Ticks Resist Skin Commensals with Immune Factor of Bacterial Origin.

Ticks transmit a diverse array of microbes to vertebrate hosts, including human pathogens, which has led to a human-centric focus in this vector system. Far less is known about pathogens of ticks themselves. Here, we discover that a toxin in blacklegged ticks (Ixodes scapularis) horizontally acquired from bacteria-called domesticated amidase effector 2 (dae2)-has evolved to kill mammalian skin microbes with remarkable efficiency. Secreted into the saliva and gut of ticks, Dae2 limits skin-associated staphylococci in ticks while feeding. In contrast, Dae2 has no intrinsic ability to kill Borrelia burgdorferi, the tick-borne Lyme disease bacterial pathogen. These findings suggest ticks resist their own pathogens while tolerating symbionts. Thus, just as tick symbionts can be pathogenic to humans, mammalian commensals can be harmful to ticks. Our study underscores how virulence is context-dependent and bolsters the idea that "pathogen" is a status and not an identity.

An Affair to Remember: How an Endosymbiont Partners with Its Host to Build a Cell Envelope.

Studying endosymbionts gives us insight into early cellular mechanisms that led to the emergence of eukaryotic organelles. In this issue of Cell, Bublitz et al. (2019) report on how a nested bacterial endosymbiont of mealybugs builds its cell wall peptidoglycan through a biosynthetic pathway that is dependent on transported host enzymes.

Imaging Bacterial Cell Wall Biosynthesis.

Peptidoglycan is an essential component of the cell wall that protects bacteria from environmental stress. A carefully coordinated biosynthesis of peptidoglycan during cell elongation and division is required for cell viability. This biosynthesis involves sophisticated enzyme machineries that dynamically synthesize, remodel, and degrade peptidoglycan. However, when and where bacteria build peptidoglycan, and how this is coordinated with cell growth, have been long-standing questions in the field. The improvement of microscopy techniques has provided powerful approaches to study peptidoglycan biosynthesis with high spatiotemporal resolution. Recent development of molecular probes further accelerated the growth of the field, which has advanced our knowledge of peptidoglycan biosynthesis dynamics and mechanisms. Here, we review the technologies for imaging the bacterial cell wall and its biosynthesis activity. We focus on the applications of fluorescent d-amino acids, a newly developed type of probe, to visualize and study peptidoglycan synthesis and dynamics, and we provide direction for prospective research.

Triple serine loop region regulates the aspartate racemase activity of the serine/aspartate racemase family.

Recently, we cloned and characterized eleven serine and aspartate racemases (SerR and AspR, respectively) from animals. These SerRs and AspRs are not separated by their racemase functions and form a serine/aspartate racemase family cluster based on phylogenetic analysis. Moreover, we have proposed that the AspR-specific triple serine loop region at amino acid positions 150-152 may be responsible for the large AspR activity. In the present study, to test this hypothesis, we prepared and characterized fourteen mutants in this region of animal SerRs and AspRs. The large AspR activity in Acropora and Crassostrea AspR was reduced to <0.04% of wild-type after substitution of the triple serine loop region. Conversely, introducing the triple serine loop region into Acropora, Crassostrea, and Penaeus SerR drastically increased the AspR activity. Those mutants showed similar or higher substrate affinity for aspartate than serine and showed 11-683-fold higher k cat and 28-351-fold higher k cat/K m values for aspartate than serine racemization. Furthermore, we introduced serine residues in all combinations at position 150-152 in mouse SerR. These mutants revealed that a change in the enzyme function from SerR to AspR can be caused by introduction of Ser151 and Ser152, and addition of the third serine residue at position 150 further enhances the enzyme specificity for aspartate due to a decrease in the serine racemase and serine dehydratase activity. Here, we provide convincing evidence that the AspR gene has evolved from the SerR gene by acquisition of the triple serine loop region.

Distribution and evolution of the serine/aspartate racemase family in invertebrates.

Free D-amino acids have been found in various invertebrate phyla, while amino acid racemase genes have been identified in few species. The purpose of this study is to elucidate the distribution, function, and evolution of amino acid racemases in invertebrate animals. We searched the GenBank databases, and found 11 homologous serine racemase genes from eight species in eight different invertebrate phyla. The cloned genes were identified based on their maximum activity as Acropora millepora (Cnidaria) serine racemase (SerR) and aspartate racemase (AspR), Caenorhabditis elegans (Nematoda) SerR, Capitella teleta (Annelida) SerR, Crassostrea gigas (Mollusca) SerR and AspR, Dugesia japonica (Platyhelminthes) SerR, Milnesium tardigradum (Tardigrada) SerR, Penaeus monodon (Arthropoda) SerR and AspR and Strongylocentrotus purpuratus (Echinodermata) AspR. We found that Acropora, Aplysia, Capitella, Crassostrea and Penaeus had two amino acid racemase paralogous genes and these paralogous genes have evolved independently by gene duplication at their recent ancestral species. The transcriptome analyses using available SRA data and enzyme kinetic data suggested that these paralogous genes are expressed in different tissues and have different functions in vivo. Phylogenetic analyses clearly indicated that animal SerR and AspR are not separated by their particular racemase functions and form a serine/aspartate racemase family cluster. Our results revealed that SerR and AspR are more widely distributed among invertebrates than previously known. Moreover, we propose that the triple serine loop motif at amino acid positions 150-152 may be responsible for the large aspartate racemase activity and the AspR evolution from SerR.

Bacterial synthesis of D-amino acids.

Recent work has shed light on the abundance and diversity of D-amino acids in bacterial extracellular/periplasmic molecules, bacterial cell culture, and bacteria-rich environments. Within the extracellular/periplasmic space, D-amino acids are necessary components of peptidoglycan, and disruption of their synthesis leads to cell death. As such, enzymes responsible for D-amino acid synthesis are promising targets for antibacterial compounds. Further, bacteria are shown to incorporate a diverse collection of D-amino acids into their peptidoglycan, and differences in D-amino acid incorporation may occur in response to differences in growth conditions. Certain D-amino acids can accumulate to millimolar levels in cell culture, and their synthesis is proposed to foretell movement from exponential growth phase into stationary phase. While enzymes responsible for synthesis of D-amino acids necessary for peptidoglycan (D-alanine and D-glutamate) have been characterized from a number of different bacteria, the D-amino acid synthesis enzymes characterized to date cannot account for the diversity of D-amino acids identified in bacteria or bacteria-rich environments. Free D-amino acids are synthesized by racemization or epimerization at the α-carbon of the corresponding L-amino acid by amino acid racemase or amino acid epimerase enzymes. Additionally, D-amino acids can be synthesized by stereospecific amination of α-ketoacids. Below, we review the roles of D-amino acids in bacterial physiology and biotechnology, and we describe the known mechanisms by which they are synthesized by bacteria.

Amino acid racemization in Pseudomonas putida KT2440.

D-Amino acids have been shown to play an increasingly diverse role in bacterial physiology, yet much remains to be learned about their synthesis and catabolism. Here we used the model soil- and rhizosphere-dwelling organism Pseudomonas putida KT2440 to elaborate on the genomics and enzymology of d-amino acid metabolism. P. putida KT2440 catabolized the d-stereoisomers of lysine, phenylalanine, arginine, alanine, and hydroxyproline as the sole carbon and nitrogen sources. With the exception of phenylalanine, each of these amino acids was racemized by P. putida KT2440 enzymes. Three amino acid racemases were identified from a genomic screen, and the enzymes were further characterized in vitro. The putative biosynthetic alanine racemase Alr showed broad substrate specificity, exhibiting measurable racemase activity with 9 of the 19 chiral amino acids. Among these amino acids, activity was the highest with lysine, and the k(cat)/K(m) values with l- and d-lysine were 3 orders of magnitude greater than the k(cat)/K(m) values with l- and d-alanine. Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. However, DadX did show 6- and 9-fold higher k(cat)/K(m) values than Alr with l- and d-alanine, respectively. The annotated proline racemase ProR of P. putida KT2440 showed negligible activity with either stereoisomer of the 19 chiral amino acids but exhibited strong epimerization activity with hydroxyproline as the substrate. Comparative genomic analysis revealed differences among pseudomonads with respect to alanine racemase genes that may point to different roles for these genes among closely related species.

Distribution and evolution of the serine/aspartate racemase family in plants.

Previous studies have shown that several d-amino acids are widely present in plants, and serine racemase (SerR), which synthesizes d-serine in vivo, has already been identified from three plant species. However, the full picture of the d-amino acid synthesis pathway in plants is not well understood. To clarify the distribution of amino acid racemases in plants, we have cloned, expressed and characterized eight SerR homologous genes from five plant species, including green alga. These SerR homologs exhibited racemase activity towards serine or aspartate and were identified on the basis of their maximum activity as SerR or aspartate racemase (AspR). The plant AspR gene is identified for the first time from Medicago truncatula, Manihot esculenta, Solanum lycopersicum, Sphagnum girgensohnii and Spirogyra pratensis. In addition to the AspR gene, three SerR genes are identified in the former three species. Phylogenetic tree analysis showed that SerR and AspR are widely distributed in plants and form a serine/aspartate racemase family cluster. The catalytic efficiency (kcat/Km) of plant AspRs was more than 100 times higher than that of plant SerRs, suggesting that d-aspartate, as well as d-serine, can be synthesized in vivo by AspR. The amino acid sequence alignment and comparison of the chromosomal gene arrangement have revealed that plant AspR genes independently evolved from SerR in each ancestral lineage of plant species by gene duplication and acquisition of two serine residues at position 150 to 152.

Cloning and characterization of a novel aspartate/glutamate racemase from the acorn worm Saccoglossus kowalevskii.

Previously, we demonstrated that the animal aspartate racemase (AspR) gene has evolved from the serine racemase (SerR) gene by acquisition of three consecutive serine residues (Ser155-Ser156-Ser157) involved in the strong AspR activity, and this event has occurred independently and frequently during animal evolution. In the present study, we cloned and characterized two mammalian SerR homologous genes from the hemichordate acorn worm (Saccoglossus kowalevskii). The enzymes have been identified as an AspR and an aspartate/glutamate racemase (Asp/GluR) on the basis of their kinetic parameters. The S. kowalevskii Asp/GluR shows comparable substrate affinity and high catalytic efficiency (kcat/Km) for both aspartate and glutamate and is the first reported enzyme from animals that can synthesize d-glutamate. Amino acid sequence alignment analysis and site-directed mutagenesis studies have revealed that the amino acid residue at position 156, which is serine in AspR and alanine in Asp/GluR, is associated with binding and recognition of glutamate and aspartate. Phylogenetic analysis suggests that the S. kowalevskii AspR gene has evolved from the SerR gene after the divergence of hemichordata and vertebrate lineages by acquisition of the three serine residues at position 155 to 157 as in the case of other animal AspR genes. Furthermore, the S. kowalevskii Asp/GluR gene is the result of AspR gene duplication and several amino acid substitutions including that of the 156th serine residue with alanine. The fact that SerR has acquired substrate specificity towards aspartate or glutamate raises the possibility that synthesis of other d-amino acids is carried out by enzymes evolved from SerR.

Fluorogenic D-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays.

Peptidoglycan is an essential cell wall component that maintains the morphology and viability of nearly all bacteria. Its biosynthesis requires periplasmic transpeptidation reactions, which construct peptide crosslinkages between polysaccharide chains to endow mechanical strength. However, tracking the transpeptidation reaction in vivo and in vitro is challenging, mainly due to the lack of efficient, biocompatible probes. Here, we report the design, synthesis and application of rotor-fluorogenic D-amino acids (RfDAAs), enabling real-time, continuous tracking of transpeptidation reactions. These probes allow peptidoglycan biosynthesis to be monitored in real time by visualizing transpeptidase reactions in live cells, as well as real-time activity assays of D,D- and L,D-transpeptidases and sortases in vitro. The unique ability of RfDAAs to become fluorescent when incorporated into peptidoglycan provides a powerful new tool to study peptidoglycan biosynthesis with high temporal resolution and prospectively enable high-throughput screening for inhibitors of peptidoglycan biosynthesis.

A Broad Spectrum Racemase in Pseudomonas putida KT2440 Plays a Key Role in Amino Acid Catabolism.

The broad-spectrum amino acid racemase (Alr) of Pseudomonas putida KT2440 preferentially interconverts the l- and d-stereoisomers of Lys and Arg. Despite conservation of broad-spectrum racemases among bacteria, little is known regarding their physiological role. Here we explore potential functional roles for Alr in P. putida KT2440. We demonstrate through cellular fractionation that Alr enzymatic activity is found in the periplasm, consistent with its putative periplasm targeting sequence. Specific activity of Alr is highest during exponential growth, and this activity corresponds with an increased accumulation of d-Lys in the growth medium. An alr gene knockout strain (Δalr) was generated and used to assess potential roles for the alr gene in peptidoglycan structure, producing soluble signaling compounds, and amino acid metabolism. The stationary phase peptidoglycan structure did not differ between wild-type and Δalr strains, indicating that products resulting from Alr activity are not incorporated into peptidoglycan under these conditions. RNA-seq was used to assess differences in the transcriptome between the wild-type and Δalr strains. Genes undergoing differential expression were limited to those involved in amino acid metabolism. The Δalr strain exhibited a limited capacity for catabolism of l-Lys and l-Arg as the sole source of carbon and nitrogen. This is consistent with a predicted role for Alr in catabolism of l-Lys by virtue of its ability to convert l-Lys to d-Lys, which is further catabolized through the l-pipecolate pathway. The metabolic profiles here also implicate Alr in catabolism of l-Arg, although the pathway by which d-Arg is further catabolized is not clear at this time. Overall, data presented here describe the primary role of Alr as important for basic amino acid metabolism.

D-Amino Acid Catabolism Is Common Among Soil-Dwelling Bacteria.

Soil and rhizosphere environments were examined in order to determine the identity and relative abundance of bacteria that catabolize d- and l-amino acids as the sole source of carbon and nitrogen. All substrates were readily catabolized by bacteria from both environments, with most d-amino acids giving similar CFU counts to their l-amino acid counterparts. CFU count ratios between l- and d-amino acids typically ranged between 2 and 1. Isolates were phylogenetically typed in order to determine the identity of d-amino acid catabolizers. Actinobacteria, specifically the Arthrobacter genus, were abundant along with members of the α- and ß-Proteobacteria classes.