Spatial patterns of DNA replication, protein synthesis, and oxygen concentration within bacterial biofilms reveal active and inactive regions

Abstract

Biofilms harbor both active and inactive cells and it is a challenge to characterize the spatial and population heterogeneity of specific activities within a biofilm. Spatial patterns of DNA replication and protein synthetic activity were imaged by techniques developed using staphylococcal systems. The first technique measures DNA synthetic activity by pulse-labeling with the thymidine analog 5-bromo-2-deoxyuridine (BrdU) followed by immunofluorescent detection of brominated DNA. The second technique makes use of an inducible green fluorescent protein construct that can be used to detect the capacity for de novo protein synthesis. These techniques were applied to biofilms grown in three different reactor systems. In all cases, measurements revealed that even in simple single-species biofilms, complex spatial distributions of anabolic activity occur. In a colony biofilm system, two distinct regions of DNA synthetic activity were observed, one close to the nutrient interface and another adjacent to the air interface. A similar pattern was measured by GFP induction.

The dimensions of DNA synthetic activity ranged from 25 to 31 um and the average protein synthetic activity ranged from 36 to 38 um at the air interface. When pure oxygen was introduced, a wider zone of active DNA replication (45 um) and GFP synthesis (59 um) was measured at the gas interface. Oxygen penetration calculated (26um) corresponds with the zones of respiratory activity (19 to 38 um), DNA synthetic activity and protein synthetic activity measured at the air interface. The dimensions of DNA synthetic activity and protein synthesis activity at the nutrient interface ranged from 13 um to 19 um. The addition of glucose to the media increased the zone of protein synthesis at the nutrient interface to 33 um. Stratified patterns of activity were also observed in biofilms developed in two continuous flow reactors. While biofilms harbor regions of active anabolism, the techniques also demonstrate that these biofilms contain regions of complete inactivity. Such inactive zones may contribute to the special ecology of biofilms and tolerance to antimicrobial agents. The techniques, particularly BrdU labeling, are generic and may find application to many microbial biofilm systems.