Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Microbial Genomics
70
The Genome of the Ammonia Oxidizing Bacterium Nitrosomonas europaea: Iron Metabolism and Barriers to Heterotrophy
Xueming Wei, Neeraja Vajrala, Norman Hommes, Luis Sayavedra-Soto*, and Daniel Arp (arpd@science.oregonstate.edu)
Oregon State University, Corvallis, OR
Nitrosomonas europaea is an aerobic lithoautotrophic bacterium that uses ammonia (NH3) as its energy source (3). As a nitrifier, it is an important participant in the N cycle, which can also influence the C cycle. The genome sequence of N. europaea has been annotated and consists of approximately 2460 protein-encoding genes (1). We are continuing to use the genome sequence to explore the genetic structure and mechanisms underlying the lithoautotrophic growth style of N. europaea. Currently, we are investigating its Fe requirements and its possible barriers to utilizing carbon sources different from CO2.
Because N. europaea has a relatively high content of hemes, sufficient Fe must be available in the medium for it to grow. The genome revealed that approximately 5% of the coding genes in N. europaea are dedicated to Fe transport and assimilation. Nonetheless, with the exception of citrate, N. europaea lacks genes for siderophore production (1). We have initiated the study on this intriguing facet by determining the Fe requirements for growth and are characterizing the expression of the putative membrane siderophore receptors.
N. europaea changes its heme composition when Fe is at a relatively low concentration. Biochemical analyses showed that cytochrome and heme contents of cells grown in Fe-limited medium were 4 fold lower than those from Fe-rich medium. Cellular Fe contents (in both membrane and soluble fractions) showed the same trend. The activity of hydroxylamine oxidoreductase was over three fold lower in cells grown in Fe-limited medium than that in full medium. The growth yields at 0.1 µM Fe and at 0.2 µM Fe were about 35% and 65% respectively of that observed at 10 µM Fe (full medium). N. europaea has the mechanisms to cope and grow under Fe limitation.
The N. europaea genome revealed that there are over 26 sets of genes that are organized similarly to the genes in a fecR/fecI system. Through similarity searches, we have identified possible TonB-dependent receptor genes up- or downstream of these sets. Some of these are similar to genes encoding the siderophore receptors for desferrioxamine (desferal), ferrichrome, and coprogen.
The addition of desferal in Fe-limiting medium promoted the growth of N. europaea, though with a longer lag phase, suggesting a necessary induction period of the corresponding receptor. A gene for the putative desferal outer membrane receptor was identified by similarity searches (NE1097, a foxA homologue). NE1097 was expressed at a higher level (>10 fold) in Fe-limiting, desferal-containing cultures than in Fe-sufficient cultures. The expression of NE1097 required the presence of desferal, since typical lag phases were observed when inoculants from desferal cultures were used. Several membrane proteins detected only in the cells grown in Fe-limited medium may be involved in Fe transport. For example, a membrane peptide with the calculated MW of the putative desferal receptor was observed only in the cells grown in desferal-containing medium. Ferric citrate had an effect similar to that of desferal on N. europaea growth in Fe-limiting medium, but with a longer lag phase and a higher final cell density than that in the full medium. Ferrichrome, on the other hand, did not prolong the lag phase, yet increased total cell growth, suggesting that the genes for the ferrichrome receptors were expressed constitutively.
Consistent with the genome sequence data, no siderophores were detected in N. europaea culture filtrates under either Fe-limiting or Fe-sufficient conditions using a standard siderophore assay. We considered the possibility that citrate serves as a Fe chelator/siderophore, since N. europaea has the necessary genes to produce it. Citrate was detected (2 to 5 µM) in cell-free filtrates from both, low- and full Fe cultures. Surprisingly, cell-free filtrates from full Fe cultures had relatively higher concentrations (5 µM) of citrate than in low Fe cultures (2 to 3 µM). The role of citrate in Fe acquisition, if any, is yet to be determined. N. europaea apparently expresses siderophore receptors (i.e. NE1097) under low Fe conditions to scavenge Fe more efficiently. These results reinforce the notion that N. europaea uses siderophores produced by other organisms in natural habitats.
Genes encoding the putative outer membrane desferal receptor (NE1097 and NE1088, foxA homologues) have been cloned, insertional mutant constructs made, and mutant strains obtained through homologous recombination. Physiological and genetic characterization of these mutants is in progress.
In addition to the Fe experiments, analysis of the N. europaea genome has led to experiments probing the possible barriers to heterotrophy in N. europaea. The genome contains genes that are similar to the genes encoding fructose transport systems (PTS-type) in other bacteria. Furthermore, N. europaea can use fructose as the only source of carbon for growth (2). However, not all the genes required for an active PTS system are present in the genome. The inactivation of the two identified PTS genes did not affect growth on fructose or cause any other growth phenotype. Fructose may enter the cells by some other means. The role of the existing PTS genes remains unclear.
Historically, the activity of the enzyme 2-oxoglutarate dehydrogenase has not been detected in N. europaea. The lack of this activity was believed to be the cause for the obligate autotrophy of N. europaea. However, the genomic sequence reveals that the three genes necessary to encode this enzyme are present. We inactivated the first gene (odhA) in the operon of this enzyme. The mutant strain grew similarly to wild-type cells during exponential growth. However, in late stationary phase or under ammonia starvation (i.e. energy-limiting conditions), mutant strains lost viability faster and recovered more slowly upon addition of more ammonia as compared to the wild type. This suggests that 2-oxoglutarate dehydrogenase may be involved in processes occurring during the stationary growth phase of N. europaea.
A gene encoding a putative ammonia transporter (amt) is present in the genome. However, the strain with this gene inactivated showed no difference in growth to wild-type cells over a wide range of ammonium concentrations. The function of amt in N. europaea is still unknown.
We are exploring the idea that one barrier to heterotrophic growth in N. europaea may be due to a lack of transporters for alternative growth substrates. The genes encoding the enzymes to utilize glycerol as the carbon source are present, but the genes encoding a glycerol transporter are not. The heterologous expression of the gene for the glycerol permease from E. coli in N. europaea permits N. europaea to utilize glycerol as the carbon source.
Acknowledgment: This research is funded by grants from the Department of Energy (ER63149-1017305-0007065 and ER63781-1023295-0009824) and resources from the Oregon Agricultural Experiment Station.
References
- Chain, P., J. Lamerdin, F. Larimer, W. Regala, V. Lao, M. Land, L. Hauser, A. Hooper, M. Klotz, J. Norton, L. Sayavedra-Soto, D. Arciero, N. Hommes, M. Whittaker, and D. Arp. 2003. Complete genome sequence of the ammonia-oxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea. J Bacteriol 185:2759-2773.
- Hommes, N. G., L. A. Sayavedra-Soto, and D. J. Arp. 2003. Chemolithoorganotrophic growth of Nitrosomonas europaea on fructose. J Bacteriol 185:6809-6814.
- Wood, P. M. 1986. Nitrification as a bacterial energy source, p. 39-62. In J. I. Prosser (ed.), Nitrification. Society for General Microbiology, IRL Press, Oxford.
* Presenting author
