Transcript Lead-resistant Microbial Isolates from a Chat Pile.

Abstract
Microorganisms were isolated from a chat pile and soil in the vicinity. Chat is a byproduct of lead
mining that is high in Pb content, >16,000 ppm, a natural environment which would enrich for lead
tolerant microbes. Soil samples contained 2,000 – 4,000 ppm, of which 56-67% was bioavailable
as determined by differential extraction procedures. Lead tolerant bacteria and one yeast were
isolated on Roane minimal media with 1 mM total Pb concentration. The microbes were tested for
tolerance to lead on media with 1, 2.5, 5, and 10 mM total lead. From soil, the yeast, isolate Pb3,
was the most resistant, growing on media with 5 mM lead. Bacterial isolates Pb1, Pb4, and Pb5
grew at concentrations of 2.5 mM. Pb1 was the only isolate to grow at 37 oC and was identified as
a Pseudomonas species by the Vitek ID test panel, either stutzeri (55% confidence) or fluorescens
(55% confidence). From chat, distinct colony morphotypes CPC2, CPC3, CPC4, CPC5, CPA1,
CPA2, and CPA6 all grew at 2.5 mM lead. As a control 29 species of known bacteria and 4 species
of fungi were tested on the same medium. Of these, 11 bacteria and all the fungi were able to grow,
albeit slowly, on plates containing 1 mM lead. Interestingly, several bacteria, not normally known
to produce pigments, displayed reddish brown coloration suggesting Pb3O4 precipitation. Only
isolates CPC3 and CPA1 showed similar coloration. To identify bacteria that do not grow at 37 oC,
chromosomal DNA was extracted from each isolate and the 16S rDNA genes are being PCR
amplified. Products have been cloned and sequenced using M13 forward and reverse universal
primers. Sequences were compared to those in NCBI databases using the BLAST protocol.
Ribosomal data has obtained from two isolates to date. Pb4 most closely resembles Ochrobactrum,
a denitrifying soil organism which is known to be metal resistant and have significant
bioremediation potential. Partial CPA2 sequence suggests that it is an Arthrobacter species,
perhaps oxydans. This study extends our knowledge of the ecology and diversity of Pb resistant
microorganisms, and they are already being studied for the physiology of their interaction with lead.
Lead Mining in Missouri




East central Missouri is known
as the lead-belt region.
Chat is the byproduct of mining
lead ore. Large mounds of the
material dot the landscape in
the lead belt.
Although the lead
concentration is considered
hazardous, little has been
done to contain it.
Hypothesis: Chat represents a
natural enrichment for leadresistant microorganisms.
Lead Content, ppm, of Soil and Chat,
and Origin of Isolates
Fraction
Bioavailablea
Total
Isolates
a
Sample 12
Soil
2,725
Sample 14
Chat
16,733
Sample 15
Soil
1,289
4,023
Pb4, Pb5
16,733
CPA 1,2,6
CPC 2,3,4,5
2,294
Pb1, Pb3
method of Shuman, 1985. Soil Science 140:11-22
Isolation




Bacteria were eluteda from
chat and applied to the
surface of agar plates.
Defined chemical media
containing 1 mM Pb lead
called “Roane Media”b
This media is formulated to
minimize precipitation of
lead.
[Pb]soluble = 0.045[Pb]total-0.12 mM
a
Konopka, et al. 1999. Applied and Environmental Microbiology 65:2256-2259
b Roane
1999. Microbial Ecology 37:218-224
Pb3 = Unidentified yeast, Perhaps
Rhodospora toruloides

Imagesa





a. Grown in Roane media with no lead
b. Grown in Roane media with 1.5 mM
Pb, not stained
c. Grown in Roane media with 1.5 mM
stained
FAME analysis unable to
match any known yeast
WEB IDb Rhodospora
toruloides
a
Seabaugh et al., Manuscript in preparation; ASM Q-312
b www.2.cbs.knaw.nl/yeast.asp
CPA2 = CPC2 =Arthrobacter oxydans
or Arthrobacter polychromogenes
TSA
Roane






Roane
1 mM Pb
Roane
2.5 mM Pb
a
Common soil bacterium
Gram (+) irregular rods
CPA2 - 0.466 FAME
similarity index
CPC2 - 0.774 FAME
similarity index
CPA2 – Score 2742
(1521 bases) Blasta
CPA2 – Nearest neighbor
by RDPb /Phylip Interface
A. polychromogenes
http://www.ncbi.nlm.nih.gov/blast
b http://rdp.cme.msu.edu/html/
Pb4 = Ochrobactrum




Organism known to be
tolerant of high heavy
metal and salt
concentrations.
Gram (–) rod shaped
bacterium.
Score 2773 (1479 bases)
Blast
Nearest neighbor by
RDPb /Phylip Interface
Ochrobactrum sp. Str. S1
TSA
Roane
Roane
1 mM Pb
Roane
2.5 mM Pb
CPA1=CPC3=New Isolate
Roane



Roane
1 mM Pb
Gram (+) bacterium
not previously
described
Color variation may
be due to lead
precipitation
Poor match (0.04) to
Microbacterium by
FAME
CPA6 = CPC5 = Pb5 =
Rhodococcus fascians (luteus)




Gram (+) GC
Subgroup B
Common soil isolate
CPA6 - 0.912 FAME
similarity index
Misidentified as
Colwellia by short
DNA sequence
Appearance of R. fascians on Pb gradient plate
5 mM
Pb
No Growth
0 mM
Pb
Color Variation in Lead-grown Controls and
Isolates
Enterobacter aerogenes
Roane Media No lead



Roane Media –
1 mM lead
Various Lead Species
Species of known bacteria, including Enterobacter, Klebsiella, and
Bacillus, displayed a morphological shift to a reddish brown color
A similar effect was seen with the yeast
CPA1(CPC3)
Pb3Pb3
O4 and PbO
2
The contending hypothesis include;


1) production of a secondary metabolite, such as a chelator
2) cell association of a lead precipitate, which is seen with Pb3
Phylogeny of Lead Resistant Bacteria
Pseudomonas marginalis LMG2210
Pseudomonas putida F1
Escherichia coli K12
Rubrivivax gelatinosus ATCC 17011
Ochrobactrum sp. S1
Ochrobactrum sp. Pb4
Rhodopseudomonas palustris GH
Rhodospirillum rubrum 11170
Geobacter metallireducens GS15
Campylobacter jejuni ATCC 33560
Bacillus megaterium DSM32
Clostridium sordelli ATCC 9714
Rhodococcus fascians
Arthrobacter polychromogenes 2568
Arthrobacter CPA2
Deinococcus radiodurans ATCC 35073
Phylogenetic Analysis of Lead
Resistant Strains




16S rDNA genes were amplified with primers A and Ha. Amplicons were
ligated into pCR2.1, and inserts were sequence using M13 forward and
reverse primers (BioResource Center, Cornell University).
Sequences for isolates Pb4 and CPA2 were compared with those in
GenBank using the Blast protocol, and were identified as Ochrobactrum and
Arthrobacter species respectively.
The phylogenetic tree below was constructed using the Phylip Interface
available through the Ribosomal Database Project. The distance matrix
was calculated using the Kimura 2-parameter method with empirical base
frequencies and a transition/transversion ratio of 2. The tree was calculated
using the Neighbor-joining method with randomized input of strains and
Deinococcus as the out group.
Color coding




Names in red are those sequences obtained in this research
Names in blue are surrogate sequences for strains identified by FAME
Names in green are surrogate sequences for strains identified by Roane
Names in black are reference strains for various phylogenetic groupings
a
Massol-Deya, et al. 1995 In, Methods in Molecular Microbial Ecology
FAME Results
Isolate
tested
Major fatty acids
present
Closest match,
similarity index
Next match, similarity
index
CPA1
15:0 ANTEISO
16:0 ISO
17:0 ANTEISO
Microobacterium
laevaniformans, 0.045
None
CPA2
15:0 ANTEISO
15:0 ISO
16:0 ISO
Arthrobacter oxydans,
0.466
Arthrobacter pascens,
0.355
CPC2
15:0 ANTEISO
15:0 ISO
16:0 ISO
Arthrobacter oxydans,
0.774
Arthrobacter globiformis,
0.670
CPA6
18:1 w9c
16:0
15:0 ISO 2OH/16:1 w7c
Rhodococcus luteus,
0.912
Rhodococcus fascians,
0.725
Pb1
16:0
15:0 Iso 2OH/16; 1w7c
16:1 w7c/15 iso 2OH
Pseudomonas putida –
biotype A, 0.821
Pseudomonas
fluorescens – biotype B,
0.750
Pb3
18:1(w8)
18:1cis9(w9)
18:2 cis9, 12/18:0a
Yeast Species,
Unidentified
None
Identification of Lead-Resistant Microorganisms
Isolate
Cell Morphology
Genus and species
Method
CPA1 =
CPC3
Gram+ rods
?(Microbacterium)
FAME
CPA2 =
CPC2
Irregular Gram+
rod
Arthrobacter oxydans
A. polychromogenes
GenBank 16S
rDNA and FAME
RDP 16S rDNA
CPA6 =
CPC5 = Pb5
Gram+
Rhodococcus fascians
FAME
Pb1
Gram - rod
Pseudomonas stutzeri
Pseudomonas putida
Vitek ID Test Strip
FAME
Pb3
Yeast
Unidentified
Rhodospora toruloides
FAME
Web ID
Pb4
Gram + rod
Ochrobactrum
GenBank and
RDP 16S rDNA
Sequence
Acknowledgements




Funding for this project came from the Southeast Missouri State University Undergraduate
Research Program. The presenting authors (pictured above) would like to thank Dr. Allan
Bornstein and Provost Dr. Jane Stephens for their support of undergraduate research.
James Collins would like to thank members of his Graduation with Distinction advisory committee
Drs. Allen Gathman, Bjorn Olesen, John Kraemer, and Philip Crawford for their guidance. Dr. Rex
strange provided insight on the phylogenetic analysis.
Funding for student travel was made available through the Southeast Missouri State University
Student Professional Development program (Drs. Rick Burns and Christina Frazier), and the
Southeast Research Conference (Dr. Martha Zlokovich).
The authors would like to thank the following people for their excellent technical assistance: Maija
Bluma, Jennifer Arnold, and Andrew Corcoran. Also, Vicki Howell and Joanna Kubik provided
administrative support.