Temporal and stochastic control of Staphylococcus aureus biofilm development

Abstract

Biofilm communities contain distinct microniches that result in metabolic heterogeneity and variability in gene expression. Previously, these niches were visualized within Staphylococcus aureus biofilms by observing differential expression of the cid and lrg operons during tower formation. In the present study, we examined early biofilm development and identified two new stages (designated "multiplication" and "exodus") that were associated with changes in matrix composition and a distinct reorganization of the cells as the biofilm matured. The initial attachment and multiplication stages were shown to be protease sensitive but independent of most cell surface-associated proteins. Interestingly, after 6 h of growth, an exodus of the biofilm population that followed the transition of the biofilm to DNase I sensitivity was demonstrated. Furthermore, disruption of the gene encoding staphylococcal nuclease (nuc) abrogated this exodus event, causing hyperproliferation of the biofilm and disrupting normal tower development. Immediately prior to the exodus event, S. aureus cells carrying a nuc::gfp promoter fusion demonstrated Sae-dependent expression but only in an apparently random subpopulation of cells. In contrast to the existing model for tower development in S. aureus, the results of this study suggest the presence of a Sae-controlled nuclease-mediated exodus of biofilm cells that is required for the development of tower structures. Furthermore, these studies indicate that the differential expression of nuc during biofilm development is subject to stochastic regulatory mechanisms that are independent of the formation of metabolic microniches. Importance: In this study, we provide a novel view of four early stages of biofilm formation by the human pathogen Staphylococcus aureus. We identified an initial nucleoprotein matrix during biofilm development that is DNase I insensitive until a critical point when a nuclease-mediated exodus of the population is induced prior to tower formation. Unlike the previously described dispersal of cells that occurs after tower development, we found that the mechanism controlling this exodus event is dependent on the Sae regulatory system and independent of Agr. In addition, we revealed that the gene encoding the secreted staphylococcal nuclease was expressed in only a subpopulation of cells, consistent with a model in which biofilms exhibit multicellular characteristics, including the presence of specialized cells and a division of labor that imparts functional consequences to the remainder of the population.

FIG 1

Early stages of Staphylococcus aureus biofilm development. S. aureus UAMS-1 (wild-type) biofilms were grown in a BioFlux microfluidics system, and bright-field images were captured throughout an 18-h time course experiment. (A) Representative images at the indicated time points of a typical UAMS-1 S. aureus biofilm depicting four stages of development: attachment (stage 1), multiplication (stage 2), exodus (stage 3), and biofilm maturation (stage 4). Attachment of cells to the glass bottom plate is quickly followed by the multiplication of the cell population into a confluent “lawn.” An exodus event after multiplication is followed by robust tower formation. Scale bar, 50 µm. (B) Quantification of typical S. aureus biofilm development presented as a percentage of biofilm coverage plotted versus time. Labels indicate the duration during which each biofilm stage is occurring. See Movie S1 in the supplemental material for a video depiction of the developmental stages of S. aureus biofilm formation.

FIG 2

Effects of exogenous proteinase K and DNase I on biofilm attachment and multiplication. S. aureus wild-type (UAMS-1) biofilms were grown in the BioFlux system with (open circles) or without (closed circles) exogenously added (A) DNase I (0.5 U ml⁻¹) or (B) proteinase K (100 µg ml⁻¹) at 0, 2, 4, and 6 h after the initiation of the experiment. Arrows in graphs indicate time points at which either proteinase K or DNase I was added to developing biofilm. Each graph shows the mean percentage of biofilm coverage in 2-h intervals. The data represent the means from two independent experiments, each containing at least two technical replicates. Error bars show the standard errors of the means (SEM) from the two independent experiments.

FIG 3

Effect of agr quorum sensing on early biofilm development. The S. aureus agr mutant strain, UAMS-155 (agr::tet), was inoculated in parallel with UAMS-1 (wild type) in the BioFlux system and allowed to form a biofilm for 18 h (A) Images selected at 4 h and 8 h are representative of wild-type (WT) and agr::tet biofilms from multiple experiments. Scale bar, 50 µm. (B) The graph depicts the percentage of biofilm coverage in 15-min intervals of wild-type (WT) and agr::tet mutant biofilms over 8 h of growth. The data represent the means from two independent experiments, each containing three technical replicates. Error bars show the SEM from the two independent experiments.

FIG 4

Exodus requires staphylococcal nuclease. Biofilms of the S. aureus wild-type (UAMS-1) and Δnuc mutant (UAMS-1471) containing pRMC2 or pRMC2-nuc were grown in the BioFlux system. (A) Selected bright-field images at 4 h and 8 h are representative of bioflims of the wild-type (UAMS-1) or Δnuc mutant (UAMS-1471) containing pRMC2 or pRMC2-nuc from multiple experiments. Scale bar, 50 µm. (B) The graph shows the mean percentage of biofilm coverage in 15-min intervals of biofilms of the wild-type (UAMS-1) and Δnuc mutant (UAMS-1471) containing pRMC2 or pRMC2-nuc over 8 h of growth. The data represent the means from two independent experiments, each containing at least three technical replicates. Error bars show the SEM from the two independent experiments. For a video compilation of the Δnuc mutant containing pRMC2 over 10 h of biofilm growth, see Movie S2 in the supplemental material.

FIG 5

Functional complementation of the nuc mutant biofilm phenotype by addition of DNase I. S. aureus wild-type (UAMS-1) and Δnuc mutant (UAMS-1471) cells were grown in the BioFlux with or without DNase I (0.5 U ml⁻¹). The graph shows the mean percentage of biofilm coverage in 15-min intervals of wild-type (WT) and Δnuc biofilms grown in the presence or absence of DNase I. The data represent the means from two independent experiments, each containing three technical replicates. Error bars show the SEM from the two independent experiments. For a video compilation of the Δnuc mutant biofilm grown in the presence of DNase I, see Movie S3 in the supplemental material.

FIG 6

Expression of nuc precedes exodus of a biofilm subpopulation. S. aureus wild-type (UAMS-1) cells containing the nuc::gfp reporter plasmid (pCM20) were grown in the BioFlux system. Bright-field and epifluorescence microscopic images were acquired in 5-min intervals at ×200 magnification. (A) Bright-field and epifluorescence (GFP) images at 2 h and 5 h are representative of multiple experiments. Scale bar, 20 µm. (B) The plot depicts biofilm growth as the mean percentage of biofilm coverage and the nuc-expressing cells as the mean fluorescence coverage in 15-min intervals over 8 h of growth. The data represent the means from two independent experiments, each containing at least two technical replicates. Error bars were omitted for clarity. For a video compilation observing nuc expression, see Movie S4 in the supplemental material.

FIG 7

Nuclease-mediated exodus is regulated by Sae. S. aureus wild-type (AH1263) and saeQRS::spc mutant (AH1558) strains carrying the nuc::gfp reporter plasmid (pCM20) were grown in the BioFlux system. The plot depicts biofilm growth as the mean percentage of biofilm coverage and the nuc-expressing cells as the mean fluorescence coverage in 15-min intervals over 8 h of growth. The data represent the means from two independent experiments, each containing at least two technical replicates. Error bars were omitted for clarity. For representative images, see Fig. S4 and for a video compilation of a saePQRS::spc mutant biofilm see Movie S5 in the supplemental material.