YfmK is a Novel Nε-lysine Acetyltransferase that Directly Acetylates the Histone-like Protein HBsu in Bacillus Subtilis

Recently, Ne-lysine acetylation was realized to be a prevalent bacterial post-translational modification (PTM), contrary to the historical notion that this was a rare occurrence. Acetylation can impact protein function in multiple ways, by modifying conformation, interactions, subcellular localization or activity. In bacteria, hundreds of proteins are known to be acetylated, including those involved essential processes such as DNA replication, nucleoid organization, translation, cell shape, central carbon metabolism, and even several virulence factors. Despite the growing recognition that numerous proteins are acetylated, the biological significance of the vast majority of these modifications in any bacteria remains largely unknown. Previously, we characterized the Bacillus subtilis acetylome and found that the essential histone-like protein HBsu contains seven novel acetylation sites in vivo. HBsu is a bacterial nucleoid-associated protein, which is largely responsible for chromosome compaction and the coordination of DNA processes. Despite the lack of sequence or structural homology, it is generally considered to be a functional homolog of eukaryotic histones. We investigated whether acetylation is a regulatory component of the function of HBsu in nucleoid compaction. Using mutations that mimic the acetylated and unacetylated forms of the protein, we showed that the inability to acetylate key HBsu lysine residues results in a more compacted nucleoid. We further investigated the mechanism of HBsu acetylation. By screening knockouts of the ~50 putative Gcn5-N-acetyltransferase (GNAT)-domain encoding genes in B. subtilis for their effects on DNA compaction, five candidates were identified that may encode transacetylases acting on HBsu. Genetic bypass experiments demonstrated that two of these, YfmK and YdgE, can acetylate HBsu and their potential sites of action on HBsu were identified. Additionally, purified YfmK was able to directly acetylate HBsu in vitro,suggesting that it is the second identified protein acetyltransferase in B. subtilis. We propose that at least one physiological function of the acetylation of HBsu at key lysine residues is to regulate nucleoid compaction, analogously to the role of histone acetylation in eukaryotes. With the alarming rise in antibiotic resistance, the need to develop novel therapeutics is critical. Bacterial protein acetylation represents a world of untapped potential that may uncover new drug targets to replace or supplement our antiquated antibiotic arsenal. With proper study, the enzymes involved in regulation (i.e. acetylases and deacetylases) or the acetylated form of a key protein (i.e. virulence factors, essential genes, etc.) may provide valuable, druggable targets. The targeting of bacterial protein acetylation is a practical option, as targeting enzymes involved in acetylation regulation has been successful in treatment of certain cancers, latent viral and fungal infections

LrhA Regulates rpoS Translation in Response to the Rcs Phosphorelay System in Escherichia coli

Regulation of the Escherichia coli stationary-phase sigma factor RpoS is complex and occurs at multiple levels in response to different environmental stresses. One protein that reduces RpoS levels is the transcription factor LrhA, a global regulator of flagellar synthesis. Here we clarify the mechanism of this repression and provide insight into the signaling pathways that feed into this regulation. We show that LrhA represses RpoS at the level of translation in a manner that is dependent on the small RNA (sRNA) chaperone Hfq. Although LrhA also represses the transcription of the sRNA RprA, its regulation of RpoS mainly occurs independently of RprA. To better understand the physiological signals affecting this pathway, a transposon mutagenesis screen was carried out to find factors affecting LrhA activity levels. The RcsCDB phosphorelay system, a cell envelope stress-sensing pathway, was found to repress lrhA synthesis. In addition, mutations in the gene encoding the DNA motor protein FtsK induce lrhA synthesis, which may explain why such strains fail to accumulate RpoS in stationary phase

The Response Regulator SprE (RssB) Is Required for Maintaining Poly(A) Polymerase I-Degradosome Association during Stationary Phase▿ †

Poly(A) polymerase I (PAP I) is the enzyme responsible for the addition of poly(A) tails onto RNA molecules in Escherichia coli. Polyadenylation is believed to facilitate the destruction of such RNAs by the mRNA degradosome. Recently, it was discovered that the stationary-phase regulatory protein SprE (RssB) has a second function in the control of polyadenylation that is distinct from its known function in the regulated proteolysis of RpoS. In the work presented herein, we used a targeted proteomic approach to further investigate SprE's involvement in the polyadenylation pathway. Specifically, we used cryogenic cell lysis, immunopurifications on magnetic beads, and mass spectrometry to identify interacting partners of PAP I-green fluorescent protein. We provide the first in vivo evidence that PAP I interacts with the mRNA degradosome during both exponential and stationary phases and find that the degradosome can contain up to 10 different proteins under certain conditions. Moreover, we demonstrate that the majority of these PAP I interactions are formed via protein-protein interactions and that SprE plays an important role in the maintenance of the PAP I-degradosome association during stationary phase

The Response Regulator SprE (RssB) Modulates Polyadenylation and mRNA Stability in Escherichia coli▿

In Escherichia coli, the adaptor protein SprE (RssB) controls the stability of the alternate sigma factor RpoS (σ38 and σS). When nutrients are abundant, SprE binds RpoS and delivers it to ClpXP for degradation, but when carbon sources are depleted, this process is inhibited. It also has been noted that overproduction of SprE is toxic. Here we show that null mutations in pcnB, encoding poly(A) polymerase I (PAP I), and in hfq, encoding the RNA chaperone Hfq, suppress this toxicity. Since PAP I, in conjunction with Hfq, is responsible for targeting RNAs, including mRNAs, for degradation by adding poly(A) tails onto their 3′ ends, these data indicate that SprE helps modulate the polyadenylation pathway in E. coli. Indeed, in exponentially growing cells, sprE deletion mutants exhibit significantly reduced levels of polyadenylation and increased stability of specific mRNAs, similar to what is observed in a PAP I-deficient strain. In stationary phase, we show that SprE changes the intracellular localization of PAP I. Taken together, we propose that SprE plays a multifunctional role in controlling the transcriptome, regulating what is made via its effects on RpoS, and modulating what is degraded via its effects on polyadenylation and turnover of specific mRNAs

Evaluating the Efficacy of Eravacycline and Omadacycline against Extensively Drug-Resistant <i>Acinetobacter baumannii</i> Patient Isolates

For decades, the spread of multidrug-resistant (MDR) Acinetobacter baumannii has been rampant in critically ill, hospitalized patients. Traditional antibiotic therapies against this pathogen have been failing, leading to rising concerns over management options for patients. Two new antibiotics, eravacycline and omadacycline, were introduced to the market and have shown promising results in the treatment of Gram-negative infections. Since these drugs are newly available, there is limited in vitro data about their effectiveness against MDR A. baumannii or even susceptible strains. Here, we examined the effectiveness of 22 standard-of-care antibiotics, eravacycline, and omadacycline against susceptible and extensively drug-resistant (XDR) A. baumannii patient isolates from Cooper University Hospital. Furthermore, we examined selected combinations of eravacycline or omadacycline with other antibiotics against an XDR strain. We demonstrated that this collection of strains is largely resistant to monotherapies of carbapenems, fluoroquinolones, folate pathway antagonists, cephalosporins, and most tetracyclines. While clinical breakpoint data are not available for eravacycline or omadacycline, based on minimum inhibitory concentrations, eravacycline was highly effective against these strains. The aminoglycoside amikacin alone and in combination with eravacycline or omadacycline yielded the most promising results. Our comprehensive characterization offers direction in the treatment of this deadly infection in hospitalized patients

Evaluating the Effectiveness of Hospital Antiseptics on Multidrug-Resistant Acinetobacter baumannii: Understanding the Relationship between Microbicide and Antibiotic Resistance

Acinetobacter baumannii hospital infections are difficult to treat due to the rapid emergence of multidrug-resistant (MDR) strains. In addition, A. baumannii can survive in numerous adverse environments, including in the presence of common hospital antiseptics. We hypothesized that in addition to accumulating drug resistance determinants, MDR A. baumannii strains also accumulate mutations that allow for greater microbicide tolerance when compared to pan-susceptible (PS) strains. To test this hypothesis, we compared the survival of five MDR and five PS patient isolates when exposed to bleach, ethanol, quaternary ammonium compounds, chlorhexidine gluconate, and povidone. We evaluated bacteria in a free-living planktonic state and under biofilm conditions. Each disinfectant eliminated 99.9% of planktonic bacteria, but this was not the case for bacterial biofilms. Next, we characterized strains for the presence of the known microbicide-resistance genes cepA, qacE&Delta;1, qacE, and qacA. MDR strains did not survive more than PS strains in the presence of microbicides, but microbicide-resistant strains had higher survival rates under some conditions. Interestingly, the PS strains were more likely to possess microbicide-resistance genes. Microbicide resistance remains an important topic in healthcare and may be independent of antimicrobial resistance. Hospitals should consider stricter isolation precautions that take pan-susceptible strains into account

Recent Advances in Antimicrobial Peptide Hydrogels

Advances in the number and type of available biomaterials have improved medical devices such as catheters, stents, pacemakers, prosthetic joints, and orthopedic devices. The introduction of a foreign material into the body comes with a risk of microbial colonization and subsequent infection. Infections of surgically implanted devices often lead to device failure, which leads to increased patient morbidity and mortality. The overuse and improper use of antimicrobials has led to an alarming rise and spread of drug-resistant infections. To overcome the problem of drug-resistant infections, novel antimicrobial biomaterials are increasingly being researched and developed. Hydrogels are a class of 3D biomaterials consisting of a hydrated polymer network with tunable functionality. As hydrogels are customizable, many different antimicrobial agents, such as inorganic molecules, metals, and antibiotics have been incorporated or tethered to them. Due to the increased prevalence of antibiotic resistance, antimicrobial peptides (AMPs) are being increasingly explored as alternative agents. AMP-tethered hydrogels are being increasingly examined for antimicrobial properties and practical applications, such as wound-healing. Here, we provide a recent update, from the last 5 years of innovations and discoveries made in the development of photopolymerizable, self-assembling, and AMP-releasing hydrogels