Abstract
CodY, a nutritional regulator highly conserved in low G+C Gram-positive bacteria, is essential in Streptococcus pneumoniae (the pneumococcus). A published codY mutant possessed suppressing mutations inactivating the fatC and amiC genes, respectively belonging to iron (Fat/Fec) and oligopeptide (Ami) ABC permease operons, which are directly repressed by CodY. Here we analyzed two additional published codY mutants to further explore the essentiality of CodY. We show that one, in which the regulator of glutamine/glutamate metabolism glnR had been inactivated by design, had only a suppressor in fecE (a gene in the fat/fec operon), while the other possessed both fecE and amiC mutations. Independent isolation of three different fat/fec suppressors thus establishes that reduction of iron import is crucial for survival without CodY. We refer to these as primary suppressors, while inactivation of ami, which is not essential for survival of codY mutants and acquired after initial fat/fec inactivation, can be regarded as a secondary suppressor. The availability of codY - ami + cells allowed us to establish that CodY activates competence for genetic transformation indirectly, presumably by repressing ami which is known to antagonize competence. The glnR codY fecE mutant was then found to be only partially viable on solid medium and hypersensitive to peptidoglycan (PG) targeting agents such as the antibiotic cefotaxime and the muramidase lysozyme. While analysis of PG and teichoic acid composition uncovered no alteration in the glnR codY fecE mutant compared to wildtype, electron microscopy revealed altered ultrastructure of the cell wall in the mutant, establishing that co-inactivation of GlnR and CodY regulators impacts pneumococcal cell wall physiology. In light of rising levels of resistance to PG-targeting antibiotics of natural pneumococcal isolates, GlnR and CodY constitute potential alternative therapeutic targets to combat this debilitating pathogen, as co-inactivation of these regulators renders pneumococci sensitive to iron and PG-targeting agents.
Highlights
The global nutritional regulator CodY is highly conserved in low G+C Gram-positive bacteria [1], and regulates up to 200 genes in Bacillus subtilis [2]
CodY was suggested to be an essential protein in S. pneumoniae primarily because repression of the fat/fec operon by CodY was required to avoid uncontrolled iron import resulting in toxicity [8]
The wildtype recipient was transformed with similar efficiency using as donor TD196 or the previously characterized codY::trim socY strain (Fig 1C). Since in the latter case survival of codY- transformants required the simultaneous co-transfer of two independent suppressors [8], explaining the very low frequency of TrimR clones recovered, these results suggested that yet uncharacterized suppressing mutations could be present in the glnR codY mutant to allow tolerance of codY inactivation
Summary
The global nutritional regulator CodY is highly conserved in low G+C Gram-positive bacteria [1], and regulates up to 200 genes in Bacillus subtilis [2]. A first suppressing mutation was identified in the fatC gene by whole-genome sequencing of the codY mutant [8] This gene belongs to the fatD–fatC–fecE–fatB operon; this operon ( called piuBCDA or pit1) [9], which is directly repressed by CodY [7], encodes the major ferric iron ABC permease of S. pneumoniae [10], with FatB shown to bind heme [11]. It was concluded that the three different amiC mutations identified in the codY- population arose subsequently to fatC in an otherwise codY fatC mutant lineage, presumably providing a selective advantage over ami+ cells Based on these data, CodY was suggested to be an essential protein in S. pneumoniae primarily because repression of the fat/fec operon by CodY was required to avoid uncontrolled iron import resulting in toxicity [8]
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