Image: Bacterial induced formation of Cd crystals by A. eutrophus on Zirfon M5 membranes in the continuous tubular membrane reactor. The figure shows the bacterial formed CdCO3 crystals. The bar represents 1 mm. (S.Taghavi) We thank S. Taghavi for providing this image.

     Alcaligenes eutrophus CH34, recently renamed Ralstonia eutrophus is a gram-negative, non-spore forming bacterium which thrives in the presence of millimolar concentrations of several heavy-metals (Zn, Cd, Co,Pb,Hg, Ni & Cr). The heavy-metal resistance is conferred by two large megaplasmids (pMOL28=180 kbp and pMOL30=240 kbp) carrying gene clusters that encode cation-efflux machinery spanning both bacterial membranes. These low-copy number plasmids are stably maintained in the presence or absence of selective pressure and are self-transferable at relatively low frequencies.

     A. eutrophus uses a variety of substrates as its carbon source or it can grow chemo-lithotropically using molecular hydrogen as the energy source and carbon dioxide as a carbon source. When nitrate is present A. eutrophus can grow anaerobically.

     Regions of the megaplasmid DNA relevant to the cation-efflux pump were sequenced; however, to the best of our knowledge, none of the groups is systematically sequencing any of the plasmids. The complete sequence of the megaplasmids will be instrumental in understanding gene organization, especially mechanism of plasmid replication, partition and transfer, and allowing the construction of environmentally-friendly bacteria e.g. E.coli with stable and efficient mechanism for heavy-metal resistance.

For more in depth information on biology of this bacterium, please refer to some of the following publications and references therein.

1. M. Mergeay et. al. "Alcaligenes eutrophus CH34 Is a Facultative Chemolithotroph with Plasmid-Bound Resistance to Heavy Metals" J. Bact. Vol. 162 (1) 328-334, 1985.
2. A. Nies et. al. "Nucleotide Sequence and Expression of a Plasmid-encoded Chromate Resistance Determinant from Alcaligenes eutrophus" J. Biol. Chem. Vol. 265 (10):5648-5653, 1990.
3. S. Taghavi et. al. "Genetic and Physical Maps of the Alcaligenes eutrophus CH34 Megaplasmid pMOL28 and its Derivative pMOL50 Obtained after Temperature-Induced Mutagenesis and Mortality" Plasmid Vol. 37:22-34, 1997.

In collaboration with Dr. M. Mergeay mergeaym@vito.be and Dr. D. van der Lelie vdlelied@vito.be we set out to accomplish in the first phase the following goals:

1. Complete the sequence of the entire megaplasmid pMOL28 (160 kbp) and pMOL30 (240 kbp) using both standard approach (shotgun) and more directed high-throughput strategies developed in our group.
2. Analyze the plasmid nucleotide sequences to locate all genes/gene families, particularly those responsible for heavy heavy-metal resistance. Of interest will also be, any genetic regulatory elements like replication origins, partition loci, plasmid maintenance, etc.
3. Locate and remove/modify the regions of plasmid responsible for transfer to prevent plasmid spontaneous plasmid exchange under natural conditions.

Note: Initially we started sequencing on the libraries prepared from DNA isolated from wild type CH34 strain which has two megaplasmids. Later on we obtained cured strains with one (pMOL28) or another (pMOL30) plasmids. In addition some sequencing was done on a plasmid which has approximately 40,000 bases of chromosomal DNA incorporated into pMOL28 (called pMOL50=strain AE3).

Partial list of long-term goals includes:

1. Solve the structure of antiporters membrane proteins using X-ray crystallography.
2. Use site-directed mutagenesis to locate key amino acid determinants of heavy-metal resistance and selectivity.
3. Modify and/or broaden the metal selectivity through amino acid substitution.

Some of the genes located in CH34 megaplasmids are shown below in table 1 .

Table 1.

DNA fragment	Homologous to--from:	P(n)
Fragment 01	Phosphoribosylformylglycineamide synthase--E.coli	5.7e-62
Fragment 02	Nickel & cobalt reistance protein--Alcaligenes denitrificans	5.3e-54
Fragment 03	Nitrogen fixation protein--Haemophilus influenzae	2.2e-45
Fragment 04	Glycine dehydrogenase--E.coli	4.0e-45
Fragment 05	Putative L-lactate permease--E.coli	2.2e-43
Fragment 06	Long-chain-fatty-acid-CoA--Archeoglobus fulgidus	1.7e-24
Fragment 07	Phosphate binding protein--Xanthomonas oryzae	7.1e-20
Fragment 08	Hydrogenase expression/formation protein HYPC--Rhizobium leguminosarum	3.7e-13
Fragment 09	TniBdelta1 transposon Tn21--Pseudomonas aeruginosa	1.7e-11
Fragment 10	Transposase tnpA protein--Pseudomonas putida	1.8e-11
Fragment 11	Aconitate hydratase--Bacillus subtilis	5.3e-11
Fragment 12	Anthranilate synthase component I--Aquifex aeolicus	5.4e-09
Fragment 13	MocB protein--Agrobacterium tumefaciens	6.7e-09
Fragment 14	Proline dehydrogenase--E.coli	1.9e-08
Fragment 15	Oligopeptide transport ATP-binding protein OppD.-E.coli	6.7e-07

Summary information for all sequenced megaplasmids is provided in table 2. "No. of base sequenced" represents edited sequence.

Table 2.