AbstractMicroorganisms 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 lead-resistant microorganisms.

Lead Content, ppm, of Soil and Chat, and Origin of Isolates

Fraction Sample 12 Soil

Sample 14 Chat

Sample 15 Soil

Bioavailablea 2,725 16,733 1,289

Total 4,023 16,733 2,294

Isolates Pb4, Pb5 CPA 1,2,6

CPC 2,3,4,5

Pb1, Pb3

a 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 oxydansor Arthrobacter polychromogenes

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

TSA Roane

Roane1 mM Pb

Roane2.5 mM Pb

a http://www.ncbi.nlm.nih.gov/blastb 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

Roane1 mM Pb

Roane2.5 mM Pb

CPA1=CPC3=New Isolate

Gram (+) bacterium not previously described

Color variation may be due to lead precipitation

Poor match (0.04) to Microbacterium by FAME

Roane

Roane1 mM Pb

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 No Growth

5 mM Pb

0 mM Pb

Appearance of R. fascians on Pb gradient plate

Color Variation in Lead-grown Controls and Isolates

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 Pb3 and CPA1(CPC3) 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

Roane Media - No lead

Roane Media – 1 mM lead

Pb3O4 PbO2

Enterobacter aerogenes

Various Lead Species

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 ResultsIsolate 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.