The Scientist - USA (2020-01 & 2020-02)

20 THE SCIENTIST | the-scientist.com

© GEORGE RETSECK

B

acteria and archaea make up most of

the living world, but the vast majority

of species, including some that are

intimately associated with humans, have

never been isolated or cultured.

Sequencing of DNA from natural microbe

populations has allowed the identification

of previously unknown taxa and in some

cases provided detailed genomic infor-

mation about the organisms. But having

sequence data is “like having the parts list”

for a machine, says microbiologist Karsten

Zengler of the University of California, San

Diego. This alone “does not tell you what

this machine will do.”

For a better understanding of a microbe’s

physiology and functions, researchers need

to study living specimens, or at least whole

cells. To that end, microbial geneticist Mircea

Podar of Oak Ridge National Laboratory and

colleagues are examining the sequence data

of uncultured microbes to design tools with

which to capture the bugs.

Focusing on bacteria present in the human

mouth, Podar’s team compared available

sequence data from uncultured organisms

therein with sequences of previously cultured

bacteria to identify codes for potential cell-

surface proteins with regions unique to the

uncultured organisms’ DNA. The researchers

then generated peptides from these candidate

codes, injected them into rabbits to create

antibodies against them, and labeled the

antibodies with fluorescent markers. Adding

these fluorescent antibodies to microbe

samples from human saliva enabled the

detection of bacteria with corresponding

surface proteins and their isolation via

fluorescence-activated cell sorting (FACS).

The team isolated two different taxa of

previously uncultured oral bacteria—TM7

and SR1—both of which the team was able

to cultivate using carefully selected media.

In addition to enriching for the target organ-

isms themselves, the antibody-driven tech-

nique pulled out specific types of bacteria

with which the TM7 and SR1 organisms were

physically associated. In fact, the success of

subsequent cultivation of TM7 and SR1 may

be due in part to the co-isolation of microbes

required for their growth.

While in principle the technique could

be applied to any bacteria, it will be neces-

sary with each new target strain to carefully

design the peptides used for antibody genera-

tion, says Podar. The design is a careful bal-

ance between having similarities to known

surface proteins in other microbes and being

specific for the desired organism. So “it’s

never going to be a kit,” he says. Rather,

“it’s a guided approach that enhances your

chance of success.”

Zengler, who was not involved in the

research, says the technique “will help us to

get more organisms in culture and to learn

more about organisms that we only know

from their genomic fingerprint.” (Nat Bio-

technol, 37:1314–21, 2019) g

STUDYING UNCULTURED

BACTERIA

Single-cell sequencing

Reverse genomics–enabled

isolation

ISOLATING CELLS

Individual cells from a mixed

population are sorted into droplets

for genomic amplification and

sequencing.

Bacterial DNA sequences are used

to generate peptides for antibody

production. The antibodies are then

used to isolate the bacteria of

interest from a mixed population.

ADVANTAGES

The approach can be high-throughput

and uses the increasingly commonplace

techniques of microfluidics and single-cell

sequencing.

Target organisms are greatly enriched,

facilitating genomic, morphological, and

physiological characterization. Associated

organisms may also be isolated, enabling

insights into relationships.

DISADVANTAGES

Yields no morphological or direct physio-

logical information. Difficult to gather data

from especially rare organisms. Little or

no information on interspecies interactions.

Choosing peptides for antibody generation

requires a tricky balancing act between

homology and specificity.

AT A GLANCE

MODUS OPERANDI

CAPTURE AND CHARACTERIZE: To isolate an uncultured microbe of interest, researchers can

examine available sequence data to find likely cell-surface proteins  1 and select a peptide region

as unique to the desired organism as possible  2. This peptide is then used to generate antibodies

in rabbits  3 , and the antibodies are labeled with a fluorescent tag  4. Next, researchers add the

labeled antibodies to the microbial sample  5 and use flow cytometry to isolate those that bind  6.

The isolated bacteria can then be sequenced, characterized, and ideally, cultured.

Using reverse genetics, researchers create antibodies to reel in previously uncultured bacteria.

BY RUTH WILLIAMS

Capturing Elusive Microbes

Membrane protein-

encoding gene

CULTURING POTENTIAL

None

Culturing involves some trial and

error, but an abundance of the

target organism and isolation of

associated microbes improves

chances of success.

 (^1)  2
 3
 4
 5
 6
Sequencing
Bacterial Antibody
DNA
Fluorescent tag
Flow
cytometer
Unique membrane-
protein epitope Fluorescent antibodies added
to sample of mixed microbes
Target
microbes
isolated