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The emerging view of Nε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA) to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that Nε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of Escherichia coli proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP)-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial Nε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase.

Dental plaque is a complex oral biofilm responsible for periodontal diseases. Bacterial biofilms are often regulated by Quorum Sensing (QS) mediated by N -acyl homoserine lactones (AHLs). While their presence and roles in oral microbiota have been debated, emerging evidence suggests AHLs influence oral biofilm development. AHLs are detectable in a microbial community derived from human dental plaque cultured under 5% CO 2 but not under anaerobic conditions. Manipulating QS in this community via AHL lactonases enriched commensals and pioneer colonizers under 5% CO₂, whereas in anaerobic conditions exogenous AHLs promoted late colonizers. QS disruption reduced biofilm formation, enhanced sucrose fermentation to lactate, and altered metabolic profiles of the community depending on the lactonase substrate specificity. Our findings highlight the importance of AHL-mediated QS in oral biofilm development and suggest its differential roles under aerobic versus anaerobic conditions. Targeting QS may offer a novel strategy for managing oral biofilms and preventing periodontal disease.

To successfully colonize the oral cavity, bacteria must directly or indirectly adhere to available oral surfaces. Fusobacterium nucleatum plays an important role in oral biofilm community development due to its broad adherence abilities, serving as a bridge between members of the oral biofilm that cannot directly bind to each other. In our efforts to characterize the molecular mechanisms utilized by F. nucleatum to physically bind to key members of the oral community, we investigated the involvement of F. nucleatum outer membrane proteins in its ability to bind to the pioneer biofilm colonizer, Streptococcus gordonii. Here, we present evidence that in addition to the previously characterized fusobacterial adhesin RadD, the interaction between F. nucleatum ATCC 23726 and S. gordonii V288 involves a second outer membrane protein, which we named coaggregation mediating protein A (CmpA). We also characterized the role of CmpA in dual‐species biofilm formation with S. gordonii V288, evaluated growth‐phase‐dependent as well as biofilm expression profiles of radD and cmpA, and confirmed an important role for CmpA, especially under biofilm growth conditions. Our findings underscore the complex set of specific interactions involved in physical binding and thus community integration of interacting bacterial species. This complex set of interactions could have critical implications for the formation and maturation of the oral biofilms in vivo, and could provide clues to the mechanism behind the distribution of organisms inside the human oral cavity. Fusobacterium nucleatum utilizes multiple surface adhesins to directly bind and form biofilm with Streptococcus gordonii.

Surface attachment is the first step in biofilm formation, and the ability of bacteria to adhere to surfaces and develop a biofilm is directly influenced by electrostatic interactions between the bacteria and the chemical composition of material surfaces. Here, we investigated the influence of physical and chemical characteristics of titanium (Ti) and zirconia (ZrO 2 ) as implant abutment surfaces on the bacterial adhesion phase and compared the results to bovine enamel (BE) simulating a human tooth. To achieve this goal, we used 2 common pathogens of the oral cavity, Streptococcus mutans UA140 and Porphyromonas gingivalis 33277 . To investigate the influence of material surfaces on bacterial adhesion, we studied the surface free energy as well as the topography by atomic force microscopy, and the chemical elements composition by scanning electron microscopy equipped with an energy dispersive X-ray spectroscope. Our results indicated a hydrophobic characteristic for all of the materials; however, the presence of polar and nonpolar components could aid in understanding why greater numbers of bacteria had adhered to BE compared to the other surfaces. Our confocal microscopy data support the proposition that electrostatic interactions, indeed, affected the initial adhesion phase. Within the limitations of a laboratory study, the results revealed bacterial adhered on BE and no bacteria could be observed by confocal images on Ti and ZrO 2 implant abutment surfaces.

Discovered over a half-century ago, lipoteichoic acid (LTA) is an abundant polymer found on the surface of Gram-positive bacteria. Although LTA is essential for the survival of many Gram-positive species, knowledge of how LTA contributes to bacterial physiology has remained elusive. Recently, LTA-deficient strains have been generated in some Gram-positive species, including the human oral commensal Streptococcus gordonii . The significance of our research is that we utilized an LTA-deficient strain of S. gordonii to address why LTA is physiologically important to Gram-positive bacteria. We demonstrate that in S. gordonii , LTA plays an important role in the presentation of many cell surface-associated proteins, contributing to cell envelope homeostasis, cell-to-cell interactions in biofilms, and adhesion to eukaryotic cells. These data may broadly reflect a physiological role of LTA in Gram-positive bacteria. Lipoteichoic acid (LTA) is an abundant polymer of the Gram-positive bacterial cell envelope and is essential for many species. Whereas the exact function of LTA has not been elucidated, loss of LTA in some species affects hydrophobicity, biofilm formation, and cell division. Using a viable LTA-deficient strain of the human oral commensal Streptococcus gordonii , we demonstrated that LTA plays an important role in surface protein presentation. Cell wall fractions derived from the wild-type and LTA-deficient strains of S. gordonii were analyzed using label-free mass spectroscopy. Comparisons showed that the abundances of many proteins differed, including (i) SspA, SspB, and S. gordonii 0707 (SGO_0707) (biofilm formation); (ii) FtsE (cell division); (iii) Pbp1a and Pbp2a (cell wall biosynthesis and remodeling); and (iv) DegP (envelope stress response). These changes in cell surface protein presentation appear to explain our observations of altered cell envelope homeostasis, biofilm formation, and adhesion to eukaryotic cells, without affecting binding and coaggregation with other bacterial species, and provide insight into the phenotypes revealed by the loss of LTA in other species of Gram-positive bacteria. We also characterized the chemical structure of the LTA expressed by S. gordonii . Similarly to Streptococcus suis , S. gordonii produced a complex type I LTA, decorated with multiple d -alanylations and glycosylations. Hence, the S. gordonii LTA appears to orchestrate expression and presentation of cell surface-associated proteins and functions. IMPORTANCE Discovered over a half-century ago, lipoteichoic acid (LTA) is an abundant polymer found on the surface of Gram-positive bacteria. Although LTA is essential for the survival of many Gram-positive species, knowledge of how LTA contributes to bacterial physiology has remained elusive. Recently, LTA-deficient strains have been generated in some Gram-positive species, including the human oral commensal Streptococcus gordonii . The significance of our research is that we utilized an LTA-deficient strain of S. gordonii to address why LTA is physiologically important to Gram-positive bacteria. We demonstrate that in S. gordonii , LTA plays an important role in the presentation of many cell surface-associated proteins, contributing to cell envelope homeostasis, cell-to-cell interactions in biofilms, and adhesion to eukaryotic cells. These data may broadly reflect a physiological role of LTA in Gram-positive bacteria.

Staphylococcus aureus, an opportunistic pathogen member of the nasal and skin microbiota, can also be found in human oral samples and has been linked to infectious diseases of the oral cavity. As the nasal and oral cavities are anatomically connected, it is currently unclear whether S. aureus can colonize the oral cavity and become part of the oral microbiota, or if its presence in the oral cavity is simply transient. To start addressing this question, we assessed S. aureus ability to directly bind selected members of the oral microbiota as well as its ability to integrate into a human-derived complex oral microbial community in vitro. Our data show that S. aureus forms aggregates with Fusobacterium nucleatum and Porphyromonas gingivalis and that it can incorporate into the human-derived in vitro oral community. Further analysis of the F. nucleatum-S. aureus interaction revealed that the outer-membrane adhesin RadD is partially involved in aggregate formation and that the RadD-mediated interaction leads to an increase in expression of the staphylococcal global regulator gene sarA. Our findings lend support to the notion that S. aureus can become part of the complex microbiota of the human mouth, which could serve as a reservoir for S. aureus. Furthermore, direct interaction with key members of the oral microbiota could affect S. aureus pathogenicity contributing to the development of several S. aureus associated oral infections.

Objective To analyze if the microbiome community composition in primary endodontic infections is associated with clinical or radiographic factors. Materials and methods Seventy-one patients with primary endodontic infections were evaluated for percussion tenderness, presence of a sinus tract, presence of caries, sex, probing depth > 4 mm, and age. Samples from the root canals were obtained and the microbiome was subsequently characterized by 16 S rRNA amplicon sequencing. For the radiographic analysis, a subset of 12 samples with a periapical index (PAI) ≤ 2 were compared with 19 samples with PAI of 5. The Shannon and Chao1 indices were used to measure alpha diversity. Differences in abundances of genera were evaluated using the Kruskal-Wallis test with Bonferroni’s correction. Differences in community composition were evaluated using analysis of similarity (ANOSIM) with Bray-Curtis dissimilarity matrices. Results No significant differences in microbiome composition relative to clinical factors were found using ANOSIM. Teeth within the two categories of periapical index showed a similar number of species richness, and alpha diversity values P  > 0.05. Community composition was significantly affected by the periapical index (ANOSIM P  = 0.039, R  = 0.10). Larger radiographic lesions demonstrated significant increase in Prevotellaceae , Olsenella , and the motile bacteria Oribacterium , Selenomonadaceae spp ., and Treponema . Conclusion Clinical factors associated with apical periodontitis have a limited impact on the root canal microbiome composition. Community composition appears to be affected in teeth with large apical lesions.

As queer and trans scientists, we face varied and systemic barriers to our professional success, resulting in our relative absence from faculty ranks at many institutions. In this Perspective, we call for a change in faculty hiring practices and present concrete guidance to make it a more inclusive process.

The central metabolite acetyl phosphate (acP) has long been proposed to influence transcription regulation by directly transferring its phosphoryl group to a number of response regulators in many bacterial species. Here, we provide in vitro evidence for this proposition and demonstrate, using an in vitro transcription system, that acP-dependent phosphorylation of aspartate 51 of CpxR induces transcription of one of its regulon members in E. coli, cpxP. We also used this in vitro transcription system to extend our previously reported in vivo data that hypothesized that acetylation of RNA polymerase (RNAP) influences acP-dependent cpxP transcription, using glutamine as a genetic mimic for acetylated arginine 291 of the carboxy-terminal domain of RNAP α subunit. The data we present here lend strong support to the hypothesis that acP has a direct effect on transcription regulation in E. coli via phosphorylation of CpxR, and that RNAP acetylation can modulate this response. In vitro assessment of effects of phosphorylation and acetylation on transcriptional regulation in E. coli.

-acyl homoserine lactones (AHLs) are small diffusible signaling molecules that mediate a cell density-dependent bacterial communication system known as quorum sensing (QS). AHL-mediated QS regulates gene expression to control many critical bacterial behaviors including biofilm formation, pathogenicity, and antimicrobial resistance. Dental plaque is a complex multispecies oral biofilm formed by successive colonization of the tooth surface by groups of commensal, symbiotic, and pathogenic bacteria, which can contribute to tooth decay and periodontal diseases. While the existence and roles of AHL-mediated QS in oral microbiota have been debated, recent evidence indicates that AHLs play significant roles in oral biofilm development and community dysbiosis. The underlying mechanisms, however, remain poorly characterized. To better understand the importance of AHL signaling in dental plaque formation, we manipulated AHL signaling by adding AHL lactonases or exogenous AHL signaling molecules. We find that AHLs can be detected in dental plaque grown under 5% CO conditions, but not when grown under anaerobic conditions, and yet anaerobic cultures are still responsive to AHLs. QS signal disruption using lactonases leads to changes in microbial population structures in both planktonic and biofilm states, changes that are dependent on the substrate preference of the used lactonase but mainly result in the increase in the abundance of commensal and pioneer colonizer species. Remarkably, the opposite manipulation, that is the addition of exogenous AHLs increases the abundance of late colonizer bacterial species. Hence, this work highlights the importance of AHL-mediated QS in dental plaque communities, its potential different roles in anaerobic and aerobic parts of dental plaque, and underscores the potential of QS interference in the control of periodontal diseases.