Spatial patterns of DNA replication, protein synthesis, and oxygen concentration within bacterial biofilms reveal diverse physiological states

Abstract

It has long been suspected that microbial biofilms harbor cells in a variety of activity states, but there have been few direct experimental visualizations of this physiological heterogeneity. Spatial patterns of DNA replication and protein synthetic activity were imaged and quantified in staphylococcal biofilms using immunofluorescent detection of pulse-labeled DNA and also an inducible green fluorescent protein (GFP) construct. Stratified patterns of DNA synthetic and protein synthetic activity were observed in all three biofilm systems to which the techniques were applied. In a colony biofilm system, the dimensions of the zone of anabolism at the air interface ranged from 16 to 38 microm and corresponded with the depth of oxygen penetration measured with a microelectrode. A second zone of activity was observed along the nutrient interface of the biofilm. Much of the biofilm was anabolically inactive. Since dead cells constituted only 10% of the biofilm population, most of the inactive cells in the biofilm were still viable. Collectively, these results suggest that staphylococcal biofilms contain cells in at least four distinct states: growing aerobically, growing fermentatively, dead, and dormant. The variety of activity states represented in a biofilm may contribute to the special ecology and tolerance to antimicrobial agents of biofilms.

FIG. 1.

Patterns of DNA synthesis in S. epidermidis colony biofilms. Panel A shows an unlabeled (no BrdU) colony biofilm probed with antibody. Panel B shows a colony biofilm labeled with BrdU under aerobic conditions. Panel C shows a colony biofilm labeled with BrdU under anaerobic conditions. Panel D shows a colony biofilm labeled with BrdU in an environment of pure oxygen. Green areas are due to BrdU incorporation into DNA and indicate active replication. Red areas are due to rhodamine B counterstaining that reveals the extent of the biomass independent of its activity. In each image, the membrane interface of the colony is on the bottom and the air interface is on the top.

FIG. 2.

Patterns of DNA synthesis in S. epidermidis drip-flow (A) and capillary (B) biofilms and overlay of DNA synthetic activity and protein synthetic activity in the same S. aureus colony biofilm (C and D). In panels A and B, green areas are due to BrdU labeling and red areas are due to rhodamine B counterstain. In panel A, the substratum was on the bottom and the air interface was on the top. Flow was from top to bottom in panel B. In panel C, green areas are due to GFP expression and red areas are due to BrdU labeling. In panel C, the substratum was on the bottom, with the air interface on the top. Panel D shows the average fluorescence intensity along a linear transect across the section for the two signals. The position indicated on the x axis is arbitrary. The intensities indicated on the y axis of panel D are arbitrary as they result from automatic scaling by the digital image acquisition system.

FIG. 3.

Patterns of protein synthetic activity (green) imaged in S. aureus colony biofilms induced for GFP expression in air (A), in an anaerobic environment (B), in pure oxygen (C), or in air on medium supplemented with glucose (D) and in a S. aureus drip-flow reactor-grown biofilm (E). The green fluorescent image is overlaid with a transmitted light image of the section to allow visualization of the extent of the biomass independent of activity. In panel E, the substratum is on the bottom and the air interface is on the top.

FIG. 4.

Time series of GFP expression in S. aureus biofilm during tetracycline induction in a capillary reactor biofilm. The numeric label in the lower left corner of a panel is the time, in hours, after the first appearance of tetracycline in the flow cell. Green areas are due to GFP expression. A movie of this sequence can be viewed at http://www.erc.montana.edu/Res-Lib99-SW/Movies/Database/MD_DisplayScript.asp. Panel A shows a transmission image of the same region. Flow was from top to bottom. Panel B quantifies the variable dynamics of GFP expression during induction in S. aureus biofilm. Each numbered curve corresponds to a different spot in the biofilm as indicated by the boxes in panel A. The pixel intensity in the area indicated by the box was averaged at each time point.

FIG. 5.

Oxygen concentration profiles in S. epidermidis (A) and S. aureus (B) colony biofilms. Zero on the x axis corresponds to the biofilm-air interface. Negative positions are in air, and positive distances are in the biofilm. Representative measurements from two different colonies are plotted in each panel.