1. 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 o C 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.

2. 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.

3. Lead Content, ppm, of Soil and Chat, and Origin of Isolates FractionSample 12 Soil Sample 14 Chat Sample 15 Soil Bioavailable a 2,72516,7331,289 Total4,02316,7332,294 IsolatesPb4, Pb5CPA 1,2,6 CPC 2,3,4,5 Pb1, Pb3 a method of Shuman, 1985. Soil Science 140:11-22

4. Isolation  Bacteria were eluted a 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

5. Pb3 = Unidentified yeast, Perhaps Rhodospora toruloides Images a  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 ID b Rhodospora toruloides a Seabaugh et al., Manuscript in preparation; ASM Q-312 b www.2.cbs.knaw.nl/yeast.asp

6. CPA2 = CPC2 =Arthrobacter oxydans or 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) Blast a CPA2 – Nearest neighbor by RDP b /Phylip Interface A. polychromogenes TSA Roane 1 mM Pb Roane 2.5 mM Pb a http://www.ncbi.nlm.nih.gov/blast b http://rdp.cme.msu.edu/html/

7. 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 RDP b /Phylip Interface Ochrobactrum sp. Str. S1 TSA Roane 1 mM Pb Roane 2.5 mM Pb

8. 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 1 mM Pb

9. 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

10. 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 Pb 3 O 4 PbO 2 Enterobacter aerogenes Various Lead Species

11. 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

12. Phylogenetic Analysis of Lead Resistant Strains 16S rDNA genes were amplified with primers A and H a. 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

13. 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

14. Identification of Lead-Resistant Microorganisms Isolate Cell Morphology Genus and speciesMethod 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 fasciansFAME Pb1 Gram - rod Pseudomonas stutzeri Pseudomonas putida Vitek ID Test Strip FAME Pb3 Yeast Unidentified Rhodospora toruloides FAME Web ID Pb4 Gram + rod OchrobactrumGenBank and RDP 16S rDNA Sequence

15. 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.