Occurrence of mutations impairing sigma factor B (SigB) function upon inactivation of Listeria monocytogenes genes encoding surface proteins

JUAN J. QUEREDA; M. GRACIELA PUCCIARELLI; LAURA BOTELLO-MORTE; ENRIQUE CALVO; FILIPE CARVALHO; CHRISTIANE BOUCHIER; ANA VIEIRA; JAVIER FERNANDO MARISCOTTI; TRINAD CHAKRABORTY; PASCALE COSSART; TORSTEN HAIN; DIDIER CABANES; FRANCISCO GARCÍA-DEL PORTILLO

MICROBIOLOGY-UK

SOC GENERAL MICROBIOLOGY

Lugar: London; Año: 2013 vol. 159 p. 1328 - 1339

1350-0872

Bacteria of the genus Listeria contain the largest family of LPXTG surface proteins covalently anchored to the peptidoglycan. The extent at which these proteins may function or be regulated cooperatively is at present unknown. Because of their unique cellular location, we reasoned that distinct LPXTG proteins could act as elements contributing to cell wall homeostasis or influencing the stability of other surface proteins bound to peptidoglycan. To test this hypothesis, we analysed by proteomics mutants of the intracellular pathogen L. monocytogenes lacking distinct LPXTG proteins implicated in pathogen-host interactions as InlA, InlF, InlG, InlH, InlJ, LapB and Vip. Changes in the cell wall proteome were found in inlG and vip mutants, which exhibited reduced levels of the LPXTG proteins InlH, Lmo0610, Lmo0880 and Lmo2085, all regulated by the stress-related sigma factor SigB. The ultimate basis of this alteration was uncovered by genome sequencing, which revealed that these inlG and vip mutants carried loss-of-function mutations in the rsbS, rsbU and rsbV genes encoding regulatory proteins that control SigB activity. Attempts to recapitulate this negative selection of SigB in large series of new inlG or vip mutants constructed for this purpose were however unsuccessful. These results indicate that inadvertent secondary mutations affecting SigB functionality can randomly arise in L. monocytogenes when using widely used genetic procedures or during sub-culturing. Testing of SigB activity could be therefore valuable when manipulating genetically L. monocytogenes prior to any subsequent phenotypic analysis. This test may be even more justified when generating deletions affecting cell envelope components.