Glycerol metabolism contributes to competition by oral streptococci through production of hydrogen peroxide.

As a biological byproduct from both humans and microbes, glycerol's contribution to microbial homeostasis in the oral cavity remains understudied. In this study, we examined glycerol metabolism by Streptococcus sanguinis, a commensal associated with oral health. Genetic mutants of glucose-PTS enzyme II (manL), glycerol metabolism (glp and dha pathways), and transcriptional regulators were characterized with regard to glycerol catabolism, growth, production of hydrogen peroxide (H2O2), transcription, and competition with Streptococcus mutans. Biochemical assays identified the glp pathway as a novel source for H2O2 production by S. sanguinis that is independent of pyruvate oxidase (SpxB). Genetic analysis indicated that the glp pathway requires glycerol and a transcriptional regulator, GlpR, for expression and is negatively regulated by PTS, but not the catabolite control protein, CcpA. Conversely, deletion of either manL or ccpA increased the expression of spxB and a second, H2O2-non-producing glycerol metabolic pathway (dha), indicative of a mode of regulation consistent with conventional carbon catabolite repression (CCR). In a plate-based antagonism assay and competition assays performed with planktonic and biofilm-grown cells, glycerol greatly benefited the competitive fitness of S. sanguinis against S. mutans. The glp pathway appears to be conserved in several commensal streptococci and actively expressed in caries-free plaque samples. Our study suggests that glycerol metabolism plays a more significant role in the ecology of the oral cavity than previously understood. Commensal streptococci, though not able to use glycerol as a sole carbohydrate source for growth, benefit from the catabolism of glycerol through production of both ATP and H2O2. IMPORTANCE: Glycerol is an abundant carbohydrate in the oral cavity. However, little is understood regarding the metabolism of glycerol by commensal streptococci, some of the most abundant oral bacteria. This was in part because most streptococci cannot grow on glycerol as the sole carbon source. In this study, we show that Streptococcus sanguinis, a commensal associated with dental health, can degrade glycerol for persistence and competition through two pathways, one of which generates hydrogen peroxide at levels capable of inhibiting Streptococcus mutans. Preliminary studies suggest that several additional commensal streptococci are also able to catabolize glycerol, and glycerol-related genes are actively expressed in human dental plaque samples. Our findings reveal the potential of glycerol to significantly impact microbial homeostasis, which warrants further exploration.

Glucose Phosphotransferase System Modulates Pyruvate Metabolism, Bacterial Fitness, and Microbial Ecology in Oral Streptococci.

Spontaneous mutants with defects in the primary glucose phosphotransferase permease (manLMNO) of Streptococcus sanguinis SK36 showed enhanced fitness at low pH. Transcriptomics and metabolomics with a manL deletion mutant (SK36/manL) revealed redirection of pyruvate to production of acetate and formate, rather than lactate. These observations were consistent with measurements of decreased lactic acid accumulation and increased excretion of acetate, formate, pyruvate, and H2O2. Genes showing increased expression in SK36/manL included those encoding carbohydrate transporters, extracellular glycosidases, intracellular polysaccharide metabolism, and arginine deiminase and pathways for metabolism of acetoin, ethanolamine, ascorbate, and formate, along with genes required for membrane biosynthesis and adhesion. Streptococcus mutans UA159 persisted much better in biofilm cocultures with SK36/manL than with SK36, an effect that was further enhanced by culturing the biofilms anaerobically but dampened by adding arginine to the medium. We posited that the enhanced persistence of S. mutans with SK36/manL was in part due to excess excretion of pyruvate by the latter, as addition of pyruvate to S. mutans-S. sanguinis cocultures increased the proportions of UA159 in the biofilms. Reducing the buffer capacity or increasing the concentration of glucose benefited UA159 when cocultured with SK36, but not with SK36/manL, likely due to the altered metabolism and enhanced acid tolerance of the mutant. When manL was deleted in S. mutans or Streptococcus gordonii, the mutants presented altered fitness characteristics. Our study demonstrated that phosphotransferase system (PTS)-dependent modulation of central metabolism can profoundly affect streptococcal fitness and metabolic interactions, revealing another dimension in commensal-pathogen relationships influencing dental caries development. IMPORTANCE Dental caries is underpinned by a dysbiotic microbiome and increased acid production. As beneficial bacteria that can antagonize oral pathobionts, oral streptococci such as S. sanguinis and S. gordonii can ferment many carbohydrates, despite their relative sensitivity to low pH. We characterized the molecular basis for why mutants of glucose transporter ManLMNO of S. sanguinis showed enhanced production of hydrogen peroxide and ammonia and improved persistence under acidic conditions. A metabolic shift involving more than 300 genes required for carbohydrate transport, energy production, and envelope biogenesis was observed. Significantly, manL mutants engineered in three different oral streptococci displayed altered capacities for acid production and interspecies antagonism, highlighting the potential for targeting the glucose-PTS to modulate the pathogenicity of oral biofilms.

A single system detects and protects the beneficial oral bacterium Streptococcus sp. A12 from a spectrum of antimicrobial peptides.

The commensal bacterium Streptococcus sp. A12 has multiple properties that may promote the stability of health-associated oral biofilms, including overt antagonism of the dental caries pathogen Streptococcus mutans. A LanFEG-type ABC transporter, PcfFEG, confers tolerance to the lantibiotic nisin and enhances the ability of A12 to compete against S. mutans. Here, we investigated the regulation of pcfFEG and adjacent genes for a two-component system, pcfRK, to better understand antimicrobial peptide resistance by A12. Induction of pcfFEG-pcfRK was the primary mechanism to respond rapidly to nisin. In addition to nisin, PcfFEG conferred tolerance by A12 to a spectrum of lantibiotic and non-lantibiotic antimicrobial peptides produced by a diverse collection of S. mutans isolates. Loss of PcfFEG resulted in the altered spatio-temporal arrangement of A12 and S. mutans in a dual-species biofilm model. Deletion of PcfFEG or PcfK resulted in constitutive activation of pcfFEG and expression of pcfFEG was inhibited by small peptides in the pcfK mutant. Transcriptional profiling of pcfR or pcfK mutants combined with functional genomics revealed peculiarities in PcfK function and a novel panel of genes responsive to nisin. Collectively, the results provide fundamental insights that strengthen the foundation for the design of microbial-based therapeutics to control oral infectious diseases.

Activation of TnSmu1, an integrative and conjugative element, by an ImmR-like transcriptional regulator in Streptococcus mutans.

Integrative and conjugative elements (ICEs) are chromosomally encoded mobile genetic elements that can transfer DNA between bacterial strains. Recently, as part of efforts to determine hypothetical gene functions, we have discovered an important regulatory module encoded on an ICE known as TnSmu1 on the Streptococcus mutans chromosome. The regulatory module consists of a cI-like repressor with a helix-turn-helix DNA binding domain immR Smu (immunity repressor) and a metalloprotease immA Smu (anti-repressor). It is not possible to create an in-frame deletion mutant of immR Smu and repression of immR Smu with CRISPRi (CRISPR interference) causes substantial cell defects. We used a bypass of essentiality (BoE) screen to discover genes that allow deletion of the regulatory module. This revealed that conjugation genes, located within TnSmu1, can restore the viability of an immR Smu mutant. Deletion of immR Smu also leads to production of a circular intermediate form of TnSmu1, which is also inducible by the genotoxic agent mitomycin C. To gain further insights into potential regulation of TnSmu1 by ImmRSmu and broader effects on S. mutans UA159 physiology, we used CRISPRi and RNA-seq. Strongly induced genes included all the TnSmu1 mobile element, genes involved in amino acid metabolism, transport systems and a type I-C CRISPR-Cas system. Lastly, bioinformatic analysis shows that the TnSmu1 mobile element and its associated genes are well distributed across S. mutans isolates. Taken together, our results show that activation of TnSmu1 is controlled by the immRA Smu module, and that activation is deleterious to S. mutans, highlighting the complex interplay between mobile elements and their host.

Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function.

A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.

Spontaneous Mutants of Streptococcus sanguinis with Defects in the Glucose-Phosphotransferase System Show Enhanced Post-Exponential-Phase Fitness.

Genetic truncations in a gene encoding a putative glucose-phosphotransferase system (PTS) protein (manL, EIIABMan) were identified in subpopulations of two separate laboratory stocks of Streptococcus sanguinis SK36; the mutants had reduced PTS activities on glucose and other monosaccharides. To understand the emergence of these mutants, we engineered deletion mutants of manL and showed that the ManL-deficient strain had improved bacterial viability in the stationary phase and was better able to inhibit the growth of the dental caries pathogen Streptococcus mutans. Transcriptional analysis and biochemical assays suggested that the manL mutant underwent reprograming of central carbon metabolism that directed pyruvate away from production of lactate, increasing production of hydrogen peroxide (H2O2) and excretion of pyruvate. Addition of pyruvate to the medium enhanced the survival of SK36 in overnight cultures. Meanwhile, elevated pyruvate levels were detected in the cultures of a small but significant percentage (∼10%) of clinical isolates of oral commensal bacteria. Furthermore, the manL mutant showed higher expression of the arginine deiminase system than the wild type, which enhanced the ability of the mutant to raise environmental pH when arginine was present. To our surprise, significant discrepancies in genome sequence were identified between strain SK36 obtained from ATCC and the sequence deposited in GenBank. As the conditions that are likely associated with the emergence of spontaneous manL mutations, i.e., excess carbohydrates and low pH, are those associated with caries development, we propose that glucose-PTS strongly influences commensal-pathogen interactions by altering the production of ammonia, pyruvate, and H2O2. IMPORTANCE A health-associated dental microbiome provides a potent defense against pathogens and diseases. Streptococcus sanguinis is an abundant member of a health-associated oral flora that antagonizes pathogens by producing hydrogen peroxide. There is a need for a better understanding of the mechanisms that allow bacteria to survive carbohydrate-rich and acidic environments associated with the development of dental caries. We report the isolation and characterization of spontaneous mutants of S. sanguinis with impairment in glucose transport. The resultant reprograming of the central metabolism in these mutants reduced the production of lactic acid and increased pyruvate accumulation; the latter enables these bacteria to better cope with hydrogen peroxide and low pH. The implications of these discoveries in the development of dental caries are discussed.

Amino Sugars Reshape Interactions between Streptococcus mutans and Streptococcus gordonii.

Amino sugars, particularly glucosamine (GlcN) and N-acetylglucosamine (GlcNAc), are abundant carbon and nitrogen sources supplied in host secretions and in the diet to the biofilms colonizing the human oral cavity. Evidence is emerging that these amino sugars provide ecological advantages to beneficial commensals over oral pathogens and pathobionts. Here, we performed transcriptome analysis on Streptococcus mutans and Streptococcus gordonii growing in single-species or dual-species cultures with glucose, GlcN, or GlcNAc as the primary carbohydrate source. Compared to glucose, GlcN caused drastic transcriptomic shifts in each species of bacteria when it was cultured alone. Likewise, cocultivation in the presence of GlcN yielded transcriptomic profiles that were dramatically different from the single-species results from GlcN-grown cells. In contrast, GlcNAc elicited only minor changes in the transcriptome of either organism in single- and dual-species cultures. Interestingly, genes involved in pyruvate metabolism were among the most significantly affected by GlcN in both species, and these changes were consistent with measurements of pyruvate in culture supernatants. Differing from what was found in a previous report, growth of S. mutans alone with GlcN inhibited the expression of multiple operons required for mutacin production. Cocultivation with S. gordonii consistently increased the expression of two manganese transporter operons (slo and mntH) and decreased expression of mutacin genes in S. mutans Conversely, S. gordonii appeared to be less affected by the presence of S. mutans but did show increases in genes for biosynthetic processes in the cocultures. In conclusion, amino sugars profoundly alter the interactions between pathogenic and commensal streptococci by reprogramming central metabolism.IMPORTANCE Carbohydrate metabolism is central to the development of dental caries. A variety of sugars available to dental microorganisms influence the development of caries by affecting the physiology, ecology, and pathogenic potential of tooth biofilms. Using two well-characterized oral bacteria, one pathogen (Streptococcus mutans) and one commensal (Streptococcus gordonii), in an RNA deep-sequencing analysis, we studied the impact of two abundant amino sugars on bacterial gene expression and interspecies interactions. The results indicated large-scale remodeling of gene expression induced by GlcN in particular, affecting bacterial energy generation, acid production, protein synthesis, and release of antimicrobial molecules. Our study provides novel insights into how amino sugars modify bacterial behavior, information that will be valuable in the design of new technologies to detect and prevent oral infectious diseases.

Discovering candidate genes that regulate resin canal number in Pinus taeda stems by integrating genetic analysis across environments, ages, and populations.

Genetically improving constitutive resin canal development in Pinus stems may enhance the capacity to synthesize terpenes for bark beetle resistance, chemical feedstocks, and biofuels. To discover genes that potentially regulate axial resin canal number (RCN), single nucleotide polymorphisms (SNPs) in 4027 genes were tested for association with RCN in two growth rings and three environments in a complex pedigree of 520 Pinus taeda individuals (CCLONES). The map locations of associated genes were compared with RCN quantitative trait loci (QTLs) in a (P. taeda × Pinus elliottii) × P. elliottii pseudo-backcross of 345 full-sibs (BC1). Resin canal number was heritable (h(2) ˜ 0.12-0.21) and positively genetically correlated with xylem growth (rg ˜ 0.32-0.72) and oleoresin flow (rg ˜ 0.15-0.51). Sixteen well-supported candidate regulators of RCN were discovered in CCLONES, including genes associated across sites and ages, unidirectionally associated with oleoresin flow and xylem growth, and mapped to RCN QTLs in BC1. Breeding is predicted to increase RCN 11% in one generation and could be accelerated with genomic selection at accuracies of 0.45-0.52 across environments. There is significant genetic variation for RCN in loblolly pine, which can be exploited in breeding for elevated terpene content.

Posteruptive Loss of Enamel Proteins Concurs with Gain in Enamel Hardness.

Tooth enamel maturation requires the removal of proteins from the mineralizing enamel matrix to allow for crystallite growth until full hardness is reached to meet the mechanical needs of mastication. While this process takes up to several years in humans before the tooth erupts, it is greatly accelerated in in the faster developing pig. As a result, pig teeth erupt with softer, protein-rich enamel that is similar to hypomineralized human enamel but continues to harden quickly after eruption.Proteins, such as albumin, that bind to enamel crystals and prevent crystal growth and enamel hardening have been suggested as cause for hypomineralized human enamel that does not naturally harden after eruption. However, albumin is abundant in pig enamel. It is unclear whether fast posteruptive enamel hardening in pigs occurs despite the high protein content or requires a facilitated protein loss to allow for crystal growth. This study asked how the protein content in porcine enamel changes after eruption in relation to saliva. Based on previous data demonstrating the high albumin content in erupted porcine enamel, we hypothesize that following pre-eruptive maturation, enamel and saliva derived enzymes facilitate protein removal from porcine enamel after eruption. We analyzed enamel and the saliva proteome at three critical timepoints: at the time of tooth eruption, 2 weeks after eruption, and enamel 6 weeks after eruption. We used only fourth deciduous premolars and saliva samples from animals sacrificed at the respective time points to determine the organic content in tooth enamel, saliva, and saliva proteins within enamel. We found a decrease in the number of proteins and their abundancy in enamel with posteruptive time, including a decrease in serum albumin within enamel. The rapid decrease in the first two weeks is in line with previously reported rapid increase in mineral density of porcine enamel after eruption. In addition to the enamel proteases KLK-4 and MMP-20, we identified serine-, cysteine-, aspartic-, and metalloproteases. Some of these were only identified in enamel, while almost half of the enzymes are in common with saliva at all timepoints. Our findings suggest that the fast posteruptive enamel maturation in the porcine model coincides with saliva exchange and influx of saliva enzymes into porous enamel.

Glycerol Metabolism Contributes to Competition by Oral Streptococci through Production of Hydrogen Peroxide.

As a biological byproduct from both humans and microbes, glycerol's contribution to microbial homeostasis in the oral cavity remains understudied. Here we examined glycerol metabolism by Streptococcus sanguinis, a commensal associated with oral health. Genetic mutants of glucose-PTS enzyme II ( manL ), glycerol metabolism ( glp and dha pathways), and transcriptional regulators were characterized with regard to glycerol catabolism, growth, production of hydrogen peroxide (H 2 O 2 ), transcription, and competition with Streptococcus mutans . Biochemical assays identified the glp pathway as a novel source of H 2 O 2 production by S. sanguinis that is independent of pyruvate oxidase (SpxB). Genetic analysis indicated that the glp pathway requires glycerol and a transcriptional regulator, GlpR, for expression and is negatively regulated by PTS, but not the catabolite control protein, CcpA. Conversely, deletion of either manL or ccpA increased expression of spxB and a second, H 2 O 2 -non-producing glycerol metabolic pathway ( dha ), indicative of a mode of regulation consistent with conventional carbon catabolite repression (CCR). In a plate-based antagonism assay and competition assays performed with planktonic and biofilm-grown cells, glycerol greatly benefited the competitive fitness of S. sanguinis against S. mutans. The glp pathway appears to be conserved in several commensal streptococci and actively expressed in caries-free plaque samples. Our study suggests that glycerol metabolism plays a more significant role in the ecology of the oral cavity than previously understood. Commensal streptococci, though not able to use glycerol as a sole carbohydrate for growth, benefit from catabolism of glycerol through production of both ATP and H 2 O 2 . Importance: Glycerol is an abundant carbohydrate found in oral cavity, both due to biological activities of humans and microbes, and as a common ingredient of foods and health care products. However, very little is understood regarding the metabolism of glycerol by some of the most abundant oral bacteria, commensal streptococci. This was in part because most streptococci cannot grow on glycerol as the sole carbon source. Here we show that Streptococcus sanguinis , an oral commensal associated with dental health, can degrade glycerol for persistence and competition through two independent pathways, one of which generates hydrogen peroxide at levels capable of inhibiting a dental pathobiont, Streptococcus mutans . Preliminary studies suggest that several other commensal streptococci are also able to catabolize glycerol, and glycerol-related genes are being actively expressed in human dental plaque samples. Our findings reveal the potential of glycerol to significantly impact microbial homeostasis which warrants further exploration.

Association genetics of oleoresin flow in loblolly pine: discovering genes and predicting phenotype for improved resistance to bark beetles and bioenergy potential.

Rapidly enhancing oleoresin production in conifer stems through genomic selection and genetic engineering may increase resistance to bark beetles and terpenoid yield for liquid biofuels. We integrated association genetic and genomic prediction analyses of oleoresin flow (g 24 h(-1)) using 4854 single nucleotide polymorphisms (SNPs) in expressed genes within a pedigreed population of loblolly pine (Pinus taeda) that was clonally replicated at three sites in the southeastern United States. Additive genetic variation in oleoresin flow (h(2) ≈ 0.12-0.30) was strongly correlated between years in which precipitation varied (r(a) ≈ 0.95), while the genetic correlation between sites declined from 0.8 to 0.37 with increasing differences in soil and climate among sites. A total of 231 SNPs were significantly associated with oleoresin flow, of which 81% were specific to individual sites. SNPs in sequences similar to ethylene signaling proteins, ABC transporters, and diterpenoid hydroxylases were associated with oleoresin flow across sites. Despite this complex genetic architecture, we developed a genomic prediction model to accelerate breeding for enhanced oleoresin flow that is robust to environmental variation. Results imply that breeding could increase oleoresin flow 1.5- to 2.4-fold in one generation.

Growth of Porphyromonas gingivalis on human serum albumin triggers programmed cell death.

Aims: Gingival crevicular fluid (GCF) constitutes the primary growth substrate for Porphyromonas gingivalis in vivo. The goal of this work was to evaluate the growth of different strains of P. gingivalis on human serum albumin (HSA), a major constituent of GCF. Methods: Growth of five different strains of P. gingivalis in the HSA medium was examined and, surprisingly, three of the strains underwent autolysis within 24 h. Comparative transcriptomic analysis was used to identify genes involved in autolysis. Results: Two highly related reference strains (W50 and W83) differed dramatically in their survival when grown on HSA. Strain W83 grew fast and lysed within 24 h, while W50 survived for an additional 20 h. Differential gene expression analysis led us to a gene cluster containing enzymes involved in arginine metabolism and a gene predicted to be lytic murein transglycosylase, which are known to play a role in autolysis. Deletion of this gene (PG0139) resulted in a mutant that did not lyse, and complementation restored the HSA lysis phenotype, indicating that this enzyme plays a central role in the autolysis of P. gingivalis. Conclusions: P. gingivalis undergoes autolysis when provided with HSA as a substrate for growth.

Investigating CRISPR spacer targets and their impact on genomic diversification of Streptococcus mutans.

CRISPR-Cas is a bacterial immune system that restricts the acquisition of mobile DNA elements. These systems provide immunity against foreign DNA by encoding CRISPR spacers that help target DNA if it re-enters the cell. In this way, CRISPR spacers are a type of molecular tape recorder of foreign DNA encountered by the host microorganism. Here, we extracted ∼8,000 CRISPR spacers from a collection of over three hundred Streptococcus mutans genomes. Phage DNA is a major target of S. mutans spacers. S. mutans strains have also generated immunity against mobile DNA elements such as plasmids and integrative and conjugative elements. There may also be considerable immunity generated against bacterial DNA, although the relative contribution of self-targeting versus bona fide intra- or inter-species targeting needs to be investigated further. While there was clear evidence that these systems have acquired immunity against foreign DNA, there appeared to be minimal impact on horizontal gene transfer (HGT) constraints on a species-level. There was little or no impact on genome size, GC content and 'openness' of the pangenome when comparing between S. mutans strains with low or high CRISPR spacer loads. In summary, while there is evidence of CRISPR spacer acquisition against self and foreign DNA, CRISPR-Cas does not act as a barrier on the expansion of the S. mutans accessory genome.

Identification and Analysis of Essential Genes in Streptococcus mutans with Transposon Sequencing.

Transposon sequencing (Tn-seq) has greatly accelerated the rate at which gene function can be profiled in microbial organisms. This technique has been applied to the study of the dental caries pathogen Streptococcus mutans where it has been used to generate large transposon mutant libraries. Coupled with high-throughput sequencing and bioinformatics tools, culture of these transposon mutant libraries has facilitated the identification of essential and conditional essential genes. In this chapter, we describe a procedure for performing Tn-seq studies in S. mutans that covers pooled transposon mutant construction, in vitro culture, and DNA library sequencing and data analysis.

Characterization of a Bacterial Kinase That Phosphorylates Dihydrosphingosine to Form dhS1P.

Like other members of the phylum Bacteroidetes, the oral anaerobe Porphyromonas gingivalis synthesizes a variety of sphingolipids, similar to its human host. Studies have shown that synthesis of these lipids (dihydroceramides [DHCs]) is involved in oxidative stress resistance, the survival of P. gingivalis during stationary phase, and immune modulation. Here, we constructed a deletion mutant of P. gingivalis strain W83 with a deletion of the gene encoding DhSphK1, a protein that shows high similarity to a eukaryotic sphingosine kinase, an enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate. Our data show that deletion of the dhSphK1 gene results in a shift in the sphingolipid composition of P. gingivalis cells; specifically, the mutant synthesizes higher levels of phosphoglycerol DHCs (PG-DHCs) than the parent strain W83. Although PG1348 shows high similarity to the eukaryotic sphingosine kinase, we discovered that the PG1348 enzyme is unique, since it preferentially phosphorylates dihydrosphingosine, not sphingosine. Besides changes in lipid composition, the W83 ΔPG1348 mutant displayed a defect in cell division, the biogenesis of outer membrane vesicles (OMVs), and the amount of K antigen capsule. Taken together, we have identified the first bacterial dihydrosphingosine kinase whose activity regulates the lipid profile of P. gingivalis and underlies a regulatory mechanism of immune modulation. IMPORTANCE Sphingoid base phosphates, such as sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (dhS1P), act as ligands for S1P receptors, and this interaction is known to play a central role in mediating angiogenesis, vascular stability and permeability, and immune cell migration to sites of inflammation. Studies suggest that a shift in ratio to higher levels of dhS1P in relation to S1P alters downstream signaling cascades due to differential binding and activation of the various S1P receptor isoforms. Specifically, higher levels of dhS1P are thought to be anti-inflammatory. Here, we report on the characterization of a novel kinase in Porphyromonas gingivalis that phosphorylates dihydrosphingosine to form dhS1P.

Unraveling city-specific signature and identifying sample origin locations for the data from CAMDA MetaSUB challenge.

BACKGROUND: Composition of microbial communities can be location-specific, and the different abundance of taxon within location could help us to unravel city-specific signature and predict the sample origin locations accurately. In this study, the whole genome shotgun (WGS) metagenomics data from samples across 16 cities around the world and samples from another 8 cities were provided as the main and mystery datasets respectively as the part of the CAMDA 2019 MetaSUB "Forensic Challenge". The feature selecting, normalization, three methods of machine learning, PCoA (Principal Coordinates Analysis) and ANCOM (Analysis of composition of microbiomes) were conducted for both the main and mystery datasets. RESULTS: Features selecting, combined with the machines learning methods, revealed that the combination of the common features was effective for predicting the origin of the samples. The average error rates of 11.93 and 30.37% of three machine learning methods were obtained for main and mystery datasets respectively. Using the samples from main dataset to predict the labels of samples from mystery dataset, nearly 89.98% of the test samples could be correctly labeled as "mystery" samples. PCoA showed that nearly 60% of the total variability of the data could be explained by the first two PCoA axes. Although many cities overlapped, the separation of some cities was found in PCoA. The results of ANCOM, combined with importance score from the Random Forest, indicated that the common "family", "order" of the main-dataset and the common "order" of the mystery dataset provided the most efficient information for prediction respectively. CONCLUSIONS: The results of the classification suggested that the composition of the microbiomes was distinctive across the cities, which could be used to identify the sample origins. This was also supported by the results from ANCOM and importance score from the RF. In addition, the accuracy of the prediction could be improved by more samples and better sequencing depth.

Unraveling City-Specific Microbial Signatures and Identifying Sample Origins for the Data From CAMDA 2020 Metagenomic Geolocation Challenge.

The composition of microbial communities has been known to be location-specific. Investigating the microbial composition across different cities enables us to unravel city-specific microbial signatures and further predict the origin of unknown samples. As part of the CAMDA 2020 Metagenomic Geolocation Challenge, MetaSUB provided the whole genome shotgun (WGS) metagenomics data from samples across 28 cities along with non-microbial city data for 23 of these cities. In our solution to this challenge, we implemented feature selection, normalization, clustering and three methods of machine learning to classify the cities based on their microbial compositions. Of the three methods, multilayer perceptron obtained the best performance with an error rate of 19.60% based on whether the correct city received the highest or second highest number of votes for the test data contained in the main dataset. We then trained the model to predict the origins of samples from the mystery dataset by including these samples with the additional group label of "mystery." The mystery dataset compromised of samples collected from a subset of the cities in the main dataset as well as samples collected from new cities. For samples from cities that belonged to the main dataset, error rates ranged from 18.18 to 72.7%. For samples from new cities that did not belong to the main dataset, 57.7% of the test samples could be correctly labeled as "mystery" samples. Furthermore, we also predicted some of the non-microbial features for the mystery samples from the cities that did not belong to main dataset to draw inferences and narrow the range of the possible sample origins using a multi-output multilayer perceptron algorithm.

Direct interactions with commensal streptococci modify intercellular communication behaviors of Streptococcus mutans.

The formation of dental caries is a complex process that ultimately leads to damage of the tooth enamel from acids produced by microbes in attached biofilms. The bacterial interactions occurring within these biofilms between cariogenic bacteria, such as the mutans streptococci, and health-associated commensal streptococci, are thought to be critical determinants of health and disease. To better understand these interactions, a Streptococcus mutans reporter strain that actively monitors cell-cell communication via peptide signaling was cocultured with different commensal streptococci. Signaling by S. mutans, normally highly active in monoculture, was completely inhibited by several species of commensals, but only when the bacteria were in direct contact with S. mutans. We identified a novel gene expression pattern that occurred in S. mutans when cultured directly with these commensals. Finally, mutant derivatives of commensals lacking previously shown antagonistic gene products displayed wild-type levels of signal inhibition in cocultures. Collectively, these results reveal a novel pathway(s) in multiple health-associated commensal streptococci that blocks peptide signaling and induces a common contact-dependent pattern of differential gene expression in S. mutans. Understanding the molecular basis for this inhibition will assist in the rational design of new risk assessments, diagnostics, and treatments for the most pervasive oral infectious diseases.

A Novel Regulation of K-antigen Capsule Synthesis in Porphyromonas gingivalis Is Driven by the Response Regulator PG0720-Directed Antisense RNA.

The periodontal pathogen Porphyromonas gingivalis strain W83 displays at least three different surface glycans, specifically two types of lipopolysaccharides (O-LPS and A-LPS) and K-antigen capsule. Despite the importance of K-antigen capsule to the virulence of P. gingivalis, little is known as to how expression of genes involved in the synthesis of this surface glycan is regulated. The genes required for K-antigen capsule synthesis are located in a locus that encodes a number of transcripts, including an operon (PG0104 to PG0121, generating ~19.4-kb transcript) which contains a non-coding 77-bp inverted repeat (77 bpIR) region near the 5'-end. Previously, we identified a 550-nucleotide antisense RNA molecule (designated asSuGR for antisense Surface Glycan Regulator) encoded within the 77-bpIR element that influences the synthesis of surface glycans. In this study, we demonstrate that the DNA-binding response regulator PG0720 can bind the promoter region of asSuGR and activate expression of asSuGR, indicating that PG0720 may indirectly influence transcript levels of the K-antigen capsule operon expressed from the sense strand. The data show that deletion of the PG0720 gene confers a defect in the presentation of surface polysaccharides compared with the parent strain and quantitative RT-PCR (qPCR) analysis determined that the overall expression of genes involved in K-antigen capsule synthesis were down-regulated in the PG0720 mutant. Furthermore, the defects of the PG0720 deletion mutant were restored by complementation. Importantly, the PG0720 deletion mutant showed reduced virulence. Altogether, our data show that the response regulator PG0720 regulates expression of asSuGR, a trans-acting antisense RNA molecule involved in modulating the production of surface polysaccharides in P. gingivalis strain W83. The data provide further evidence that surface glycans are key virulence determinants and significantly advances our understanding of the molecular mechanisms controlling the synthesis of P. gingivalis K-antigen capsule, a key virulence determinant.

Genome-Wide Association Study of Wood Anatomical and Morphological Traits in Populus trichocarpa.

To understand the genetic mechanisms underlying wood anatomical and morphological traits in Populus trichocarpa, we used 869 unrelated genotypes from a common garden in Clatskanie, Oregon that were previously collected from across the distribution range in western North America. Using GEMMA mixed model analysis, we tested for the association of 25 phenotypic traits and nine multitrait combinations with 6.741 million SNPs covering the entire genome. Broad-sense trait heritabilities ranged from 0.117 to 0.477. Most traits were significantly correlated with geoclimatic variables suggesting a role of climate and geography in shaping the variation of this species. Fifty-seven SNPs from single trait GWAS and 11 SNPs from multitrait GWAS passed an FDR threshold of 0.05, leading to the identification of eight and seven nearby candidate genes, respectively. The percentage of phenotypic variance explained (PVE) by the significant SNPs for both single and multitrait GWAS ranged from 0.01% to 6.18%. To further evaluate the potential roles of candidate genes, we used a multi-omic network containing five additional data sets, including leaf and wood metabolite GWAS layers and coexpression and comethylation networks. We also performed a functional enrichment analysis on coexpression nearest neighbors for each gene model identified by the wood anatomical and morphological trait GWAS analyses. Genes affecting cell wall composition and transport related genes were enriched in wood anatomy and stomatal density trait networks. Signaling and metabolism related genes were also common in networks for stomatal density. For leaf morphology traits (leaf dry and wet weight) the networks were significantly enriched for GO terms related to photosynthetic processes as well as cellular homeostasis. The identified genes provide further insights into the genetic control of these traits, which are important determinants of the suitability and sustainability of improved genotypes for lignocellulosic biofuel production.