Growth control of small-colony variants by genetic regulation of the hemin uptake system

Small-colony variants (SCVs) are slow-growing variants of human bacterial pathogens. They are associated with chronic persistent infections, and their biochemical identification and antimicrobial treatment are impaired by altered metabolic properties. To contribute to the understanding of SCV-mediated infections, we analyzed a clinical SCV isolate derived from a chronic prosthetic hip infection. A sequence analysis of housekeeping genes identified an Enterobacter hormaechei-like organism. The SCV phenotype, with growth as microcolonies, was caused by a block within the heme biosynthesis pathway through deletion of the hemB locus, as shown by hybridization and complementation experiments. At a low frequency, large-colony variants (LCVs) arose that were dependent on exogenous hemin. To investigate this phenomenon, we cloned and sequenced the 5.8-kb hemin uptake system, denoted ehu. Gene expression analysis indicated regulation of this locus in wild-type bacteria by the global iron regulator Fur. Inactivation of Fur in LCVs caused the derepression of ehu expression and facilitated bacterial growth. Genetic alterations of the fur locus in LCVs were identified as insertions of IS1A elements and point mutations. In contrast, SCVs could utilize exogenous hemin only in the absence of iron. Thus, we provide the first molecular characterization of the growth properties of a clinical SCV isolate, which may help to improve the diagnostic and therapeutic management of patients with chronic persistent infections.