Activation of the antibiotic resistance factor WhiB7 can stimulate aggregate biofilm formation in stationary phase Mycobacterium smegmatis by reinitiating translation

TY - JOUR

T1 - Activation of the antibiotic resistance factor WhiB7 can stimulate aggregate biofilm formation in stationary phase Mycobacterium smegmatis by reinitiating translation

AU - Meyer, Mitchell D

AU - Bergkessel, Megan

AU - DePas, William H

N1 - Publisher Copyright: Copyright © 2026 Meyer et al.

PY - 2026/7/23

Y1 - 2026/7/23

N2 - There is a growing understanding that slow growth and dormancy due to nutrient deprivation are very common physiological states exhibited by bacterial communities in a myriad of environments. However, very little is known about the role of slow growth and dormancy in biofilm regulation. Here, we utilize tractable dormancy and aggregation assays in nontuberculous mycobacteria (NTM) to ask the fundamental question of how growth arrest impacts the processes of aggregation and dispersal. First, we show that the well-conserved dormancy regulator DosSR affects biofilm formation in Mycobacterium smegmatis, as a dosR deletion mutant undergoes spurious re-aggregation and dispersal during aerobic late stationary phase. Identification of a suppressor mutation blocking re-aggregation in the ΔdosR mutant allowed us to determine a role for the antibiotic resistance factor WhiB7 in driving re-aggregation in M. smegmatis. We utilized BioOrthogonal NonCanonical Amino acid Tagging (BONCAT), qPCR, and quantitative aggregation assays to build a model wherein reductive stress in ΔdosR potentiates stationary phase translation in a WhiB7-dependent manner, permitting aggregation in dormant stationary phase cells. In addition, during stationary phase, WhiB7-activating reducing agents and antibiotics could trigger re-aggregation in both wild-type M. smegmatis and clinical isolates of the opportunistic NTM pathogen Mycobacterium abscessus. Finally, we determined that, in contrast to aerobic stationary phase, M. smegmatis does not aggregate or disperse in response to chemical cues or antibiotics under the Wayne model of hypoxic dormancy. Our work reveals a regulatory interaction between dormancy and aggregation that could have broad implications for treating and preventing NTM biofilms.IMPORTANCEMycobacteria aggregate to form multicellular biofilms that provide protection from external stressors and increase antibiotic tolerance. Understanding the pathways regulating biofilm formation can aid the identification of useful targets for developing new drugs. With a growing appreciation that pathogens are often in a slow growth/dormant state during infection, we investigate how dormancy affects biofilm formation and dispersal in two nontuberculous mycobacteria (NTM) species: Mycobacterium smegmatis and the opportunistic pathogen Mycobacterium abscessus. We find that activation of the WhiB7-mediated antibiotic response permits biofilm formation in aerobic stationary phase by reinitiating protein synthesis; however, cells under hypoxic dormancy are unresponsive. Our work provides important context to combatting biofilm formation in infection sites, informing future studies and aiding design of biofilm dispersal agents.

AB - There is a growing understanding that slow growth and dormancy due to nutrient deprivation are very common physiological states exhibited by bacterial communities in a myriad of environments. However, very little is known about the role of slow growth and dormancy in biofilm regulation. Here, we utilize tractable dormancy and aggregation assays in nontuberculous mycobacteria (NTM) to ask the fundamental question of how growth arrest impacts the processes of aggregation and dispersal. First, we show that the well-conserved dormancy regulator DosSR affects biofilm formation in Mycobacterium smegmatis, as a dosR deletion mutant undergoes spurious re-aggregation and dispersal during aerobic late stationary phase. Identification of a suppressor mutation blocking re-aggregation in the ΔdosR mutant allowed us to determine a role for the antibiotic resistance factor WhiB7 in driving re-aggregation in M. smegmatis. We utilized BioOrthogonal NonCanonical Amino acid Tagging (BONCAT), qPCR, and quantitative aggregation assays to build a model wherein reductive stress in ΔdosR potentiates stationary phase translation in a WhiB7-dependent manner, permitting aggregation in dormant stationary phase cells. In addition, during stationary phase, WhiB7-activating reducing agents and antibiotics could trigger re-aggregation in both wild-type M. smegmatis and clinical isolates of the opportunistic NTM pathogen Mycobacterium abscessus. Finally, we determined that, in contrast to aerobic stationary phase, M. smegmatis does not aggregate or disperse in response to chemical cues or antibiotics under the Wayne model of hypoxic dormancy. Our work reveals a regulatory interaction between dormancy and aggregation that could have broad implications for treating and preventing NTM biofilms.IMPORTANCEMycobacteria aggregate to form multicellular biofilms that provide protection from external stressors and increase antibiotic tolerance. Understanding the pathways regulating biofilm formation can aid the identification of useful targets for developing new drugs. With a growing appreciation that pathogens are often in a slow growth/dormant state during infection, we investigate how dormancy affects biofilm formation and dispersal in two nontuberculous mycobacteria (NTM) species: Mycobacterium smegmatis and the opportunistic pathogen Mycobacterium abscessus. We find that activation of the WhiB7-mediated antibiotic response permits biofilm formation in aerobic stationary phase by reinitiating protein synthesis; however, cells under hypoxic dormancy are unresponsive. Our work provides important context to combatting biofilm formation in infection sites, informing future studies and aiding design of biofilm dispersal agents.

KW - Mycobacterium smegmatis/genetics

KW - Biofilms/growth & development

KW - Bacterial Proteins/genetics

KW - Gene Expression Regulation, Bacterial

KW - Protein Biosynthesis

KW - Drug Resistance, Bacterial

KW - dormancy

KW - NTM

KW - antibiotic tolerance

KW - two-component systems

KW - biofilms

UR - https://www.scopus.com/pages/publications/105045663267

U2 - 10.1128/jb.00076-26

DO - 10.1128/jb.00076-26

M3 - Article

C2 - 42328874

SN - 0021-9193

VL - 208

SP - e0007626

JO - Journal of Bacteriology

JF - Journal of Bacteriology

IS - 7

M1 - e00076-26

ER -