiology
Universitas Gadjah Mada

Introduction (Terminology)
Microbial interactions
Symbiosis
An association of two or more different species
Ectosymbisis
One organism can be located on the surface of another,
as an ectosymbiont. In this case, the ectosymbiont usually
is a smaller organism located on the surface of a larger
organism.
Endosymbiosis
One organism can be located within another organism as
an endosymbiont
Ecto/endosymbiosis.
Microorganisms live on both the inside and the outside of
another organism

Examples (Ecto/endosymbiosis)

Thiothrix species, a sulfur-using bacterium, which is at- ached to the surface

of a mayfly larva and which itself contains a parasitic bacterium.

mycorrhizal fungi are surrounded by complex microbial communities, which

modulate the mycorrhizal symbiosis. Here, the focus is on the so-called
mycorrhiza helper bacteria (MHB).

Persistence of symbiosis
Permanent Symbiotic

Cyclic Symbiotic

Coptotermes formosanus

Pseudotrichonympha
grassii

P. bursaria +algae

CfPt1-2

Hongoh et al. 2008. Sci. 322:1108-09

Takahashi, 2016)

P. Bursaria algae free

Persistence of symbiosis
Intermittent Symbiotic

Type of interaction
Microorganisms (including viruses, bacteria, archaea and
protists) do not exist in isolation but form complex ecological
interaction webs.

Mutualism
[Latin mutuus, borrowed or reciprocal] defines the relationship in which
some reciprocal benefit accrues to both partners.
Relationship with some degree of obligation
partners cannot live separately
Mutualist and host are dependent on each other

Hodotermopsis sjoestedti

Lower termite group

harbor unique flagellated
protists in their guts

The protist: Eucomonympha

A schematic view of the endosymbiotic relationships

Lichens
Lichens are the association between specific
ascomycetes (my-cobiont) and either green
algae or cyanobacteria phycobiont.
Morphology : Resultant of mutualistic
association, not symbiont individually.
Phycobiont: Photosynthetic and provide food
for mycobiont
Mycobiont:
Protect
phycobiont
from
environmental stress
Soredia
are
common
reproductive
structures
of
lichens
Recently: Lichens is not a
dwipartit symbiotic between the
algae and ascomycetes, but
tripartit, asco, algae, and
basidiomycetes yeast (Spribille et
al., 2016. Sci. 353(6298):488-92

Object used in research

Both contain the pulvinic acid
(derivative of vulvinic acids)

Bryoria fremontii (non toxic)

Brown

Bryoria tortuosa (toxic)

Yellow

(Causing yellow color)

Restricted to to the
sorelia and apothecia

Bryoria fremontii (non toxic)

Sorelium

Thallus

Bryoria tortuosa (toxic)

Bryoria fremontii (non toxic)

Phylogenetic analysis fail to detect any

fixed sequence differences the two
species

hypothesized
that
differential
gene
expression might account for the increased
production of vulpinic acid in B. tortuosa.
Bryoria tortuosa (toxic)

Metatranscriptomic analysis

Results
Initial transcriptome-wide analysis of single-nucleotide polymorphisms (SNPs)
for Ascomycota (fungi) and Viridiplantae (algae) transcript subsets.

There is no difference in tranascrptomic SNPs of two symbionts (fungi and alga)

in two lichens. The diversity of two symbiont in the two lichens are similar.

Fail to detect the differences among the two lichens

Reconfirmation by
estimated transcript abundances by mapping raw reads back to a single,
pooled metatranscriptome assembly and binning by taxon.

Only little difference either in fungus or algal

metatranscriptomic
(indicated by arrow in E, P<0.05)

In order to find the discriminant of the two lichens, analysis was focused
on the total fungi
Found 506 contigs with
significantly
higher
abundances in vulpinic acid
rich B. tortuosa thalli.
Ascomycota

Basidiomycota group!

Anlysis of 349 locus

phylogenomic tree

Cyphobasidium (yeast)

Basidiomycota

Red dots indicate a log fold change

with P < 0.05 in (C), the inset of (D),

FISH Analysis of Cyphobasidium

Both contains the

yeast
but
B.
tortuosa is more
abundant

Probe: From 16S rRNA gene sequence of Cyphobasidiumycete

Syntrophism
Syntrophism [Greek syn, together, and trophe, nourishment] is an association in
which the growth of one organism either depends on or is improved by growth
factors, nutrients, or substrates provided by another organism growing nearby.
Sometimes both organisms benefit. This type of mutualism is also known as
cross- feeding or the satellite phenomenon.

Compound
Compound A
A Population 1

Compound
Compound B
B

Population 2

Compound
Compound C
C

Population 1
and 2

Energy
Energy and
and
end
end product
product

Cell Research (2003); 13(4):229-238

http://www.cell-research.com

Quorum sensing (autoinduction)

a system of stimuli and response correlated to
population density

Vibrio fischeri

Diagram depicting a simplified quorum sensing circuit involving LuxI (light

blue) and LuxR (dark blue) in Vibrio fischeri.

Some of LuxI and LuxR homologue

Both LuxI and LuxR were discovered initially in Vibrio fischeri.

Commensalism
Commensalism [Latin com, together, and mensa, table] is a
relationship in which one symbiont, the commensal, benefits
while the other (sometimes called the host) is neither harmed
nor helped (neutral)

Commensal - organism that benefits

When the commensal is separated from its host experimentally, it can
survive without being provided some factor or factors of host origin.
Commensalistic relationships between microorganisms include
situations in which the waste product of one microorganism is the
substrate for another species.
Commensalistic associations also occur when one microbial group
modifies the environment to make it more suited for another organism.

Commensalism Example
Intestinal microorganisms in the human colon, when
oxygen is used up by the facultatively anaerobic E. coli,
obligate anaerobes such as Bacteroides are able to grow
in the colon.
Microbial succession during spoilage of milk
fermenting bacteria promote growth of acid tolerant
species
Formation of biofilms
initial colonizer helps other microorganisms attach
Skin or surface microbes on plants or animals
host plant or animal releases volatile, soluble, and
particulate organic compounds used by commensals

Negative Interaction

Negative Interaction

Predation

Parasitism

Kill the opponent

Do not kill the

host

Amensalism

One is inhibited
by other but no
advantage obtain
from the inhibition

Competition

Both get
negative impact

-proteobacteria that enter and kill diverse

pathogenic
Gram-negative
bacterial
species.

Bdellovibrio bacteriovorus

Produce Endopeptidase
Decrosslink cell wall and
wasteful for entry second
predator

Bdellovibrio bacteriovorus is a periplasmic

predator that enters through the outer
membrane of prey and metabolizes the
infected cell from within.

Predatory and protection mechanism of Bdellovibrio

rin
ky
An
(B
0)
46
d3

Arrow indicates the predator position

Endopeptidase (Bd3459)

Ankyrin functions to protect the

predator from self attacking
by their peptidase
Arrow indicates the predator position

Lambert et al., 2012

MATHEMATICAL MODEL IN MICROBIAL

ECOLOGY
M. Saifur R., PhD
Laboratory of Agricultural Microbiology
Department of Agricultural Microbiology
Universitas Gadjah Mada

Role of mathematics
Construction
Mathematical
Ecosystem

Phenomenon
Phenomenon
describing
describing

and
Model

Phenomenon
Phenomenon
Aid
Aid
understanding
understanding

Phenomenon
Phenomenon
predictions
predictions

of
analysis
Microbial
of

Role
Role of
of
mathematics
mathematics
in
in Ecology
Ecology

The physical environment of microbial ecosystems

Physical environment

the inanimate surroundings of

microorganisms in a microbial ecosystem
System
Microbial population

Input and output

Well mix of microbial

population or not

There is material/energy
input/output or not

Systems
Closed System
Microbial growth:

X
:
population
concentration; : specific
growth rate or the capita
rate of increase

Open system
For well mixed ecosystem and
constant volume , V, and flow
rate, f, the mass balance is:
Change of biomass = input of
biomass + change due to
growth/death output of
biomass

D = f/V is dilution rate

Microbial Growth
Single-species populations
If the growth is considered to be dependent on a single growth-limiting substrate
Monod Equation :

Binary fission:

usually
decrease
population increase

as

Where S is substrate concentration; Ks is known as saturation constant and is

numerically equal to the concentration of substrate required to support the
growth at half maximum specific growth rate, m.
Microbial interactions
Predator-prey interaction
The predator specific growth rate, , will be proportional with prey concentration (H):

Microbial Growth
Microbial interactions
Predator-prey interaction
The predator specific growth rate, , will be proportional with prey concentration (H):

Where 2 is constant; Based on the Monod equation then it could be formulated as:

where 13, is the maximum specific growth rate of the predator and L, is the
saturation constant

Mathematical models of microbial ecosystems

combine the equations of mass balance with the kinetic
expressions to produce several models which will serve as
examples of the ways in which mathematical techniques of
analysis have been applied.
Unrestricted growth in a closed environment

Density-dependent growth in a closed environment

the logistic equation