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ABSTRACT Penicillin-binding proteins (PBPs) play critical roles in cell wall construction, cell shape maintenance, and bacterial replication. Bacteria maintain a diversity of PBPs, indicating that despite their apparent functional redundancy, there is differentiation across the PBP family. Apparently-redundant proteins can be important for enabling an organism to cope with environmental stressors. In this study, we evaluated the consequence of environmental pH on PBP enzymatic activity inBacillus subtilis. Our data show that a subset of PBPs inB. subtilischange activity levels during alkaline shock and that one PBP isoform is rapidly modified to generate a smaller protein (i.e., PBP1a to PBP1b). Our results indicate that a subset of the PBPs are favored for growth under alkaline conditions, while others are readily dispensable. Indeed, we found that this phenomenon could also be observed inStreptococcus pneumoniae, implying that it may be generalizable across additional bacterial species and further emphasizing the evolutionary benefit of maintaining many, seemingly-redundant periplasmic enzymes. IMPORTANCEMicrobes adapt to ever-changing environments and thrive over a vast range of conditions. While bacterial genomes are relatively small, significant portions encode for “redundant” functions. Apparent redundancy is especially pervasive in bacterial proteins that reside outside of the inner membrane. While conditions within the cytoplasm are carefully controlled, those of the periplasmic space are largely determined by the cell’s exterior environment. As a result, proteins within this environmentally exposed region must be capable of functioning under a vast array of conditions, and/or there must be several similar proteins that have evolved to function under a variety of conditions. This study examines the activity of a class of enzymes that is essential in cell wall construction to determine if individual proteins might be adapted for activity under particular growth conditions. Our results indicate that a subset of these proteins are preferred for growth under alkaline conditions, while others are readily dispensable. 

The use of engineered nanomaterials, defined as those smaller than 100 nm, in the health, energy, agricultural, and environmental sectors is expanding rapidly. As such, human and environmental exposure to these materials is increasing every day. For example, metal-based nanomaterials, such as nanosilver, have become ubiquitous in antibacterial applications ranging from socks and baby bottles to healthcare materials, such as oral fillings. Engineered nanomaterials are also used as antibacterial agents and adjuvants to improve antibiotic delivery or efficacy. However, even nanomaterials that were not designed to be antimicrobial can possess potent bactericidal activity. Alarmingly, there are clear connections between nanomaterial exposure, metal resistance, and antibiotic resistance and it is crucial that we dramatically improve our understanding of both the toxicity of these materials and their ability to permanently change the organisms that they encounter. Emerging research indicates that microbes are capable of adapting to nanomaterial toxicity, often with the same generalizable mechanisms used to overcome antibiotic toxicity. In this perspective, we highlight existing knowledge about microbial response to engineered nanomaterials and the key outstanding questions that must be addressed. 

Use of complex metal oxide nanoparticles has drastically risen in recent years, especially due to their utility in electric vehicle batteries. However, use of these materials has outpaced our understanding of how they might affect environmental organisms, which they could encounter through release during manufacture, use, and disposal. In particular, little is known about the effects of chronic exposure to complex metal oxide nanoparticles. Here, we have focused on an environmentally-relevant bacterial species, Shewanella oneidensis, which is ubiquitous in nature and responsible for bioremediation of heavy metals, and assessed the toxic effects of nanoscale lithiated nickel manganese cobalt oxide (NMC), which is an emerging battery cathode material for electronic devices. We previously reported that chronic exposure of S. oneidensis to NMC results in the emergence of an adaptive phenotype where the bacteria are able to tolerate otherwise lethal concentrations of NMC. In the present study, we aim to investigate the role of reactive oxygen species (ROS) and changes in phenotype of the NMC-adapted bacterial population. We found that NMC-exposed bacteria possess ROS-containing membrane vesicles, as well as an increased propensity to generate random DNA mutations and harbor other DNA damage. Thus, our data indicate substantial genetic-level variation in bacteria that results from chronic exposure to toxic complex metal oxide nanomaterials. 

Histidine kinases (HKs) are sensor proteins found ubiquitously in prokaryotes. They are the first protein in two-component systems (TCSs), signaling pathways that respond to a myriad of environmental stimuli. TCSs are typically comprised of a HK and its cognate response regulator (RR) which often acts as a transcription factor. RRs will bind DNA and ultimately lead to a cellular response. These cellular outputs vary widely, but HKs are particularly interesting as they are tied to antibiotic resistance and virulence pathways in pathogenic bacteria, making them promising drug targets. We anticipate that HK inhibitors could serve as either standalone antibiotics or antivirulence therapies. Additionally, while the cellular response mediated by the HKs is often well-characterized, very little is known about which stimuli trigger the sensor kinase to begin the phosphorylation cascade. Studying HK activity and enrichment of active HKs through activity-based protein profiling will enable these stimuli to be elucidated, filling this fundamental gap in knowledge. Here, we describe methods to evaluate the potency of putative HK inhibitors in addition to methods to calculate kinetic parameters of various activity-based probes designed for the HKs. 

Engineered nanoparticles are incorporated into numerous emerging technologies because of their unique physical and chemical properties. Many of these properties facilitate novel interactions, including both intentional and accidental effects on biological systems. Silver-containing particles are widely used as antimicrobial agents and recent evidence indicates that bacteria rapidly become resistant to these nanoparticles. Much less studied is the chronic exposure of bacteria to particles that were not designed to interact with microorganisms. For example, previous work has demonstrated that the lithium intercalated battery cathode nanosheet, nickel manganese cobalt oxide (NMC), is cytotoxic and causes a significant delay in growth of Shewanella oneidensis MR-1 upon acute exposure. Here, we report that S. oneidensis MR-1 rapidly adapts to chronic NMC exposure and is subsequently able to survive in much higher concentrations of these particles, providing the first evidence of permanent bacterial resistance following exposure to nanoparticles that were not intended as antibacterial agents. We also found that when NMC-adapted bacteria were subjected to only the metal ions released from this material, their specific growth rates were higher than when exposed to the nanoparticle. As such, we provide here the first demonstration of bacterial resistance to complex metal oxide nanoparticles with an adaptation mechanism that cannot be fully explained by multi-metal adaptation. Importantly, this adaptation persists even after the organism has been grown in pristine media for multiple generations, indicating that S. oneidensis MR-1 has developed permanent resistance to NMC.