Identification of a gene (arpU) controlling muramidase-2 export in Enterococcus hirae

Abstract

Muramidase-2 of Enterococcus hirae is a 74-kDa peptidoglycan hydrolase that plays a role in cell wall growth and division. To study its regulation, we isolated a mutant defective in muramidase-2 release under certain growth conditions. This mutant had cell walls which apparently lacked 74-kDa muramidase-2 but which accumulated two proteolytic fragments of 32 and 43 kDa, which exhibited muramidase-2 activity in the membrane fraction. By complementation cloning, we identified a 2.6-kb fragment of the E. hirae chromosome containing a gene cluster coding for proteins of 58 to 137 amino acids. One of these genes (arpU), which encoded a 15.9-kDa protein, was shown to complement the defect of the A9 mutant in trans. We propose that this gene may be involved in the regulation of muramidase-2 export.Peptidoglycan hydrolases are enzymes responsible for a number of important biological functions such as cell wall growth, cell separation, peptidoglycan turnover, competence for genetic transformation, sporulation, and bacteriolysis induced by beta-lactam antibiotics (7,8,14,21,23,27). Regulation of autolysin activity is believed to occur mostly at the posttranslational level. Possible regulatory mechanisms include enzyme activation by changes in substrate structure, topological restriction of enzyme distribution in the cell wall, and control at the site of export (8).Two autolytic enzymes with muramidase activity have been described in Enterococcus hirae. Muramidase-1 is synthesized in a latent 130-kDa form, which is transported to specific sites in the cell wall, where it is converted to the active 87-kDa form (10). This enzyme can hydrolyze E. hirae cell walls but not Micrococcus luteus cell walls. The second enzyme, peptidoglycan hydrolase-2, or muramidase-2, is found in the culture medium during cell growth and is active on cell walls of M. luteus and purified peptidoglycan of E. hirae (5, 11). It has been postulated that muramidase-2 may operate in conjunction with muramidase-1 in peptidoglycan hydrolysis (2) and may facilitate cell separation (14,24). The E. hirae muramidase-2 gene has recently been cloned and sequenced (3,9).In an attempt to gain insight into the mechanisms that control muramidase-2 activity in E. hirae, we have characterized a mutant (A9) with medium-dependent impairment of muramidase-2 export. By functional complementation of the mutant with an E. hirae genomic bank, we cloned a gene (arpU) that would appear to be involved in muramidase-2 export. MATERIALS AND METHODSBacterial strains and growth conditions. Unless stated otherwise, E. hirae wild-type (ATCC 9790) and mutant cells were grown at 37ЊC in N medium (1% Bacto Peptone [Difco Laboratories, Detroit, Mich.], 0.5% yeast extract [Difco], 1% Na 2 HPO 4 ⅐ 2H 2 O). For some experiments, brain heart infusion (BHI; Difco) was used. To monitor growth, the optical density at 650 nm was measured in an LKB Ultrospec II.Selection of mutants. For the isolation of mutants deficient in muramidase-2, wild-type E. hirae was mutagenized as follows. Cells in mid...

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A multi-omic analysis of an Enterococcus faecium mutant reveals specific genetic mutations and dramatic changes in mRNA and protein expression

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