The hmu locus of Yersinia pestis is essential for utilization of free haemin and haem‐protein complexes as iron sources

Abstract

Yersinia pestis strains utilize haem and several haem-protein complexes as sole sources of iron. In this study, the haemin uptake locus (hmu) of Y. pestis KIM6+ was selected from a genomic library by transduction into an Escherichia coli siderophore synthesis (entC) mutant. Recombinant plasmids containing a common 16 kb BamHI insert were isolated that allowed E. coli entC to use haemin as an iron source. An 8.6 kb region of this insert was found to be essential for haemin utilization and encoded at least five proteins with molecular masses of 79/77, 44, 37, 35, and 30/27.5 kDa. A 10.9 kb Clal fragment containing the hmu locus showed varying degrees of homology to genomic DNA from Yersinia pseudotuberculosis, Yersinia enterocolitica, and other genera of Enterobacteriaceae. An E. coli hemA aroB strain harbouring cloned hmu genes used haemin as both an iron and porphyrin source but only on iron-poor medium, suggesting that haemin uptake is tightly iron regulated. Additionally, haemoglobin and myoglobin were used as iron sources by an E. coli entC (pHMU2.2) strain. Deletion of the hmu locus from Y. pestis KIM6+ chromosome generated a mutant that grew poorly on iron-depleted medium containing free haemin as well as mammalian haem-protein complexes including haemoglobin, haemoglobin-haptoglobin, myoglobin, haem-haemopexin, and haem-albumin unless it was complemented with cloned hmu genes.

The interaction of Yersinia pestis with erythrocytes

“…Plague bacilli utilise several ways of scavenging iron from mammalian haemoproteins and haemin [1,3,17,18] which is an abundant iron-containing compound found in vivo in the form of haemoglobin [16,20]. Thus, haemoglobincontaining mammalian RBC could be attractive to Y. pestis, as sources of iron or other nutrients.…”

“…In vivo, Y. pestis is able to scavenge mainly Fe 3 but not Fe 2 [1,3,4,17,18]. However, oxygenated haemoglobin in all cases possesses only Fe 2 without any oxidation to Fe 3 [16,20].…”

“…However, oxygenated haemoglobin in all cases possesses only Fe 2 without any oxidation to Fe 3 [16,20]. In contrast, haemin has Fe 3 [16,20] and can be used easily by Y. pestis [1,3,4,17,18] although there is no free haemin in normal mammalian RBC in vivo [16,20,21]. Thus, Y. pestis must possess a special mechanism to transform haemoglobin iron to a scavengeable form.…”

“…The last supposition seems quite possible because host tissues do not contain enough free purines to support growth of Y. pestis in vivo and the haemin utilisation system but not an iron only utilisation system was found in Y. pestis [1,3,4,17,18]. These ®ndings, apparently, are consistent with the ability of Y. pestis to adsorb large amounts of exogenous haemin that cause formation of dark brown or`pigmented' colonies [1,3,4,19] due to the oxidation of any haem-containing proteins in the presence of O 2 [16].…”

“…The results of testing HRBC for biochemical activities during their combined incubation with Y. pestis strains provided strong evidence that glucose is directly involved in haemoglobin destabilisation without its conversion to organic acid (Table 3). Finally, generated Fe 3 is suitable for utilisation by Y. pestis which possesses a haemin utilisation system [1,3,4,17,18].…”