Metabolite profiling uncovers plasmid-induced cobalt limitation under methylotrophic growth conditions.
BACKGROUND
The introduction and maintenance of plasmids in cells is often associated with a reduction of growth rate. The reason for this growth reduction is unclear in many cases.


RESULTS
We observed a surprisingly large reduction in growth rate of about 50% of Methylobacterium extorquens AM1 during methylotrophic growth in the presence of a plasmid, pCM80 expressing the tetA gene, relative to the wild-type. A less pronounced growth delay during growth under non-methylotrophic growth conditions was observed; this suggested an inhibition of one-carbon metabolism rather than a general growth inhibition or metabolic burden. Metabolome analyses revealed an increase in pool sizes of ethylmalonyl-CoA and methylmalonyl-CoA of more than 6- and 35-fold, respectively, relative to wild type, suggesting a strongly reduced conversion of these central intermediates, which are essential for glyoxylate regeneration in this model methylotroph. Similar results were found for M. extorquens AM1 pCM160 which confers kanamycin resistance. These intermediates of the ethylmalonyl-CoA pathway have in common their conversion by coenzyme B(12)-dependent mutases, which have cobalt as a central ligand. The one-carbon metabolism-related growth delay was restored by providing higher cobalt concentrations, by heterologous expression of isocitrate lyase as an alternative path for glyoxylate regeneration, or by identification and overproduction of proteins involved in cobalt import.


CONCLUSIONS
This study demonstrates that the introduction of the plasmids leads to an apparent inhibition of the cobalt-dependent enzymes of the ethylmalonyl-CoA pathway. Possible explanations are presented and point to a limited cobalt concentration in the cell as a consequence of the antibiotic stress.
Kiefer
Patrick
P
Institute of Microbiology, ETH Zürich, Zürich, Switzerland.
Buchhaupt
Markus
M
Christen
Philipp
P
Kaup
Björn
B
Schrader
Jens
J
Vorholt
Julia A
JA
D000214:Acyl Coenzyme A / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport; D049109:Cell Proliferation; D003035:Cobalt / Q000737:chemistry*; D003038:Cobamides / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006038:Glyoxylates / Q000378:metabolism; D019751:Intramolecular Transferases / Q000378:metabolism; D007522:Isocitrate Lyase / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism; D000432:Methanol / Q000737:chemistry; D020580:Methylobacterium / Q000378:metabolism*; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism*
D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D003038:Cobamides; D006038:Glyoxylates; D008565:Membrane Proteins; C102829:ethylmalonyl-coenzyme A; C057616:tonB protein, Bacteria; C015357:methylmalonyl-coenzyme A; D003035:Cobalt; D007522:Isocitrate Lyase; D019751:Intramolecular Transferases; C000913:cobamamide; C031150:glyoxylic acid; D000432:Methanol