Identification of ypqP as a New Bacillus subtilis Biofilm Determinant That Mediates the Protection of Staphylococcus aureus against Antimicrobial Agents in Mixed-Species Communities

Abstract

In most habitats, microbial life is organized in biofilms, three-dimensional edifices sustained by extracellular polymeric substances that enable bacteria to resist harsh and changing environments. Under multispecies conditions, bacteria can benefit from the polymers produced by other species ("public goods"), thus improving their survival under toxic conditions. A recent study showed that a Bacillus subtilis hospital isolate (NDmed) was able to protect Staphylococcus aureus from biocide action in multispecies biofilms. In this work, we identified ypqP, a gene whose product is required in NDmed for thick-biofilm formation on submerged surfaces and for resistance to two biocides widely used in hospitals. NDmed and S. aureus formed mixed biofilms, and both their spatial arrangement and pathogen protection were mediated by YpqP. Functional ypqP is present in other natural B. subtilis biofilm-forming isolates. However, the gene is disrupted by the SP␤ prophage in the weak submerged-biofilm-forming strains NCIB3610 and 168, which are both less resistant than NDmed to the biocides tested. Furthermore, in a 168 laboratory strain cured of the SP␤ prophage, the reestablishment of a functional ypqP gene led to increased thickness and resistance to biocides of the associated biofilms. We therefore propose that YpqP is a new and important determinant of B. subtilis surface biofilm architecture, protection against exposure to toxic compounds, and social behavior in bacterial communities. Bacillus subtilis is a nonpathogenic Gram-positive bacterium that can be found in its natural habitats as free cells or associated with surfaces in biofilms. In the soil, B. subtilis strains have been shown to form surface-associated communities on plant tissues that protect them from infection by pathogens (1-3). Because of its ability to resist stress through biofilm or spore formation, the bacterium has been isolated from extreme environments, such as sand deserts, clouds, and the digestive tracts of animals (4 -6). As well as its ubiquitous presence in diverse habitats, B. subtilis has been used extensively in biotechnological applications, such as the production of natto, a traditional Japanese food made of fermented soybeans (7), and the production of industrial enzymes and pharmaceutical proteins (8, 9). As a generally recognized as safe (GRAS) organism, B. subtilis is also used as a biocontrol agent in agriculture and livestock buildings (10 -14) and as a probiotic agent to improve human and animal health by preventing gastrointestinal infections (15,16).In fundamental research, B. subtilis has emerged as the model organism for deciphering the complex genetic regulation involved in the biofilm mode of life of Gram-positive bacteria. The domesticated strain B. subtilis 168 has been widely used to dissect metabolic and cellular processes. However, this strain is not able to form robust pellicles and complex colonies like those of its parental strain, NCIB3610, a descendant of the original Marburg strain that was deposited in 1951 (17)....

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