Streptomyces
Streptomyces
sp. as
sp. as
predators of bacteria
predators of bacteria
Charushila Kumbhar
Charushila Kumbhar
Milind Watve
Milind Watve
Anujeeva Biosciences Pvt. Ltd., Pune, India
Anujeeva Biosciences Pvt. Ltd., Pune, India
and
and
Department of Microbiology,
Department of Microbiology,
Abasaheb Garware College, Pune, India
Abasaheb Garware College, Pune, India
Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007
Abstract
Abstract
Background: The ecological role of actinomycetes and
Background: The ecological role of actinomycetes and
their secondary metabolites is not yet completely
their secondary metabolites is not yet completely
understood.
understood.
Hypothesis: Actinomycetes and
Hypothesis: Actinomycetes and
Streptomyces
Streptomyces
sp. in
sp. in
particular, are non-obligate predators of bacteria in soil.
particular, are non-obligate predators of bacteria in soil.
Evidence: Ability to grow on live bacterial cells as a sole
Evidence: Ability to grow on live bacterial cells as a sole
source of nutrients. Prey cell lysis accompanying
source of nutrients. Prey cell lysis accompanying
growth. Circumstantial evidence for the involvement of
growth. Circumstantial evidence for the involvement of
antimicrobials along with enzymes.
antimicrobials along with enzymes.
Implications: This finding may open up a new source of
Implications: This finding may open up a new source of
novel secondary metabolites from the genus.
novel secondary metabolites from the genus.
Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007
Are actinomycetes primarily
Are actinomycetes primarily
K
K
selected oligophilic bacteria?
selected oligophilic bacteria?
For any soil sample the absolute number as well as
For any soil sample the absolute number as well as
proportion of actinomycete colonies on oligotrophic
proportion of actinomycete colonies on oligotrophic
medium is substantially greater than any other
medium is substantially greater than any other
conventional actinomycete media (1).
conventional actinomycete media (1).
Growth yields of actinomycetes on oligotrophic media
Growth yields of actinomycetes on oligotrophic media
are substantially greater than conventional media (2)
are substantially greater than conventional media (2)
Primary oligophilic isolates show antimicrobial activity
Primary oligophilic isolates show antimicrobial activity
significantly more frequently (3).
significantly more frequently (3).
Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007
A logical thread to predatory activity
A logical thread to predatory activity
Predation: potential benefits in oligotrophic environments
Predation: potential benefits in oligotrophic environments
All known predatory bacteria are antibiotic producers (
All known predatory bacteria are antibiotic producers (
Myxobacteria
Myxobacteria
(4), Lysobacter (5), Burkholderia (6), Bacillus
(4), Lysobacter (5), Burkholderia (6), Bacillus
sp.(7) )
sp.(7) )
Suggestion of predation based on indirect bacteriophage analysis
Suggestion of predation based on indirect bacteriophage analysis
technique (8)
technique (8)
Why are secondary metabolites expressed more frequently in
Why are secondary metabolites expressed more frequently in
oligotrophic conditions?
oligotrophic conditions?
Competition or Predation?
Competition or Predation?
Antibiotics should be produced in early
and mid growth phase so that they help
in competition.
Antibiotics should be produced when
other nutrients are scanty.
Most antibiotics are known to be produced during end of growth phase,
stationary phase or starvation. Supports predation hypothesis
.
Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007
Proportion of different classes of isolates (potential
Proportion of different classes of isolates (potential
predators) when soil is sprinkled on a bacterial
predators) when soil is sprinkled on a bacterial
lawn on water agar.
lawn on water agar.
M yxobacteria
9%
A m ebae and
slim e m olds
10%
Unidentified
14%
Lysobacter and
other gram
negative
16%
B acillus
23%
A ctinom ycetes
28%
Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007
Adaptation and selection of oligophilic
Adaptation and selection of oligophilic
isolates for predation
isolates for predation
The isolates were sub-cultured on water
The isolates were sub-cultured on water
agar with a lawn of bacterial cells as sole
agar with a lawn of bacterial cells as sole
source of nutrient. Repeated transfers on the
source of nutrient. Repeated transfers on the
same.
same.
Richer growth and larger zones of clearance
Richer growth and larger zones of clearance
with subsequent subcultures.
with subsequent subcultures.
Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007
Evidence for predation
Evidence for predation
Growth with washed live
Growth with washed live
bacterial cells as a sole
bacterial cells as a sole
source
of
organic
source
of
organic
nutrients.
nutrients.
No growth on control
No growth on control
washed agarose gel.
washed agarose gel.
Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007
Evidence for predation
Evidence for predation
The edge of an unidentified actinomycete colony
The edge of an unidentified actinomycete colony
invading microcolonies of bacteria on a oligotrophic
invading microcolonies of bacteria on a oligotrophic
medium
medium
Low resolution (40 X) time lapse pictures with 20 minute interval. Note
Low resolution (40 X) time lapse pictures with 20 minute interval. Note
that the edge grows faster where there are microcolonies
that the edge grows faster where there are microcolonies
(black arrow
(black arrow
).
).
Also note the fragmentation of a large colony surrounded by the predator
Also note the fragmentation of a large colony surrounded by the predator
(
(
white arrow
white arrow
).
).
Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007
Evidence for predation
Evidence for predation
Zones of lawn-clearing around colonies. Lysis of cells towards the
Zones of lawn-clearing around colonies. Lysis of cells towards the
predator colony visible at high resolution.
predator colony visible at high resolution.
Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007
Evidence for predation
Evidence for predation
Edge of a
Edge of a
Streptomyces
Streptomyces
colony growing by predation on
colony growing by predation on
Staphylococcus aureus
Staphylococcus aureus
. Arrows point to ghost cells of
. Arrows point to ghost cells of
recently lysed bacteria. Some cells show persistence.
recently lysed bacteria. Some cells show persistence.
Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007
How common is predatory ability in
How common is predatory ability in
Streptomyces?
Streptomyces?
From 40 oligophilic isolates 16 showed distinct
From 40 oligophilic isolates 16 showed distinct
predatory activity on first challenge.
predatory activity on first challenge.
Half of the remaining showed predation after three to six
Half of the remaining showed predation after three to six
subcultures under selective conditions.
subcultures under selective conditions.
Approximately one third of conventional
Approximately one third of conventional
Streptomyces
Streptomyces
isolates and standard strains showed evidence of
isolates and standard strains showed evidence of
predation after three to six subcultures of selection.
predation after three to six subcultures of selection.
This suggests that predatory abilities are widespread
This suggests that predatory abilities are widespread
within the genus.
within the genus.
Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007
Are antibiotics involved in predation?
Are antibiotics involved in predation?
S. aureus
culture contol
S. aureus on
water agar
S. aureus on
water agar
S. aureus
culture contol
1
2
3
5
4
6
6
1
2
3
4
5
6
Before visible lysis
appeared prey cells
were killed up to a
considerable
distance from
predator colony.
1
2
3
4
5
This suggest
involvement of
diffusible
antimicrobial
compounds.
24 hr
48 hr
Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007
Unexplored potential of
Unexplored potential of
Streptomyces
Streptomyces
species
species
Trend analysis (9) and genome analysis (10) indicate that
Trend analysis (9) and genome analysis (10) indicate that
only a minority of secondary metabolites from the genus
only a minority of secondary metabolites from the genus
have been discovered so far. A much greater number
have been discovered so far. A much greater number
remains unexpressed.
remains unexpressed.
Why are most of the secondary metabolites
Why are most of the secondary metabolites
unexpressed?
unexpressed?
The most likely answer is that we do not understand the
The most likely answer is that we do not understand the
ecological conditions under which they are expressed.
ecological conditions under which they are expressed.
Predation may be the key!
Predation may be the key!
Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007
Materials and Methods
Materials and Methods
Screening of soil for predators.
Screening of soil for predators.
Lawns of
Lawns of
Staphylococcus aureus, E.coli
Staphylococcus aureus, E.coli
and
and
Bacillus
Bacillus
subtilis
subtilis
were spread on water agar plates and fine soil
were spread on water agar plates and fine soil
sprinkled on it.
sprinkled on it.
After incubation for 72-96 hrs colonies/swarms showing
After incubation for 72-96 hrs colonies/swarms showing
visible growth and clearance of the lawn were selected
visible growth and clearance of the lawn were selected
as potential predators.
as potential predators.
Demonstration of Predation.
Demonstration of Predation.
Washed cells of
Washed cells of
S.aureus
S.aureus
were spread on washed
were spread on washed
agarose plates. Test cultures were spot inoculated on this
agarose plates. Test cultures were spot inoculated on this
lawn and on washed agarose control. Growth, lysis of
lawn and on washed agarose control. Growth, lysis of
cells on the lawn and absence of growth on control
cells on the lawn and absence of growth on control
indicated predation
indicated predation
.
.
Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007
Materials and Methods
Materials and Methods
Selection for predatory ability
Selection for predatory ability
Cells of
Cells of
S. aureus
S. aureus
were spread on water agar plates and
were spread on water agar plates and
test organisms were spot inoculated. The test organisms
test organisms were spot inoculated. The test organisms
that showed growth after 72 96 hr were subcultured on
that showed growth after 72 96 hr were subcultured on
fresh lawns. Subculturing was repeated up to 20 times.
fresh lawns. Subculturing was repeated up to 20 times.
Microscopic observations
Microscopic observations
Slide culture technique was used with a thin layer of
Slide culture technique was used with a thin layer of
water agar covered with a lawn of
water agar covered with a lawn of
S. aureus.
S. aureus.
The test
The test
culture was spot inoculated. The slide was incubated for
culture was spot inoculated. The slide was incubated for
48-96 hrs and observed under 100x oel. Time lapse
48-96 hrs and observed under 100x oel. Time lapse
pictures with a 20 min interval were taken using digital
pictures with a 20 min interval were taken using digital
imaging by Leica DFC 340 FX.
imaging by Leica DFC 340 FX.
Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007
Acknowledgements
Acknowledgements
Anagha kale, Kalyani Damle, Anuja Damle,
Anagha kale, Kalyani Damle, Anuja Damle,
Tejashree Modak, Uttara Lele, Ashwini Keskar,
Tejashree Modak, Uttara Lele, Ashwini Keskar,
Geeta Khaladkar, all from Anujeeva Biosciences
Geeta Khaladkar, all from Anujeeva Biosciences
and NCIM, Pune for isolates.
and NCIM, Pune for isolates.
LabIndia Instruments Pvt. Ltd. and Leica
LabIndia Instruments Pvt. Ltd. and Leica
Microsystems for Microscopy and
Microsystems for Microscopy and
photomicrography.
photomicrography.
Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007
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Nature Precedings : doi:10.1038/npre.2007.1263.1 : Posted 27 Oct 2007